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Oropouche fever caused by Oropouche virus (OROV) is a significant zoonosis in Central and South America. Despite its public health significance, we lack high-throughput diagnostics, therapeutics, and a comprehensive knowledge of OROV biology. Reporter viruses are valuable tools to rapidly study virus dynamics and develop neutralization and antiviral screening assays. OROV is a tri-segmented bunyavirus, which makes generating a reporter virus challenging, as introducing foreign elements into the viral genome typically affects fitness. We previously demonstrated that the non-structural gene NSm on the OROV medium (M) segment is non-essential for replication in vitro. Taking advantage of this, we have now generated a recombinant OROV expressing fluorescent protein ZsGreen in place of NSm. This reporter OROV is both stable and pathogenic in IFNAR-/- mice and provides a powerful tool for OROV pathogenesis studies and assay development.IMPORTANCEEmerging and reemerging infectious agents such as zoonotic bunyaviruses are of global health concern. Oropouche virus (OROV) causes recurring outbreaks of acute febrile illness in the Central and South American human populations. Biting midges are the primary transmission vectors, whereas sloths and non-human primates are their reservoir hosts. As global temperatures increase, we will likely see an expansion in arthropod-borne pathogens such as OROV. Therefore, developing reagents to study pathogen biology to aid in identifying druggable targets is essential. Here, we demonstrate the feasibility and use of a fluorescent OROV reporter in mice to study viral dynamics and pathogenesis. We show that this reporter OROV maintains characteristics such as growth and pathogenicity similar to the wild-type virus. Using this reporter virus, we can now develop methods to assist OROV studies and establish various high-throughput assays.
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Klebsiella pneumoniae is an opportunistic pathogen and an important cause of pneumonia, bacteremia, and urinary tract infection. K. pneumoniae infections are historically associated with diabetes mellitus. There is a fundamental gap in our understanding of how diabetes mellitus, specifically type 2 diabetes, influences K. pneumoniae pathogenesis. K. pneumoniae pathogenesis is a multifactorial process that often begins with gut colonization, followed by an escape from the gut to peripheral sites, leading to host damage and infection. We hypothesized that type 2 diabetes enhances K. pneumoniae pathogenesis. To test this, we used well-established mouse models of K. pneumoniae colonization and lung infection in conjunction with a mouse model of spontaneous type 2 diabetes mellitus (T2DM). We show that T2DM enhances susceptibility to both K. pneumoniae colonization and infection. The enhancement of gut colonization is dependent on T2DM-induced modulation of the gut microbiota community structure. In contrast, lung infection is exacerbated by the increased availability of amino acids in the lung, which is associated with higher levels of vascular endothelial growth factor. These data lay the foundation for mechanistic interrogation of the relationship between K. pneumoniae pathogenesis and type 2 diabetes mellitus, and explicitly establish T2DM as a risk factor for K. pneumoniae disease.
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Since SARS-CoV-2 emerged in late 2019, it spread from China to the rest of the world. An initial concern was the potential for vaccine- or antibody-dependent enhancement (ADE) of disease as had been reported with other coronaviruses. To evaluate this, we first developed a ferret model by exposing ferrets to SARS-CoV-2 by either mucosal inoculation (intranasal/oral/ocular) or inhalation using a small particle aerosol. Mucosal inoculation caused a mild fever and weight loss that resolved quickly; inoculation via either route resulted in virus shedding detected in the nares, throat, and rectum for 7-10 days post-infection. To evaluate the potential for ADE, we then inoculated groups of ferrets intravenously with 0.1, 0.5, or 1 mg/kg doses of a human polyclonal anti-SARS-CoV-2 IgG from hyper-immunized transchromosomic bovines (SAB-185). Twelve hours later, ferrets were challenged by mucosal inoculation with SARS-CoV-2. We found no significant differences in fever, weight loss, or viral shedding after infection between the three antibody groups or the controls. Signs of pathology in the lungs were noted in infected ferrets but no differences were found between control and antibody groups. The results of this study indicate that healthy, young adult ferrets of both sexes are a suitable model of mild COVID-19 and that low doses of specific IgG in SAB-185 are unlikely to enhance the disease caused by SARS-CoV-2.
