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2.
Ticks Tick Borne Dis ; 13(1): 101834, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-34656945

RESUMO

The species Keterah orthonairovirus is a member of the genus Orthonairovirus. Few studies have focused on this species, and there remains no treatment for Issyk-Kul fever, an infectious disease caused by a Keterah orthonairovirus. This study was performed to characterize this species using two viruses, Issyk-Kul virus (ISKV) and Soft tick bunyavirus (STBV), in cell culture and type I interferon receptor knockout (IFNAR-/-) mice and to evaluate the efficacy of serum transfusion using a mouse model of ISKV infection. The two viruses replicated in many kinds of mammal- and tick-derived cell lines but showed few different characteristics in tropism and antigenicity against anti-viral sera in cell culture. Neither virus caused clinical signs in wild-type mice, but both caused lethal infection in IFNAR-/- mice. ISKV caused more acute death than STBV in IFNAR-/- mice. In both viral infections in IFNAR-/- mice, macroscopic abnormalities were prominent in the liver. Similar levels of viral genome between ISKV- and STBV-infected IFNAR-/- mice were observed in blood, liver, lymphoid tissues and adrenal gland at moribund stages. Hematologic abnormalities in IFNAR-/- mice infected with these viruses, including leukopenia and thrombocytopenia, and biochemical abnormalities indicating liver damage were prominent. In addition, blood levels of many kinds of cytokines and chemokines such as granulocyte colony-stimulating factor, interleukin-6, tumor necrosis factor-α, interferon gamma-induced protein 10 and monocyte chemoattractant protein-1 were elevated. ISKV-immunized serum transfusion after infection delayed the time to death of IFNAR-/- mice. Thus, the present study showed that the species Keterah orthonairovirus could proliferate in most mammal-derived cell lines and cause severe liver lesions and death in IFNAR-/- mice and that serum transfusion might be effective in treatment against Issyk-Kul fever.

3.
Immunity ; 54(10): 2385-2398.e10, 2021 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-34508662

RESUMO

Potent neutralizing SARS-CoV-2 antibodies often target the spike protein receptor-binding site (RBS), but the variability of RBS epitopes hampers broad neutralization of multiple sarbecoviruses and drifted viruses. Here, using humanized mice, we identified an RBS antibody with a germline VH gene that potently neutralized SARS-related coronaviruses, including SARS-CoV and SARS-CoV-2 variants. X-ray crystallography revealed coordinated recognition by the heavy chain of non-RBS conserved sites and the light chain of RBS with a binding angle mimicking the angiotensin-converting enzyme 2 (ACE2) receptor. The minimum footprints in the hypervariable region of RBS contributed to the breadth of neutralization, which was enhanced by immunoglobulin G3 (IgG3) class switching. The coordinated binding resulted in broad neutralization of SARS-CoV and emerging SARS-CoV-2 variants of concern. Low-dose therapeutic antibody treatment in hamsters reduced the virus titers and morbidity during SARS-CoV-2 challenge. The structural basis for broad neutralizing activity may inform the design of a broad spectrum of therapeutics and vaccines.


Assuntos
Anticorpos Amplamente Neutralizantes/imunologia , Reações Cruzadas/imunologia , SARS-CoV-2/imunologia , Animais , Betacoronavirus/imunologia , Sítios de Ligação de Anticorpos , Anticorpos Amplamente Neutralizantes/química , Anticorpos Amplamente Neutralizantes/uso terapêutico , COVID-19/prevenção & controle , COVID-19/terapia , COVID-19/virologia , Cricetinae , Humanos , Switching de Imunoglobulina , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Imunoglobulina G/química , Imunoglobulina G/imunologia , Camundongos , Domínios Proteicos , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/metabolismo
4.
PLoS Pathog ; 17(7): e1009668, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34280241

RESUMO

SARS-CoV-2 infection presents clinical manifestations ranging from asymptomatic to fatal respiratory failure. Despite the induction of functional SARS-CoV-2-specific CD8+ T-cell responses in convalescent individuals, the role of virus-specific CD8+ T-cell responses in the control of SARS-CoV-2 replication remains unknown. In the present study, we show that subacute SARS-CoV-2 replication can be controlled in the absence of CD8+ T cells in cynomolgus macaques. Eight macaques were intranasally inoculated with 105 or 106 TCID50 of SARS-CoV-2, and three of the eight macaques were treated with a monoclonal anti-CD8 antibody on days 5 and 7 post-infection. In these three macaques, CD8+ T cells were undetectable on day 7 and thereafter, while virus-specific CD8+ T-cell responses were induced in the remaining five untreated animals. Viral RNA was detected in nasopharyngeal swabs for 10-17 days post-infection in all macaques, and the kinetics of viral RNA levels in pharyngeal swabs and plasma neutralizing antibody titers were comparable between the anti-CD8 antibody treated and untreated animals. SARS-CoV-2 RNA was detected in the pharyngeal mucosa and/or retropharyngeal lymph node obtained at necropsy on day 21 in two of the untreated group but undetectable in all macaques treated with anti-CD8 antibody. CD8+ T-cell responses may contribute to viral control in SARS-CoV-2 infection, but our results indicate possible containment of subacute viral replication in the absence of CD8+ T cells, implying that CD8+ T-cell dysfunction may not solely lead to viral control failure.


