RESUMEN

The blood-brain barrier (BBB) is one of several barriers between the brain and the peripheral blood system to maintain homeostasis. Understanding the interactions between infectious agents such as human immunodeficiency virus type 1 (HIV-1), which are capable of traversing the BBB and causing neuroinflammation requires modeling an authentic BBB in vitro. Such an in vitro BBB model also helps develop means of targeting viruses that reside in the brain via natural immune effectors such as antibodies. The BBB consists of human brain microvascular endothelial cells (HBMECs), astrocytes, and pericytes. Here we report in vitro methods to establish a dual-cell BBB model consisting of primary HBMECs and primary astrocytes to measure the integrity of the BBB and antibody penetration of the BBB, as well as a method to establish a single cell BBB model to study the impact of HIV-1 infected medium on the integrity of such a BBB.

Asunto(s)

Astrocitos , Barrera Hematoencefálica , Células Endoteliales , Infecciones por VIH , VIH-1 , Barrera Hematoencefálica/virología , Barrera Hematoencefálica/metabolismo , Humanos , Astrocitos/virología , Astrocitos/metabolismo , Astrocitos/inmunología , Células Endoteliales/virología , Células Endoteliales/metabolismo , Células Endoteliales/inmunología , VIH-1/inmunología , VIH-1/fisiología , Infecciones por VIH/virología , Infecciones por VIH/inmunología , Pericitos/virología , Pericitos/metabolismo , Pericitos/inmunología , Enfermedades Neuroinflamatorias/virología , Enfermedades Neuroinflamatorias/inmunología , Técnicas de Cocultivo/métodos , Células Cultivadas , Encéfalo/virología , Encéfalo/inmunología , Encéfalo/metabolismo

RESUMEN

Zika virus (ZIKV) causes significant human diseases without specific therapy. Previously we found erythrosin B, an FDA-approved food additive, inhibited viral NS2B-NS3 interactions, leading to inhibition of ZIKV infection in cell culture. In this study, we performed pharmacokinetic and in vivo studies to demonstrate the efficacy of erythrosin B against ZIKV in 3D mini-brain organoid and mouse models. Our results showed that erythrosin B is very effective in abolishing ZIKV replication in the 3D organoid model. Although pharmacokinetics studies indicated that erythrosin B had a low absorption profile, mice challenged by a lethal dose of ZIKV showed a significantly improved survival rate upon oral administration of erythrosin B, compared to vehicle control. Limited structure-activity relationship studies indicated that most analogs of erythrosin B with modifications on the xanthene ring led to loss or reduction of inhibitory activities towards viral NS2B-NS3 interactions, protease activity and antiviral efficacy. In contrast, introducing chlorine substitutions on the isobenzofuran ring led to slightly increased activities, suggesting that the isobenzofuran ring is well tolerated for modifications. Cytotoxicity studies indicated that all derivatives are nontoxic to human cells. Overall, our studies demonstrated erythrosin B is an effective antiviral against ZIKV both in vitro and in vivo.

RESUMEN

Swine influenza is an important disease for the swine industry. Currently used whole inactivated virus (WIV) vaccines can induce vaccine-associated enhanced respiratory disease (VAERD) in pigs when the vaccine strains mismatch with the infected viruses. Live attenuated influenza virus vaccine (LAIV) is effective to protect pigs against homologous and heterologous swine influenza virus infections without inducing VAERD but has safety concerns due to potential reassortment with circulating viruses. Herein, we used a chimeric bat influenza Bat09:mH3mN2 virus, which contains both surface HA and NA gene open reading frames of the A/swine/Texas/4199-2/1998 (H3N2) and six internal genes from the novel bat H17N10 virus, to develop modified live-attenuated viruses (MLVs) as vaccine candidates which cannot reassort with canonical influenza A viruses by co-infection. Two attenuated MLV vaccine candidates including the virus that expresses a truncated NS1 (Bat09:mH3mN2-NS1-128, MLV1) or expresses both a truncated NS1 and the swine IL-18 (Bat09:mH3mN2-NS1-128-IL-18, MLV2) were generated and evaluated in pigs against a heterologous H3N2 virus using the WIV vaccine as a control. Compared to the WIV vaccine, both MLV vaccines were able to reduce lesions and virus replication in lungs and limit nasal virus shedding without VAERD, also induced significantly higher levels of mucosal IgA response in lungs and significantly increased numbers of antigen-specific IFN-γ secreting cells against the challenge virus. However, no significant difference was observed in efficacy between the MLV1 and MLV2. These results indicate that bat influenza vectored MLV vaccines can be used as a safe live vaccine to prevent swine influenza.