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Anticorpos Antivirais , COVID-19 , Modelos Animais de Doenças , Furões , SARS-CoV-2 , Eliminação de Partículas Virais , Animais , Furões/virologia , COVID-19/imunologia , COVID-19/virologia , Anticorpos Antivirais/imunologia , SARS-CoV-2/imunologia , Humanos , Feminino , Masculino , Imunoglobulina G/imunologia , Anticorpos Facilitadores/imunologiaRESUMO
RNA viruses generate defective genomes naturally during virus replication. Defective genomes that interfere with the infection dynamics either through resource competition or by interferon stimulation are known as defective interfering (DI) genomes. DI genomes can be successfully packaged into virus-like-particles referred to as defective interfering particles (DIPs). Such DIPs can sustainably coexist with the full-length virus particles and have been shown to negatively impact virus replication in vitro and in vivo. Here, we describe a method to generate a clonal DI genome population by reverse genetics. This method is applicable to other RNA viruses and will enable assessment of DIPs for their antiviral properties.
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Vírus Defeituosos , Genoma Viral , Morbillivirus , Genética Reversa , Replicação Viral , Genética Reversa/métodos , Vírus Defeituosos/genética , Animais , Replicação Viral/genética , Morbillivirus/genética , Humanos , Vírion/genética , Células Vero , Chlorocebus aethiops , RNA Viral/genéticaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and is responsible for the largest human pandemic in 100 years. Thirty-four vaccines are currently approved for use worldwide, and approximately 67% of the world population has received a complete primary series of one, yet countries are dealing with new waves of infections, variant viruses continue to emerge, and breakthrough infections are frequent secondary to waning immunity. Here, we evaluate a measles virus (MV)-vectored vaccine expressing a stabilized prefusion SARS-CoV-2 spike (S) protein (MV-ATU3-S2PΔF2A; V591) with demonstrated immunogenicity in mouse models (see companion article [J. Brunet, Z. Choucha, M. Gransagne, H. Tabbal, M.-W. Ku et al., J Virol 98:e01693-23, 2024, https://doi.org/10.1128/jvi.01693-23]) in an established African green monkey model of disease. Animals were vaccinated with V591 or the control vaccine (an equivalent MV-vectored vaccine with an irrelevant antigen) intramuscularly using a prime/boost schedule, followed by challenge with an early pandemic isolate of SARS-CoV-2 at 56 days post-vaccination. Pre-challenge, only V591-vaccinated animals developed S-specific antibodies that had virus-neutralizing activity as well as S-specific T cells. Following the challenge, V591-vaccinated animals had lower infectious virus and viral (v) RNA loads in mucosal secretions and stopped shedding virus in these secretions earlier. vRNA loads were lower in these animals in respiratory and gastrointestinal tract tissues at necropsy. This correlated with a lower disease burden in the lungs as quantified by PET/CT at early and late time points post-challenge and by pathological analysis at necropsy.IMPORTANCESevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the largest human pandemic in 100 years. Even though vaccines are currently available, countries are dealing with new waves of infections, variant viruses continue to emerge, breakthrough infections are frequent, and vaccine hesitancy persists. This study uses a safe and effective measles vaccine as a platform for vaccination against SARS-CoV-2. The candidate vaccine was used to vaccinate African green monkeys (AGMs). All vaccinated AGMs developed robust antigen-specific immune responses. After challenge, these AGMs produced less virus in mucosal secretions, for a shorter period, and had a reduced disease burden in the lungs compared to control animals. At necropsy, lower levels of viral RNA were detected in tissue samples from vaccinated animals, and the lungs of these animals lacked the histologic hallmarks of SARS-CoV-2 disease observed exclusively in the control AGMs.