Assuntos
Linfócitos T CD8-Positivos/imunologia , COVID-19/veterinária , Macaca fascicularis/imunologia , Macaca fascicularis/virologia , Doenças dos Macacos/imunologia , Doenças dos Macacos/virologia , SARS-CoV-2/imunologia , SARS-CoV-2/fisiologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , COVID-19/imunologia , COVID-19/virologia , Modelos Animais de Doenças , Feminino , Humanos , Cinética , Depleção Linfocítica/veterinária , Masculino , RNA Viral/genética , RNA Viral/metabolismo , SARS-CoV-2/genética , Replicação Viral/imunologia
5.
Pharmaceuticals (Basel) ; 14(6)2021 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-34073502

RESUMO

Identified in 2012, the Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe and often fatal acute respiratory illness in humans. No approved prophylactic or therapeutic interventions are currently available. In this study, we developed chicken egg yolk antibodies (IgY Abs) specific to the MERS-CoV spike (S) protein and evaluated their neutralizing efficiency against MERS-CoV infection. S-specific IgY Abs were produced by injecting chickens with the purified recombinant S protein of MERS-CoV at a high titer (4.4 mg/mL per egg yolk) at week 7 post immunization. Western blotting and immune-dot blot assays demonstrated specific binding to the MERS-CoV S protein. In vitro neutralization of the generated IgY Abs against MERS-CoV was evaluated and showed a 50% neutralizing concentration of 51.42 µg/mL. In vivo testing using a human-transgenic mouse model showed a reduction of viral antigen positive cells in treated mice, compared to the adjuvant-only controls. Moreover, the lung cells of the treated mice showed significantly reduced inflammation, compared to the controls. Our results show efficient neutralization of MERS-CoV infection both in vitro and in vivo using S-specific IgY Abs. Clinical trials are needed to evaluate the efficiency of the IgY Abs in camels and humans.

6.
Proc Natl Acad Sci U S A ; 118(13)2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33753513

RESUMO

Helicobacter suis, a bacterial species naturally hosted by pigs, can colonize the human stomach in the context of gastric diseases such as gastric mucosa-associated lymphoid tissue (MALT) lymphoma. Because H. suis has been successfully isolated from pigs, but not from humans, evidence linking human H. suis infection to gastric diseases has remained incomplete. In this study, we successfully in vitro cultured H. suis directly from human stomachs. Unlike Helicobacter pylori, the viability of H. suis decreases significantly on neutral pH; therefore, we achieved this using a low-pH medium for transport of gastric biopsies. Ultimately, we isolated H. suis from three patients with gastric diseases, including gastric MALT lymphoma. Successful eradication of H. suis yielded significant improvements in endoscopic and histopathological findings. Oral infection of mice with H. suis clinical isolates elicited gastric and systemic inflammatory responses; in addition, progression of gastric mucosal metaplasia was observed 4 mo postinfection. Because H. suis could be isolated from the stomachs of infected mice, our findings satisfied Koch's postulates. Although further prospective clinical studies are needed, H. suis, like H. pylori, is likely a gastric pathogen in humans. Furthermore, comparative genomic analysis of H. suis using complete genomes of clinical isolates revealed that the genome of each H. suis isolate contained highly plastic genomic regions encoding putative strain-specific virulence factors, including type IV secretion system-associated genes, and that H. suis isolates from humans and pigs were genetically very similar, suggesting possible pig-to-human transmission.


Assuntos
Infecções por Helicobacter/genética , Helicobacter heilmannii/genética , Helicobacter heilmannii/patogenicidade , Gastropatias/microbiologia , Estômago/microbiologia , Fatores de Virulência/genética , Adulto , Animais , Modelos Animais de Doenças , Feminino , Genoma Bacteriano , Helicobacter heilmannii/isolamento & purificação , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Suínos , Sistemas de Secreção Tipo IV/genética , Virulência/genética
7.
PLoS Pathog ; 17(2): e1008859, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33534867