Asunto(s)

Quirópteros , Vacunas contra la Influenza , Gripe Humana , Infecciones por Orthomyxoviridae , Enfermedades de los Porcinos , Animales , Anticuerpos Antivirales , Subtipo H3N2 del Virus de la Influenza A/genética , Infecciones por Orthomyxoviridae/prevención & control , Infecciones por Orthomyxoviridae/veterinaria , Porcinos , Enfermedades de los Porcinos/prevención & control , Texas , Vacunas Atenuadas

RESUMEN

Self-splicing proteins, called inteins, are present in many human pathogens, including the emerging fungal threats Cryptococcus neoformans (Cne) and Cryptococcus gattii (Cga), the causative agents of cryptococcosis. Inhibition of protein splicing in Cryptococcus sp. interferes with activity of the only intein-containing protein, Prp8, an essential intron splicing factor. Here, we screened a small-molecule library to find addititonal, potent inhibitors of the Cne Prp8 intein using a split-GFP splicing assay. This revealed the compound 6G-318S, with IC50 values in the low micromolar range in the split-GFP assay and in a complementary split-luciferase system. A fluoride derivative of the compound 6G-318S displayed improved cytotoxicity in human lung carcinoma cells, although there was a slight reduction in the inhibition of splicing. 6G-318S and its derivative inhibited splicing of the Cne Prp8 intein in vivo in Escherichia coli and in C. neoformans Moreover, the compounds repressed growth of WT C. neoformans and C. gattii In contrast, the inhibitors were less potent at inhibiting growth of the inteinless Candida albicans Drug resistance was observed when the Prp8 intein was overexpressed in C. neoformans, indicating specificity of this molecule toward the target. No off-target activity was observed, such as inhibition of serine/cysteine proteases. The inhibitors bound covalently to the Prp8 intein and binding was reduced when the active-site residue Cys1 was mutated. 6G-318S showed a synergistic effect with amphotericin B and additive to indifferent effects with a few other clinically used antimycotics. Overall, the identification of these small-molecule intein-splicing inhibitors opens up prospects for a new class of antifungals.

Asunto(s)

Empalme de Proteína/fisiología , Proteínas de Unión al ARN/genética , Antifúngicos/farmacología , Cryptococcus neoformans/genética , Cryptococcus neoformans/metabolismo , Cryptococcus neoformans/patogenicidad , Proteínas Fúngicas/metabolismo , Humanos , Inteínas/genética , Intrones/genética , Empalme de Proteína/genética , Empalme del ARN/genética , Proteínas de Unión al ARN/metabolismo , Alineación de Secuencia/métodos

RESUMEN

Betacoronaviruses are in one genera of coronaviruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome-related coronavirus (MERS-CoV), etc. These viruses threaten public health and cause dramatic economic losses. The nucleocapsid (N) protein is a structural protein of betacoronaviruses with multiple functions such as forming viral capsids with viral RNA, interacting with viral membrane protein to form the virus core with RNA, binding to several cellular kinases for signal transductions, etc. In this review, we highlighted the potential of the N protein as a suitable antiviral target from different perspectives, including structure, functions, and antiviral strategies for combatting betacoronaviruses.

Asunto(s)

Antivirales/química , Antivirales/farmacología , Betacoronavirus/efectos de los fármacos , Infecciones por Coronavirus/tratamiento farmacológico , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Descubrimiento de Drogas , Animales , Betacoronavirus/fisiología , Proteínas de la Nucleocápside de Coronavirus/antagonistas & inhibidores , Proteínas de la Nucleocápside de Coronavirus/química , Interacciones Huésped-Patógeno/efectos de los fármacos , Humanos , Coronavirus del Síndrome Respiratorio de Oriente Medio/efectos de los fármacos , Modelos Moleculares , Terapia Molecular Dirigida , Fosfoproteínas/antagonistas & inhibidores , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Mapas de Interacción de Proteínas/efectos de los fármacos , SARS-CoV-2/efectos de los fármacos , Tratamiento Farmacológico de COVID-19

RESUMEN

Many flaviviruses including the Dengue virus (DENV), Zika virus (ZIKV), West Nile virus, Yellow Fever virus, and Japanese encephalitis virus are significant human pathogens, unfortunately without any specific therapy. Here, we demonstrate that methylene blue, an FDA-approved drug, is a broad-spectrum and potent antiviral against Zika virus and Dengue virus both in vitro and in vivo. We found that methylene blue can considerably inhibit the interactions between viral protease NS3 and its NS2B co-factor, inhibit viral protease activity, inhibit viral growth, protect 3D mini-brain organoids from ZIKV infection, and reduce viremia in a mouse model. Mechanistic studies confirmed that methylene blue works in both entry and post entry steps, reduces virus production in replicon cells and inhibited production of processed NS3 protein. Overall, we have shown that methylene blue is a potent antiviral for management of flavivirus infections, particularly for Zika virus. As an FDA-approved drug, methylene blue is well-tolerated for human use. Therefore, methylene blue represents a promising and easily developed therapy for management of infections by ZIKV and other flaviviruses.