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Vacinas contra COVID-19 , COVID-19 , Vírus do Sarampo , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus , Animais , Glicoproteína da Espícula de Coronavírus/imunologia , Glicoproteína da Espícula de Coronavírus/genética , Chlorocebus aethiops , SARS-CoV-2/imunologia , SARS-CoV-2/genética , COVID-19/prevenção & controle , COVID-19/imunologia , COVID-19/virologia , Vírus do Sarampo/imunologia , Vírus do Sarampo/genética , Vacinas contra COVID-19/imunologia , Humanos , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangue , Vetores Genéticos , Células Vero , Pandemias/prevenção & controle , Feminino , Betacoronavirus/imunologia , Betacoronavirus/genética , Pneumonia Viral/prevenção & controle , Pneumonia Viral/virologia , Pneumonia Viral/imunologia , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Infecções por Coronavirus/veterinária , Vacinas Virais/imunologia , Vacinas Virais/genética , Vacinas Virais/administração & dosagem , Modelos Animais de DoençasRESUMO
Morbilliviruses are members of the family Paramyxoviridae and are known for their ability to cause systemic disease in a variety of mammalian hosts. The prototypic morbillivirus, measles virus (MeV), infects humans and still causes morbidity and mortality in unvaccinated children and young adults. Experimental infection studies in non-human primates have contributed to the understanding of measles pathogenesis. However, ethical restrictions call for the development of new animal models. Canine distemper virus (CDV) infects a wide range of animals, including ferrets, and its pathogenesis shares many features with measles. However, wild-type CDV infection is almost always lethal, while MeV infection is usually self-limiting. Here, we made five recombinant CDVs, predicted to be attenuated, and compared their pathogenesis to the non-attenuated recombinant CDV in a ferret model. Three viruses were insufficiently attenuated based on clinical signs, fatality, and systemic infection, while one virus was too attenuated. The last candidate virus caused a self-limiting infection associated with transient viremia and viral dissemination to all lymphoid tissues, was shed transiently from the upper respiratory tract, and did not result in acute neurological signs. Additionally, an in-depth phenotyping of the infected white blood cells showed lower infection percentages in all lymphocyte subsets when compared to the non-attenuated CDV. In conclusion, infection models using this candidate virus mimic measles and can be used to study pathogenesis-related questions and to test interventions for morbilliviruses in a natural host species.IMPORTANCEMorbilliviruses are transmitted via the respiratory route but cause systemic disease. The viruses use two cellular receptors to infect myeloid, lymphoid, and epithelial cells. Measles virus (MeV) remains an important cause of morbidity and mortality in humans, requiring animal models to study pathogenesis or intervention strategies. Experimental MeV infections in non-human primates are restricted by ethical and practical constraints, and animal morbillivirus infections in natural host species have been considered as alternatives. Inoculation of ferrets with wild-type canine distemper virus (CDV) has been used for this purpose, but in most cases, the virus overwhelms the immune system and causes highly lethal disease. Introduction of an additional transcription unit and an additional attenuating point mutation in the polymerase yielded a candidate virus that caused self-limiting disease with transient viremia and virus shedding. This rationally attenuated CDV strain can be used for experimental morbillivirus infections in ferrets that reflect measles in humans.
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Modelos Animais de Doenças , Vírus da Cinomose Canina , Furões , Sarampo , Infecções por Morbillivirus , Animais , Cães , Humanos , Cinomose/virologia , Vírus da Cinomose Canina/genética , Sarampo/patologia , Vírus do Sarampo/genética , Morbillivirus/genética , Infecções por Morbillivirus/patologia , Primatas , ViremiaRESUMO
Feline morbillivirus (FeMV) is a recently discovered pathogen of domestic cats and has been classified as a morbillivirus in the Paramyxovirus family. We determined the complete sequence of FeMVUS5 directly from an FeMV-positive urine sample without virus isolation or cell passage. Sequence analysis of the viral genome revealed potential divergence from characteristics of archetypal morbilliviruses. First, the virus lacks the canonical polybasic furin cleavage signal in the fusion (F) glycoprotein. Second, conserved amino acids in the hemagglutinin (H) glycoprotein used by all other morbilliviruses for binding and/or fusion activation with the cellular receptor CD150 (signaling lymphocyte activation molecule [SLAM]/F1) are absent. We show that, despite this sequence divergence, FeMV H glycoprotein uses feline CD150 as a receptor and cannot use human CD150. We demonstrate that the protease responsible for cleaving the FeMV F glycoprotein is a cathepsin, making FeMV a unique morbillivirus and more similar to the closely related zoonotic Nipah and Hendra viruses. We developed a reverse genetics system for FeMVUS5 and generated recombinant viruses expressing Venus fluorescent protein from an additional transcription unit located either between the phospho-protein (P) and matrix (M) genes or the H and large (L) genes of the genome. We used these recombinant FeMVs to establish a natural infection and demonstrate that FeMV causes an acute morbillivirus-like disease in the cat. Virus was shed in the urine and detectable in the kidneys at later time points. This opens the door for long-term studies to address the postulated role of this morbillivirus in the development of chronic kidney disease.