RESUMO

Severe fever with thrombocytopenia syndrome (SFTS) caused by a species Dabie bandavirus (formerly SFTS virus [SFTSV]) is an emerging hemorrhagic infectious disease with a high case-fatality rate. One of the best strategies for preventing SFTS is to develop a vaccine, which is expected to induce both humoral and cellular immunity. We applied a highly attenuated but still immunogenic vaccinia virus strain LC16m8 (m8) as a recombinant vaccine for SFTS. Recombinant m8s expressing SFTSV nucleoprotein (m8-N), envelope glycoprotein precursor (m8-GPC), and both N and GPC (m8-N+GPC) in the infected cells were generated. Both m8-GPC- and m8-N+GPC-infected cells were confirmed to produce SFTSV-like-particles (VLP) in vitro, and the N was incorporated in the VLP produced by the infection of cells with m8-N+GPC. Specific antibodies to SFTSV were induced in mice inoculated with each of the recombinant m8s, and the mice were fully protected from lethal challenge with SFTSV at both 103 TCID50 and 105 TCID50. In mice that had been immunized with vaccinia virus strain Lister in advance of m8-based SFTSV vaccine inoculation, protective immunity against the SFTSV challenge was also conferred. The pathological analysis revealed that mice immunized with m8-GPC or m8-N+GPC did not show any histopathological changes without any viral antigen-positive cells, whereas the control mice showed focal necrosis with inflammatory infiltration with SFTSV antigen-positive cells in tissues after SFTSV challenge. The passive serum transfer experiments revealed that sera collected from mice inoculated with m8-GPC or m8-N+GPC but not with m8-N conferred protective immunity against lethal SFTSV challenge in naïve mice. On the other hand, the depletion of CD8-positive cells in vivo did not abrogate the protective immunity conferred by m8-based SFTSV vaccines. Based on these results, the recombinant m8-GPC and m8-N+GPC were considered promising vaccine candidates for SFTS.


Assuntos
Antígenos Virais/imunologia , Nucleoproteínas/imunologia , Phlebovirus/imunologia , Febre Grave com Síndrome de Trombocitopenia/prevenção & controle , Vacinas Atenuadas/administração & dosagem , Vacinas Sintéticas/administração & dosagem , Proteínas do Envelope Viral/imunologia , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Febre Grave com Síndrome de Trombocitopenia/imunologia , Febre Grave com Síndrome de Trombocitopenia/virologia
8.
J Infect Chemother ; 27(1): 120-122, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32988731

RESUMO

INTRODUCTION: Information on the effectiveness of personal protective equipment (PPE) for preventing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection among healthcare workers (HCWs), especially among HCWs with frequent contact with patients with SARS-CoV-2, is limited. METHODS: We conducted a prospective cohort study on 49 HCWs who worked in close contact with patients with SARS-CoV-2 infection. HCWs had blood samples taken every 2 weeks to test for SARS-CoV-2 antibodies using two different types of assay. RESULTS: Forty-nine participants (31 nurses, 15 doctors, 3 other workers) were enrolled. In total, 112 blood samples are obtained from participants. The median work days in 2 weeks was 9 (interquartile range (IQR): 5-10) days. In a single work day, 30 of the 49 participants (61.5%) had contact with patients with suspected or conformed SARS-CoV-2 at least 8 times, and approximately 60% of participants had more than 10 min of contact with a single patient. The median self-reported compliance to PPE was 90% (IQR: 80-100%). Seven participants tested positive for SARS-CoV-2 antibody using enzyme-linked immunosorbent assay (ELISA); however, none were seropositive for SARS-CoV-2 neutralizing antibody, so the positive ELISA results were assumed to be false-positive. CONCLUSIONS: The study provides evidence that appropriate PPE is sufficient to prevent infection amongHCWs. It is necessary to establish a system that provides a stable supply of PPE for HCWs to perform their duties.


Assuntos
Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/transmissão , Pessoal de Saúde , Transmissão de Doença Infecciosa do Paciente para o Profissional/prevenção & controle , Pandemias/prevenção & controle , Equipamento de Proteção Individual , Pneumonia Viral/prevenção & controle , Pneumonia Viral/transmissão , Adulto , Idoso , Anticorpos Antivirais/sangue , Betacoronavirus/imunologia , Betacoronavirus/isolamento & purificação , COVID-19 , Infecções por Coronavirus/diagnóstico , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pneumonia Viral/diagnóstico , Estudos Prospectivos , SARS-CoV-2 , Adulto Jovem
9.
Vaccines (Basel) ; 8(4)2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-33139631

RESUMO

The Middle East respiratory syndrome coronavirus (MERS-CoV) was identified in 2012 and causes severe and often fatal acute respiratory illness in humans. No approved prophylactic and therapeutic interventions are currently available. In this study, we have developed egg yolk antibodies (immunoglobulin Y (IgY)) specific for MERS-CoV spike protein (S1) in order to evaluate their neutralizing efficiency against MERS-CoV infection. S1-specific immunoglobulins were produced by injecting chickens with purified recombinant S1 protein of MERS-CoV at a high titer (5.7 mg/mL egg yolk) at week 7 post immunization. Western blotting and immune-dot blot assays demonstrated that the IgY antibody specifically bound to the MERS-CoV S1 protein. Anti-S1 antibodies were also able to recognize MERS-COV inside cells, as demonstrated by an immunofluorescence assay. Plaque reduction and microneutralization assays showed the neutralization of MERS-COV in Vero cells by anti-S1 IgY antibodies and non-significantly reduced virus titers in the lungs of MERS-CoV-infected mice during early infection, with a nonsignificant decrease in weight loss. However, a statistically significant (p = 0.0196) quantitative reduction in viral antigen expression and marked reduction in inflammation were observed in lung tissue. Collectively, our data suggest that the anti-MERS-CoV S1 IgY could serve as a potential candidate for the passive treatment of MERS-CoV infection.