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Infecções por Morbillivirus , Morbillivirus , Aminoácidos , Animais , Catepsinas/genética , Gatos , Furina , Hemaglutininas , Humanos , Rim , Morbillivirus/genética , Infecções por Morbillivirus/veterináriaRESUMO
Defective interfering (DI) genomes restrict viral replication and induce type I interferon. Since DI genomes have been proposed as vaccine adjuvants or therapeutic antiviral agents, it is important to understand their generation, delineate their mechanism of action, develop robust production capacities, assess their safety and in vivo longevity, and determine their long-term effects. To address this, we generated a recombinant canine distemper virus (rCDV) from an entirely synthetic molecular clone designed using the genomic sequence from a clinical isolate obtained from a free-ranging raccoon with distemper. rCDV was serially passaged in vitro to identify DI genomes that naturally arise during rCDV replication. Defective genomes were identified by Sanger and next-generation sequencing techniques, and predominant genomes were synthetically generated and cloned into T7-driven plasmids. Fully encapsidated DI particles (DIPs) were then generated using a rationally attenuated rCDV as a producer virus to drive DI genome replication. We demonstrate that these DIPs interfere with rCDV replication in a dose-dependent manner in vitro. Finally, we show sustained replication of a fluorescent DIP in experimentally infected ferrets over a period of 14 days. Most importantly, DIPs were isolated from the lymphoid tissues, which are a major site of CDV replication. Our established pipeline for detection, generation, and assaying DIPs is transferable to highly pathogenic paramyxoviruses and will allow qualitative and quantitative assessment of the therapeutic effects of DIP administration on disease outcome. IMPORTANCE Defective interfering (DI) genomes have long been considered inconvenient artifacts that suppressed viral replication in vitro. However, advances in sequencing technologies have led to DI genomes being identified in clinical samples, implicating them in disease progression and outcome. It has been suggested that DI genomes might be harnessed therapeutically. Negative-strand RNA virus research has provided a rich pool of natural DI genomes over many years, and they are probably the best understood in vitro. Here, we demonstrate the identification, synthesis, production, and experimental inoculation of novel CDV DI genomes in highly susceptible ferrets. These results provide important evidence that rationally designed and packaged DI genomes can survive the course of a wild-type virus infection.
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Vírus da Cinomose Canina/genética , Vírus da Cinomose Canina/fisiologia , Animais , Linhagem Celular , Chlorocebus aethiops , Vírus Defeituosos , Cães , Furões , Genoma Viral , Masculino , Guaxinins/virologia , Células Vero , Replicação Viral/genética , Replicação Viral/fisiologiaRESUMO
Globally, there is an urgency to develop effective, low-cost therapeutic interventions for coronavirus disease 2019 (COVID-19). We previously generated the stable and ultrapotent homotrimeric Pittsburgh inhalable Nanobody 21 (PiN-21). Using Syrian hamsters that model moderate to severe COVID-19 disease, we demonstrate the high efficacy of PiN-21 to prevent and treat SARS-CoV-2 infection. Intranasal delivery of PiN-21 at 0.6 mg/kg protects infected animals from weight loss and substantially reduces viral burdens in both lower and upper airways compared to control. Aerosol delivery of PiN-21 facilitates deposition throughout the respiratory tract and dose minimization to 0.2 mg/kg. Inhalation treatment quickly reverses animals' weight loss after infection, decreases lung viral titers by 6 logs leading to drastically mitigated lung pathology, and prevents viral pneumonia. Combined with the marked stability and low production cost, this innovative therapy may provide a convenient and cost-effective option to mitigate the ongoing pandemic.
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Tratamento Farmacológico da COVID-19 , COVID-19/prevenção & controle , SARS-CoV-2/efeitos dos fármacos , Anticorpos de Domínio Único/administração & dosagem , Administração por Inalação , Aerossóis/administração & dosagem , Animais , Modelos Animais de Doenças , Feminino , Masculino , Mesocricetus , Pandemias/prevenção & controle , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/prevenção & controle , Carga Viral/efeitos dos fármacosRESUMO
Globally there is an urgency to develop effective, low-cost therapeutic interventions for coronavirus disease 2019 (COVID-19). We previously generated the stable and ultrapotent homotrimeric Pittsburgh inhalable Nanobody 21 (PiN-21). Using Syrian hamsters that model moderate to severe COVID-19 disease, we demonstrate the high efficacy of PiN-21 to prevent and treat SARS-CoV-2 infection. Intranasal delivery of PiN-21 at 0.6 mg/kg protects infected animals from weight loss and substantially reduces viral burdens in both lower and upper airways compared to control. Aerosol delivery of PiN-21 facilitates deposition throughout the respiratory tract and dose minimization to 0.2 mg/kg. Inhalation treatment quickly reverses animals' weight loss post-infection and decreases lung viral titers by 6 logs leading to drastically mitigated lung pathology and prevents viral pneumonia. Combined with the marked stability and low production cost, this novel therapy may provide a convenient and cost-effective option to mitigate the ongoing pandemic.
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Rift Valley fever virus (RVFV) is a pathogen of both humans and livestock in Africa and the Middle East. Severe human disease is associated with hepatitis and/or encephalitis. Current pathogenesis studies rely on rodents and nonhuman primates, which have advantages and disadvantages. We evaluated disease progression in Mustela putorius furo (the ferret) following intradermal (i.d.) or intranasal (i.n.) infection. Infected ferrets developed hyperpyrexia, weight loss, lymphopenia, and hypoalbuminemia. Three of four ferrets inoculated intranasally with RVFV developed central nervous system (CNS) disease that manifested as seizure, ataxia, and/or hind limb weakness at 8 to 11 days postinfection (dpi). Animals with clinical CNS disease had transient viral RNAemia, high viral RNA loads in the brain, and histopathological evidence of encephalitis. The ferret model will facilitate our understanding of how RVFV accesses the CNS and has utility for the evaluation of vaccines and/or therapeutics in preventing RVFV CNS disease.IMPORTANCE Animal models of viral disease are very important for understanding how viruses make people sick and for testing out drugs and vaccines to see if they can prevent disease. In this study, we identify the ferret as a model of encephalitis caused by Rift Valley fever virus (RVFV). This novel model will allow researchers to evaluate ways to prevent RVFV encephalitis.
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Encefalite Viral/virologia , Furões/virologia , Febre do Vale de Rift/fisiopatologia , Doença Aguda , Animais , Encéfalo/patologia , Encéfalo/virologia , Modelos Animais de Doenças , Masculino , Febre do Vale de Rift/complicações , Vírus da Febre do Vale do RiftRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), emerged at the end of 2019 and by mid-June 2020 the virus had spread to at least 215 countries, caused more than 8 000 000 confirmed infections and over 450 000 deaths, and overwhelmed healthcare systems worldwide. Like severe acute respiratory syndrome coronavirus (SARS-CoV), which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low-passage (P) strains of SARS-CoV-2 (Wash1â¯:â¯P4 and Munichâ¯:â¯P1) were cultured twice in Vero E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1â¯:â¯P6 and minor variants in the Munichâ¯:â¯P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated that the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies, we investigated the development of neutralizing antibody titres in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies.
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COVID-19/metabolismo , COVID-19/virologia , Furina/metabolismo , Interações Hospedeiro-Patógeno , SARS-CoV-2/fisiologia , Replicação Viral , Adaptação Fisiológica , Animais , Anticorpos Neutralizantes/imunologia , COVID-19/epidemiologia , COVID-19/imunologia , Chlorocebus aethiops , Furina/imunologia , Variação Genética , Hospitalização , Interações Hospedeiro-Patógeno/imunologia , Humanos , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Testes de Neutralização , Proteólise , RNA Viral , Análise de Sequência de RNA , Células Vero , Carga ViralRESUMO
SARS-CoV-2, the causative agent of COVID-19, emerged at the end of 2019 and by mid-June 2020, the virus has spread to at least 215 countries, caused more than 8,000,000 confirmed infections and over 450,000 deaths, and overwhelmed healthcare systems worldwide. Like SARS-CoV, which emerged in 2002 and caused a similar disease, SARS-CoV-2 is a betacoronavirus. Both viruses use human angiotensin-converting enzyme 2 (hACE2) as a receptor to enter cells. However, the SARS-CoV-2 spike (S) glycoprotein has a novel insertion that generates a putative furin cleavage signal and this has been postulated to expand the host range. Two low passage (P) strains of SARS-CoV-2 (Wash1: P4 and Munich: P1) were cultured twice in Vero-E6 cells and characterized virologically. Sanger and MinION sequencing demonstrated significant deletions in the furin cleavage signal of Wash1: P6 and minor variants in the Munich: P3 strain. Cleavage of the S glycoprotein in SARS-CoV-2-infected Vero-E6 cell lysates was inefficient even when an intact furin cleavage signal was present. Indirect immunofluorescence demonstrated the S glycoprotein reached the cell surface. Since the S protein is a major antigenic target for the development of neutralizing antibodies we investigated the development of neutralizing antibody titers in serial serum samples obtained from COVID-19 human patients. These were comparable regardless of the presence of an intact or deleted furin cleavage signal. These studies illustrate the need to characterize virus stocks meticulously prior to performing either in vitro or in vivo pathogenesis studies.