10.
Viruses ; 12(9)2020 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-32842671

RESUMO

Filoviruses, including Ebola virus (EBOV) and Marburg virus (MARV), cause severe hemorrhagic fever in humans and nonhuman primates with high mortality rates. There is no approved therapy against these deadly viruses. Antiviral drug development has been hampered by the requirement of a biosafety level (BSL)-4 facility to handle infectious EBOV and MARV because of their high pathogenicity to humans. In this study, we aimed to establish a surrogate animal model that can be used for anti-EBOV and -MARV drug screening under BSL-2 conditions by focusing on the replication-competent recombinant vesicular stomatitis virus (rVSV) pseudotyped with the envelope glycoprotein (GP) of EBOV (rVSV/EBOV) and MARV (rVSV/MARV), which has been investigated as vaccine candidates and thus widely used in BSL-2 laboratories. We first inoculated mice, rats, and hamsters intraperitoneally with rVSV/EBOV and found that only hamsters showed disease signs and succumbed within 4 days post-infection. Infection with rVSV/MARV also caused lethal infection in hamsters. Both rVSV/EBOV and rVSV/MARV were detected at high titers in multiple organs including the liver, spleen, kidney, and lungs of infected hamsters, indicating acute and systemic infection resulting in fatal outcomes. Therapeutic effects of passive immunization with an anti-EBOV neutralizing antibody were specifically observed in rVSV/EBOV-infected hamsters. Thus, this animal model is expected to be a useful tool to facilitate in vivo screening of anti-filovirus drugs targeting the GP molecule.


Assuntos
Modelos Animais de Doenças , Ebolavirus/genética , Marburgvirus/genética , Estomatite Vesicular/virologia , Vesiculovirus/genética , Proteínas do Envelope Viral/genética , Animais , Anticorpos Antivirais/administração & dosagem , Cricetinae , Suscetibilidade a Doenças , Avaliação Pré-Clínica de Medicamentos , Ebolavirus/imunologia , Mesocricetus , Camundongos , Ratos , Vacinas Sintéticas , Estomatite Vesicular/patologia , Estomatite Vesicular/prevenção & controle , Estomatite Vesicular/terapia , Vesiculovirus/patogenicidade , Proteínas do Envelope Viral/imunologia , Carga Viral
11.
Proc Natl Acad Sci U S A ; 117(13): 7001-7003, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32165541

RESUMO

A novel betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which caused a large respiratory outbreak in Wuhan, China in December 2019, is currently spreading across many countries globally. Here, we show that a TMPRSS2-expressing VeroE6 cell line is highly susceptible to SARS-CoV-2 infection, making it useful for isolating and propagating SARS-CoV-2. Our results reveal that, in common with SARS- and Middle East respiratory syndrome-CoV, SARS-CoV-2 infection is enhanced by TMPRSS2.


Assuntos
Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Pneumonia Viral/virologia , Serina Endopeptidases/metabolismo , Animais , COVID-19 , Linhagem Celular , Chlorocebus aethiops , Surtos de Doenças , Humanos , Pandemias , RNA Viral/metabolismo , SARS-CoV-2 , Células Vero , Cultura de Vírus
12.
J Virol ; 94(6)2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31896594

RESUMO

Enterovirus 71 (EV71) is a causative agent of hand-foot-mouth disease, and it sometimes causes severe neurological disease. Development of effective vaccines and animal models to evaluate vaccine candidates are needed. However, the animal models currently used for vaccine efficacy testing, monkeys and neonatal mice, have economic, ethical, and practical drawbacks. In addition, EV71 strains prepared for lethal challenge often develop decreased virulence during propagation in cell culture. To overcome these problems, we used a mouse model expressing human scavenger receptor B2 (hSCARB2) that showed lifelong susceptibility to EV71. We selected virulent EV71 strains belonging to the subgenogroups B4, B5, C1, C2, and C4 and propagated them using a culture method for EV71 without an apparent reduction in virulence. Here, we describe a novel EV71 vaccine efficacy test based on these hSCARB2 transgenic (Tg) mice and these virulent viruses. Adult Tg mice were immunized subcutaneously with formalin-inactivated EV71. The vaccine elicited sufficient levels of neutralizing antibodies in the immunized mice. The mice were subjected to lethal challenge with virulent viruses via intravenous injection. Survival, clinical signs, and body weight changes were observed for 2 weeks. Most immunized mice survived without clinical signs or histopathological lesions. The viral replication in immunized mice was much lower than that in nonimmunized mice. Mice immunized with the EV71 vaccine were only partially protected against lethal challenge with coxsackievirus A16. These results indicate that this new model is useful for in vivo EV71 vaccine efficacy testing.IMPORTANCE The development of new vaccines for EV71 relies on the availability of small animal models suitable for in vivo efficacy testing. Monkeys and neonatal mice have been used, but the use of these animals has several drawbacks, including high costs, limited susceptibility, and poor experimental reproducibility. In addition, the related ethical issues are considerable. The new efficacy test based on hSCARB2 Tg mice and virulent EV71 strains propagated in genetically modified cell lines presented here can overcome these disadvantages and is expected to accelerate the development of new EV71 vaccines.