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Bunyaviruses pose a significant threat to human health, prosperity, and food security. In response to viral infections, interferons (IFNs) upregulate the expression of hundreds of interferon-stimulated genes (ISGs), whose cumulative action can potently inhibit the replication of bunyaviruses. We used a flow cytometry-based method to screen the ability of â¼500 unique ISGs from humans and rhesus macaques to inhibit the replication of Bunyamwera orthobunyavirus (BUNV), the prototype of both the Peribunyaviridae family and the Bunyavirales order. Candidates possessing antibunyaviral activity were further examined using a panel of divergent bunyaviruses. Interestingly, one candidate, ISG20, exhibited potent antibunyaviral activity against most viruses examined from the Peribunyaviridae, Hantaviridae, and Nairoviridae families, whereas phleboviruses (Phenuiviridae) largely escaped inhibition. Similar to the case against other viruses known to be targeted by ISG20, the antibunyaviral activity of ISG20 is dependent upon its functional RNase activity. Through use of an infectious virus-like particle (VLP) assay (based on the BUNV minigenome system), we confirmed that gene expression from all 3 viral segments is strongly inhibited by ISG20. Using in vitro evolution, we generated a substantially ISG20-resistant BUNV and mapped the determinants of ISG20 sensitivity/resistance. Taking all the data together, we report that ISG20 is a broad and potent antibunyaviral factor but that some bunyaviruses are remarkably ISG20 resistant. Thus, ISG20 sensitivity/resistance may influence the pathogenesis of bunyaviruses, many of which are emerging viruses of clinical or veterinary significance.IMPORTANCE There are hundreds of bunyaviruses, many of which cause life-threatening acute diseases in humans and livestock. The interferon (IFN) system is a key component of innate immunity, and type I IFNs limit bunyaviral propagation both in vitro and in vivo Type I IFN signaling results in the upregulation of hundreds of IFN-stimulated genes (ISGs), whose concerted action generates an "antiviral state." Although IFNs are critical in limiting bunyaviral replication and pathogenesis, much is still unknown about which ISGs inhibit bunyaviruses. Using ISG-expression screening, we examined the ability of â¼500 unique ISGs to inhibit Bunyamwera orthobunyavirus (BUNV), the prototypical bunyavirus. Using this approach, we identified ISG20, an interferon-stimulated exonuclease, as a potent inhibitor of BUNV. Interestingly, ISG20 possesses highly selective antibunyaviral activity, with multiple bunyaviruses being potently inhibited while some largely escape inhibition. We speculate that the ability of some bunyaviruses to escape ISG20 may influence their pathogenesis.
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Antivirais/farmacologia , Vírus Bunyamwera/patogenicidade , Infecções por Bunyaviridae/prevenção & controle , Exonucleases/farmacologia , Genoma Viral , Interferons/metabolismo , Infecções por Bunyaviridae/metabolismo , Infecções por Bunyaviridae/virologia , Exonucleases/genética , Exorribonucleases , Células HeLa , Ensaios de Triagem em Larga Escala , HumanosRESUMO
A number of viruses within the Peribunyaviridae family are naturally occurring reassortants, a common phenomenon for segmented viruses. Using a minigenome-reporter and virus-like particle (VLP) production assay, we have accessed the potential of Oropouche virus (OROV), Schmallenberg virus (SBV), and other orthobunyaviruses within the Simbu serogroup to reassort. We found that the untranslated region (UTR) in the medium segment is a potential contributing factor for reassortment by the tested viruses. We demonstrate that for promoter activity to occur it was essential that the viral RNA polymerase (L) and nucleocapsid (N) proteins were from the same virus, reinforcing the hypothesis that the large and small segments that encode these proteins segregate together during genome reassortment. Our results indicate that, given the right epidemiological setting, reassortment between SBV and OROV would potentially be feasible and could contribute to the emergence of a new Simbu virus.
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Infecções por Bunyaviridae/virologia , Orthobunyavirus/classificação , Orthobunyavirus/genética , Vírus Reordenados/genética , Humanos , Orthobunyavirus/isolamento & purificação , Orthobunyavirus/fisiologia , Filogenia , RNA Viral/genética , Vírus Reordenados/classificação , Vírus Reordenados/isolamento & purificação , Vírus Reordenados/fisiologia , Recombinação Genética , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Orthobunyaviruses include several recently emerging viruses of significant medical and veterinary importance. There is currently very limited understanding on what determines the host species range of these pathogens. In this study we discovered that BST-2/tetherin restricts orthobunyavirus replication in a host-specific manner. We show that viruses with human tropism (Oropouche virus and La Crosse virus) are restricted by sheep BST-2 but not by the human orthologue, while viruses with ruminant tropism (Schmallenberg virus and others) are restricted by human BST-2 but not by the sheep orthologue. We also show that BST-2 blocks orthobunyaviruses replication by reducing the amount of envelope glycoprotein into viral particles egressing from infected cells. This is the first study identifying a restriction factor that correlates with species susceptibility to orthobunyavirus infection. This work provides insight to help us dissect the adaptive changes that bunyaviruses require to cross the species barrier and emerge into new species.
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Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/metabolismo , Especificidade de Hospedeiro , Interações Hospedeiro-Patógeno , Orthobunyavirus/imunologia , Orthobunyavirus/fisiologia , Proteínas do Envelope Viral/antagonistas & inibidores , Animais , Linhagem Celular , Proteínas Ligadas por GPI/metabolismo , Humanos , Ovinos , Liberação de VírusRESUMO
UNLABELLED: Oropouche virus (OROV) is a midge-borne human pathogen with a geographic distribution in South America. OROV was first isolated in 1955, and since then, it has been known to cause recurring outbreaks of a dengue-like illness in the Amazonian regions of Brazil. OROV, however, remains one of the most poorly understood emerging viral zoonoses. Here we describe the successful recovery of infectious OROV entirely from cDNA copies of its genome and generation of OROV mutant viruses lacking either the NSm or the NSs coding region. Characterization of the recombinant viruses carried out in vitro demonstrated that the NSs protein of OROV is an interferon (IFN) antagonist as in other NSs-encoding bunyaviruses. Additionally, we demonstrate the importance of the nine C-terminal amino acids of OROV NSs in IFN antagonistic activity. OROV was also found to be sensitive to IFN-α when cells were pretreated; however, the virus was still capable of replicating at doses as high as 10,000 U/ml of IFN-α, in contrast to the family prototype BUNV. We found that OROV lacking the NSm protein displayed characteristics similar to those of the wild-type virus, suggesting that the NSm protein is dispensable for virus replication in the mammalian and mosquito cell lines that were tested. IMPORTANCE: Oropouche virus (OROV) is a public health threat in Central and South America, where it causes periodic outbreaks of dengue-like illness. In Brazil, OROV is the second most frequent cause of arboviral febrile illness after dengue virus, and with the current rates of urban expansion, more cases of this emerging viral zoonosis could occur. To better understand the molecular biology of OROV, we have successfully rescued the virus along with mutants. We have established that the C terminus of the NSs protein is important in interferon antagonism and that the NSm protein is dispensable for virus replication in cell culture. The tools described in this paper are important in terms of understanding this important yet neglected human pathogen.