Assuntos
Enterovirus Humano A/imunologia , Doença de Mão, Pé e Boca/prevenção & controle , Glicoproteínas de Membrana Associadas ao Lisossomo/imunologia , Receptores Depuradores/imunologia , Vacinas Virais/farmacologia , Animais , Linhagem Celular , Modelos Animais de Doenças , Avaliação de Medicamentos , Enterovirus Humano A/genética , Enterovirus Humano A/patogenicidade , Doença de Mão, Pé e Boca/genética , Doença de Mão, Pé e Boca/imunologia , Doença de Mão, Pé e Boca/patologia , Humanos , Glicoproteínas de Membrana Associadas ao Lisossomo/genética , Camundongos , Camundongos Transgênicos , Receptores Depuradores/genética , Vacinas de Produtos Inativados/genética , Vacinas de Produtos Inativados/imunologia , Vacinas de Produtos Inativados/farmacologia , Vacinas Virais/genética , Vacinas Virais/imunologia
13.
Microbiol Immunol ; 64(1): 33-51, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31692019

RESUMO

The spike (S) protein of coronavirus, which binds to cellular receptors and mediates membrane fusion for cell entry, is a candidate vaccine target for blocking coronavirus infection. However, some animal studies have suggested that inadequate immunization against severe acute respiratory syndrome coronavirus (SARS-CoV) induces a lung eosinophilic immunopathology upon infection. The present study evaluated two kinds of vaccine adjuvants for use with recombinant S protein: gold nanoparticles (AuNPs), which are expected to function as both an antigen carrier and an adjuvant in immunization; and Toll-like receptor (TLR) agonists, which have previously been shown to be an effective adjuvant in an ultraviolet-inactivated SARS-CoV vaccine. All the mice immunized with more than 0.5 µg S protein without adjuvant escaped from SARS after infection with mouse-adapted SARS-CoV; however, eosinophilic infiltrations were observed in the lungs of almost all the immunized mice. The AuNP-adjuvanted protein induced a strong IgG response but failed to improve vaccine efficacy or to reduce eosinophilic infiltration because of highly allergic inflammatory responses. Whereas similar virus titers were observed in the control animals and the animals immunized with S protein with or without AuNPs, Type 1 interferon and pro-inflammatory responses were moderate in the mice treated with S protein with and without AuNPs. On the other hand, the TLR agonist-adjuvanted vaccine induced highly protective antibodies without eosinophilic infiltrations, as well as Th1/17 cytokine responses. The findings of this study will support the development of vaccines against severe pneumonia-associated coronaviruses.


Assuntos
Adjuvantes Imunológicos/farmacologia , Infecções por Coronavirus/prevenção & controle , Ouro/química , Imunoglobulina G/imunologia , Pulmão/imunologia , Nanopartículas Metálicas/química , Síndrome Respiratória Aguda Grave/prevenção & controle , Glicoproteína da Espícula de Coronavírus/imunologia , Análise de Variância , Animais , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Coronavirus/imunologia , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Imunização , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/imunologia , Vírus da SARS/imunologia , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/virologia , Glicoproteína da Espícula de Coronavírus/genética , Receptores Toll-Like , Vacinação , Vacinas Sintéticas , Células Vero , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Vacinas Virais/imunologia , Vacinas Virais/farmacologia , Vacinas Virais/uso terapêutico
14.
J Neuropathol Exp Neurol ; 79(2): 209-225, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31845989

RESUMO

Coxsackievirus B (CVB) causes severe morbidity and mortality in neonates and is sometimes associated with severe brain damage resulting from acute severe viral encephalomyelitis. However, the neuropathology of CVB infection remains unclear. A prototype strain of coxsackievirus B2 (Ohio-1) induces brain lesions in neonatal mice, resulting in dome-shaped heads, ventriculomegaly, and loss of the cerebral cortex. Here, we characterized the glial pathology in this mouse model. Magnetic resonance imaging revealed an absence of the cerebral cortex within 2 weeks after inoculation. Histopathology showed that virus replication triggered activation of microglia and astrocytes, and induced apoptosis in the cortex, with severe necrosis and lateral ventricular dilation. In contrast, the brainstem and cerebellum remained morphologically intact. Immunohistochemistry revealed high expression of the coxsackievirus and adenovirus receptor (a primary receptor for CVB) in mature neurons of the cortex, hippocampus, thalamus, and midbrain, demonstrating CVB2 infection of mature neurons in these areas. However, apoptosis and neuroinflammation from activated microglia and astrocytes differed in thalamic and cortical areas. Viral antigens were retained in the brains of animals in the convalescence phase with seroconversion. This animal model will contribute to a better understanding of the neuropathology of CVB infection.