Assuntos
Interações Hospedeiro-Patógeno , Orthobunyavirus/genética , Orthobunyavirus/fisiologia , Proteínas não Estruturais Virais/deficiência , Proteínas não Estruturais Virais/metabolismo , Replicação Viral , Animais , Linhagem Celular , Humanos , Interferons/antagonistas & inibidores , Orthobunyavirus/imunologia , Genética ReversaRESUMO
Oropouche virus (OROV) is a public health threat in South America, and in particular in northern Brazil, causing frequent outbreaks of febrile illness. Using a combination of deep sequencing and Sanger sequencing approaches, we determined the complete genome sequences of eight clinical isolates that were obtained from patient sera during an Oropouche fever outbreak in Amapa state, northern Brazil, in 2009. We also report the complete genome sequences of two OROV reassortants isolatd from two marmosets in Minas Gerais state, south-east Brazil, in 2012 that contained a novel M genome segment. Interestingly, all 10 isolates possessed a 947 nt S segment that lacked 11 residues in the S-segment 3' UTR compared with the recently redetermined Brazilian prototype OROV strain BeAn19991. OROV maybe circulating more widely in Brazil and in the non-human primate population than previously appreciated, and the identification of yet another reassortant highlights the importance of bunyavirus surveillance in South America.
Assuntos
Genoma Viral , Orthobunyavirus/classificação , Orthobunyavirus/genética , RNA Viral/genética , Adolescente , Adulto , Animais , Brasil , Infecções por Bunyaviridae/veterinária , Infecções por Bunyaviridae/virologia , Callithrix , Análise por Conglomerados , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Orthobunyavirus/isolamento & purificação , Filogenia , Vírus Reordenados/classificação , Vírus Reordenados/genética , Vírus Reordenados/isolamento & purificação , Análise de Sequência de DNA , Homologia de Sequência , Adulto JovemRESUMO
Oropouche virus (OROV) is a medically important orthobunyavirus, which causes frequent outbreaks of a febrile illness in the northern parts of Brazil. However, despite being the cause of an estimated half a million human infections since its first isolation in Trinidad in 1955, details of the molecular biology of this tripartite, negative-sense RNA virus remain limited. We have determined the complete nucleotide sequence of the Brazilian prototype strain of OROV, BeAn 19991, and found a number of differences compared with sequences in the database. Most notable were that the S segment contained an additional 204 nt at the 3' end and that there was a critical nucleotide mismatch at position 9 within the base-paired terminal panhandle structure of each genome segment. In addition, we obtained the complete sequence of the Trinidadian prototype strain TRVL-9760 that showed similar characteristics to the BeAn 19991 strain. By using a T7 RNA polymerase-driven minigenome system, we demonstrated that cDNA clones of the BeAn 19991 L and S segments expressed functional proteins, and also that the newly determined terminal untranslated sequences acted as functional promoters in the minigenome assay. By co-transfecting a cDNA to the viral glycoproteins, virus-like particles were generated that packaged a minigenome and were capable of infecting naive cells.
Assuntos
Genoma Viral/genética , Genômica/métodos , Orthobunyavirus/genética , Animais , Sequência de Bases , Clonagem Molecular , Regulação Viral da Expressão Gênica/fisiologia , Humanos , Dados de Sequência Molecular , RNA Viral/genética , Análise de Sequência de RNARESUMO
Two component regulatory systems are key elements in the control of bacterial gene expression in response to environmental perturbations. The SenX3-RegX3 system is implicated in the control of phosphate uptake in Mycobacterium smegmatis and Mycobacterium tuberculosis. regX3 is reported to be essential in M. smegmatis, but not in M. tuberculosis. We attempted to construct complete senX3-regX3 operon deletion strains of M. smegmatis; initially we found that the operon could only be deleted when another functional copy was provided. Using a strain in which the only functional copy of the operon was present on an integrating plasmid, we attempted to replace the functional copy with an empty vector. Surprisingly, we obtained strains in which the functional copy had been deleted from the chromosome at a low frequency. We deleted the senX3 gene in a similar fashion, but it was not possible to delete regX3 alone. To identify possible compensatory mutations we sequenced the whole genome of two deletion strains and the wild-type. A synonymous single nucleotide polymorphism (SNP) in a lipoprotein was found in all deletion strains, but not the parental strains, and a frameshift mutation in nhaA was identified in three of the four deletion strains. Operon deletion strains were more sensitive to phosphate limitation, showing a reduced ability to grow at lower phosphate concentrations. The M. tuberculosis operon was able to functionally complement the growth phenotype in M. smegmatis under phosphate-replete conditions, but not under low phosphate conditions, reinforcing the difference between the two species. Our data show that, in contrast with previous reports, it is possible to delete the operon in M. smegmatis, possibly due to the accumulation of compensatory mutations, and that the deletion does affect growth in phosphate.