Assuntos
Encéfalo/patologia , Encéfalo/virologia , Infecções por Coxsackievirus/patologia , Enterovirus Humano B/fisiologia , Neuroglia/patologia , Neuroglia/virologia , Animais , Animais Recém-Nascidos , Apoptose , Encéfalo/metabolismo , Modelos Animais de Doenças , Encefalite Infecciosa/metabolismo , Encefalite Infecciosa/patologia , Encefalite Infecciosa/virologia , Camundongos , Receptores Virais/metabolismo
15.
Sci Rep ; 9(1): 11990, 2019 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-31427690

RESUMO

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging hemorrhagic fever caused by the SFTS phlebovirus (SFTSV). SFTS patients were first reported in China, followed by Japan and South Korea. In 2017, cats were diagnosed with SFTS for the first time, suggesting that these animals are susceptible to SFTSV. To confirm whether or not cats were indeed susceptible to SFTSV, animal subjects were experimentally infected with SFTSV. Four of the six cats infected with the SPL010 strain of SFTSV died, all showing similar or more severe symptoms than human SFTS patients, such as a fever, leukocytopenia, thrombocytopenia, weight loss, anorexia, jaundice and depression. High levels of SFTSV RNA loads were detected in the serum, eye swab, saliva, rectal swab and urine, indicating a risk of direct human infection from SFTS-infected animals. Histopathologically, acute necrotizing lymphadenitis and hemophagocytosis were prominent in the lymph nodes and spleen. Severe hemorrhaging was observed throughout the gastrointestinal tract. B cell lineage cells with MUM-1 and CD20, but not Pax-5 in the lesions were predominantly infected with SFTSV. The present study demonstrated that cats were highly susceptible to SFTSV. The risk of direct infection from SFTS-infected cats to humans should therefore be considered.


Assuntos
Doenças do Gato/virologia , Febres Hemorrágicas Virais/veterinária , Phlebovirus/fisiologia , Animais , Biomarcadores , Biópsia , Doenças do Gato/diagnóstico , Doenças do Gato/mortalidade , Doenças do Gato/transmissão , Gatos , Suscetibilidade a Doenças , Avaliação de Sintomas
16.
Sci Rep ; 9(1): 9193, 2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31235714

RESUMO

Tularemia is a severe infectious zoonotic disease caused by Francisella tularensis. Although F. tularensis is considered to be a potential biological weapon due to its high infectivity and mortality rate, no vaccine has been currently licensed. Recently, we reported that F. tularensis SCHU P9 derived ΔpdpC strain lacking the pathogenicity determinant protein C gene conferred stable and good protection in a mouse lethal model. In this study, the protective effect of ΔpdpC was evaluated using a monkey lethal model. Two cynomolgus macaques (Macaca fascicularis) intratracheally challenged with the virulent strain SCHU P9 were euthanized on 7 and 11 days post-challenge after the development of severe clinical signs. The bacterial replication in alveolar macrophages and type II epithelial cells in the lungs would cause severe pneumonia accompanied by necrosis. Conversely, two animals subcutaneously immunized with ΔpdpC survived 3 weeks after SCHU P9 challenge. Though one of the two animals developed mild symptoms of tularemia, bacterial replication was limited in the respiratory organs, which may be due to a high level of humoral and cellular immune responses against F. tularensis. These results suggest that the ΔpdpC mutant would be a safe and promising candidate as a live attenuated tularemia vaccine.


Assuntos
Proteínas de Bactérias/genética , Vacinas Bacterianas/imunologia , Francisella tularensis/imunologia , Macaca fascicularis/imunologia , Tularemia/imunologia , Fatores de Virulência/genética , Animais , Modelos Animais de Doenças , Francisella tularensis/genética , Mutação , Tularemia/prevenção & controle , Vacinação , Vacinas Atenuadas/imunologia
17.
Viruses ; 11(3)2019 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-30836639

RESUMO

Rhinoviruses (RVs) are classified into three species: RV-A, B, and C. Unlike RV-A and -B, RV-C cannot be propagated using standard cell culture systems. In order to isolate RV-Cs from clinical specimens and gain a better understanding of their biological properties and pathogenesis, we established air⁻liquid-interface (ALI) culture methods using HBEC3-KT and HSAEC1-KT immortalized human airway epithelial cells. HBEC3- and HSAEC1-ALI cultures morphologically resembled pseudostratified epithelia with cilia and goblet cells. Two fully sequenced clinical RV-C isolates, RV-C9 and -C53, were propagated in HBEC3-ALI cultures, and increases in viral RNA ranging from 1.71 log10 to 7.06 log10 copies were observed. However, this propagation did not occur in HSAEC1-ALI cultures. Using the HBEC3-ALI culture system, 11 clinical strains of RV-C were isolated from 23 clinical specimens, and of them, nine were passaged and re-propagated. The 11 clinical isolates were classified as RV-C2, -C6, -C9, -C12, -C18, -C23, -C40, and -C53 types according to their VP1 sequences. Our stable HBEC3-ALI culture system is the first cultivable cell model that supports the growth of multiple RV-C virus types from clinical specimens. Thus, the HBEC3-ALI culture system provides a cheap and easy-to-use alternative to existing cell models for isolating and investigating RV-Cs.


Assuntos
Diferenciação Celular , Enterovirus/crescimento & desenvolvimento , Células Epiteliais/virologia , Cultura de Vírus , Contagem de Células , Linhagem Celular Transformada , Enterovirus/genética , Enterovirus/fisiologia , Humanos , Sistema Respiratório/citologia
18.
J Virol ; 93(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30626685

RESUMO

Middle East respiratory syndrome coronavirus (MERS-CoV) infection can manifest as a mild illness, acute respiratory distress, organ failure, or death. Several animal models have been established to study disease pathogenesis and to develop vaccines and therapeutic agents. Here, we developed transgenic (Tg) mice on a C57BL/6 background; these mice expressed human CD26/dipeptidyl peptidase 4 (hDPP4), a functional receptor for MERS-CoV, under the control of an endogenous hDPP4 promoter. We then characterized this mouse model of MERS-CoV. The expression profile of hDPP4 in these mice was almost equivalent to that in human tissues, including kidney and lung; however, hDPP4 was overexpressed in murine CD3-positive cells within peripheral blood and lymphoid tissues. Intranasal inoculation of young and adult Tg mice with MERS-CoV led to infection of the lower respiratory tract and pathological evidence of acute multifocal interstitial pneumonia within 7 days, with only transient loss of body weight. However, the immunopathology in young and adult Tg mice was different. On day 5 or 7 postinoculation, lungs of adult Tg mice contained higher levels of proinflammatory cytokines and chemokines associated with migration of macrophages. These results suggest that the immunopathology of MERS-CoV infection in the Tg mouse is age dependent. The mouse model described here will increase our understanding of disease pathogenesis and host mediators that protect against MERS-CoV infection.IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) infections are endemic in the Middle East and a threat to public health worldwide. Rodents are not susceptible to the virus because they do not express functional receptors; therefore, we generated a new animal model of MERS-CoV infection based on transgenic mice expressing human DPP4 (hDPP4). The pattern of hDPP4 expression in this model was similar to that in human tissues (except lymphoid tissue). In addition, MERS-CoV was limited to the respiratory tract. Here, we focused on host factors involved in immunopathology in MERS-CoV infection and clarified differences in antiviral immune responses between young and adult transgenic mice. This new small-animal model could contribute to more in-depth study of the pathology of MERS-CoV infection and aid development of suitable treatments.


Assuntos
Infecções por Coronavirus/metabolismo , Dipeptidil Peptidase 4/metabolismo , Pulmão/virologia , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade , Animais , Linhagem Celular , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Macrófagos/metabolismo , Macrófagos/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infecções Respiratórias/metabolismo , Infecções Respiratórias/virologia , Células Vero
19.
J Virol ; 93(6)2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30626688

RESUMO

Transmembrane serine protease TMPRSS2 activates the spike protein of highly pathogenic human coronaviruses such as severe acute respiratory syndrome-related coronavirus (SARS-CoV) and Middle East respiratory syndrome-related coronavirus (MERS-CoV). In vitro, activation induces virus-cell membrane fusion at the cell surface. However, the roles of TMPRSS2 during coronavirus infection in vivo are unclear. Here, we used animal models of SARS-CoV and MERS-CoV infection to investigate the role of TMPRSS2. Th1-prone C57BL/6 mice and TMPRSS2-knockout (KO) mice were used for SARS-CoV infection, and transgenic mice expressing the human MERS-CoV receptor DPP4 (hDPP4-Tg mice) and TMPRSS2-KO hDPP4-Tg mice were used for MERS-CoV infection. After experimental infection, TMPRSS2-deficient mouse strains showed reduced body weight loss and viral kinetics in the lungs. Lack of TMPRSS2 affected the primary sites of infection and virus spread within the airway, accompanied by less severe immunopathology. However, TMPRSS2-KO mice showed weakened inflammatory chemokine and/or cytokine responses to intranasal stimulation with poly(I·C), a Toll-like receptor 3 agonist. In conclusion, TMPRSS2 plays a crucial role in viral spread within the airway of murine models infected by SARS-CoV and MERS-CoV and in the resulting immunopathology.IMPORTANCE Broad-spectrum antiviral drugs against highly pathogenic coronaviruses and other emerging viruses are desirable to enable a rapid response to pandemic threats. Transmembrane protease serine type 2 (TMPRSS2), a protease belonging to the type II transmembrane serine protease family, cleaves the coronavirus spike protein, making it a potential therapeutic target for coronavirus infections. Here, we examined the role of TMPRSS2 using animal models of SARS-CoV and MERS-CoV infection. The results suggest that lack of TMPRSS2 in the airways reduces the severity of lung pathology after infection by SARS-CoV and MERS-CoV. Taken together, the results will facilitate development of novel targets for coronavirus therapy.


Assuntos
Infecções por Coronavirus/imunologia , Infecções por Coronavirus/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/imunologia , Serina Endopeptidases/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Infecções por Coronavirus/virologia , Modelos Animais de Doenças , Feminino , Humanos , Pulmão/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Poli I-C/metabolismo , Vírus da SARS , Síndrome Respiratória Aguda Grave/imunologia , Síndrome Respiratória Aguda Grave/metabolismo , Síndrome Respiratória Aguda Grave/virologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Receptor 3 Toll-Like/metabolismo , Células Vero
20.
J Virol ; 92(15)2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29848582

RESUMO

Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease and sometimes causes severe or fatal neurological complications. The amino acid at VP1-145 determines the virological characteristics of EV71. Viruses with glutamic acid (E) at VP1-145 (VP1-145E) are virulent in neonatal mice and transgenic mice expressing human scavenger receptor B2, whereas those with glutamine (Q) or glycine (G) are not. However, the contribution of this variation to pathogenesis in humans is not fully understood. We compared the virulence of VP1-145E and VP1-145G viruses of Isehara and C7/Osaka backgrounds in cynomolgus monkeys. VP1-145E, but not VP1-145G, viruses induced neurological symptoms. VP1-145E viruses were frequently detected in the tissues of infected monkeys. VP1-145G viruses were detected less frequently and disappeared quickly. Instead, mutants that had a G-to-E mutation at VP1-145 emerged, suggesting that VP1-145E viruses have a replication advantage in the monkeys. This is consistent with our hypothesis proposed in the accompanying paper (K. Kobayashi, Y. Sudaka, A. Takashino, A. Imura, K. Fujii, and S. Koike, J Virol 92:e00681-18, 2018, https://doi.org/10.1128/JVI.00681-18) that the VP1-145G virus is attenuated due to its adsorption by heparan sulfate. Monkeys infected with both viruses produced neutralizing antibodies before the onset of the disease. Interestingly, VP1-145E viruses were more resistant to neutralizing antibodies than VP1-145G viruses in vitro A small amount of neutralizing antibody raised in the early phase of infection may not be sufficient to block the dissemination of VP1-145E viruses. The different resistance of the VP1-145 variants to neutralizing antibodies may be one of the reasons for the difference in virulence.IMPORTANCE The contribution of VP1-145 variants in humans is not fully understood. In some studies, VP1-145G/Q viruses were isolated more frequently from severely affected patients than from mildly affected patients, suggesting that VP1-145G/Q viruses are more virulent. In the accompanying paper (K. Kobayashi, Y. Sudaka, A. Takashino, A. Imura, K. Fujii, and S. Koike, J Virol 92:e00681-18, 2018, https://doi.org/10.1128/JVI.00681-18), we showed that VP1-145E viruses are more virulent than VP1-145G viruses in human SCARB2 transgenic mice. Heparan sulfate acts as a decoy to specifically trap the VP1-145G viruses and leads to abortive infection. Here, we demonstrated that VP1-145G was attenuated in cynomolgus monkeys, suggesting that this hypothesis is also true in a nonhuman primate model. VP1-145E viruses, but not VP1-145G viruses, were highly resistant to neutralizing antibodies. We propose the difference in resistance against neutralizing antibodies as another mechanism of EV71 virulence. In summary, VP1-145 contributes to virulence determination by controlling attachment receptor usage and antibody sensitivity.


Assuntos
Substituição de Aminoácidos , Anticorpos Neutralizantes/metabolismo , Proteínas do Capsídeo/genética , Enterovirus Humano A/patogenicidade , Infecções por Enterovirus/veterinária , Macaca fascicularis/imunologia , Animais , Anticorpos Antivirais/metabolismo , Células COS , Chlorocebus aethiops , Enterovirus Humano A/genética , Enterovirus Humano A/imunologia , Infecções por Enterovirus/imunologia , Infecções por Enterovirus/virologia , Heparitina Sulfato/metabolismo , Macaca fascicularis/virologia , Masculino , Células Vero , Virulência
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