Asunto(s)

Antivirales/administración & dosificación , Azul de Metileno/administración & dosificación , Inhibidores de Proteasas/administración & dosificación , Infección por el Virus Zika/tratamiento farmacológico , Virus Zika/crecimiento & desarrollo , Células A549 , Administración Oral , Animales , Antivirales/farmacología , Línea Celular , Virus del Dengue/efectos de los fármacos , Virus del Dengue/genética , Virus del Dengue/crecimiento & desarrollo , Modelos Animales de Enfermedad , Regulación Viral de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Azul de Metileno/farmacología , Ratones , Inhibidores de Proteasas/farmacología , Unión Proteica/efectos de los fármacos , ARN Helicasas/metabolismo , Serina Endopeptidasas/metabolismo , Carga Viral/efectos de los fármacos , Proteínas no Estructurales Virales/metabolismo , Proteínas Virales/metabolismo , Internalización del Virus/efectos de los fármacos , Virus Zika/efectos de los fármacos , Virus Zika/genética

RESUMEN

Flaviviruses causes significant human disease. Recent outbreaks of the Zika virus highlight the need to develop effective therapies for this class of viruses. Previously we identified niclosamide as a broad-spectrum inhibitor for flaviviruses by targeting the interface between viral protease NS3 and its cofactor NS2B. Here, we screened a small library of niclosamide derivatives and identified a new analogue with improved pharmacokinetic properties. Compound JMX0207 showed improved efficacy in inhibition of the molecular interaction between NS3 and NS2B, better inhibition of viral protease function, and enhanced antiviral efficacy in the cell-based antiviral assay. The derivative also significantly reduced Zika virus infection on 3D mini-brain organoids derived from pluripotent neural stem cells. Intriguingly, the compound significantly reduced viremia in a Zika virus (ZIKV) animal model. In summary, a niclosamide derivative, JMX0207, was identified, which shows improved pharmacokinetics and efficacy against Zika virus both in vitro and in vivo.

Asunto(s)

Flavivirus , Infección por el Virus Zika , Virus Zika , Animales , Humanos , Niclosamida/farmacología , Proteínas no Estructurales Virales , Infección por el Virus Zika/tratamiento farmacológico

RESUMEN

Protein-protein interactions are important in human disease. Developing and refining tools to understand physical contacts between signaling proteins is crucial. This article describes a split luciferase complementation (SLC) method designed to discover inhibitors of protein-protein interaction. Different fusion proteins with split luciferase are constructed, expressed, and purified, and then assessed to determine the best pair that generates the strongest luminescence. SLC specificity and affinity are further confirmed. Step-by-step instructions are provided for performing these assays using the NS2B-NS3 interaction as an example. NS2B is an essential cofactor for flaviviral NS3 protease function. Advantages and disadvantages of these assays are further discussed. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Expression and purification of fusion proteins Basic Protocol 2: Analysis of prey/bait pairs by SLC-based NS2B-NS3 interaction assay Support Protocol 1: Interaction specificity assay Support Protocol 2: Competition binding assay: Dose-response inhibition using cold prey or bait Support Protocol 3: Competition binding assay: Inhibition by MBP-NS3 versus irrelevant MBP tag Support Protocol 4: SLC-based NS2B-NS3 interaction assay using NS2B mutations known to disrupt NS2B-NS3 interactions.

Asunto(s)

Bioensayo/métodos , Luciferasas de Luciérnaga/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas no Estructurales Virales/metabolismo , Unión Competitiva , Clonación Molecular , Luciferasas de Luciérnaga/genética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Mutación , Unión Proteica , ARN Helicasas/genética , ARN Helicasas/metabolismo , Proteínas Recombinantes de Fusión/genética , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Proteínas no Estructurales Virales/genética

RESUMEN

Influenza viruses are important pathogens causing respiratory disease in humans and animals. In contrast to influenza A virus (IAV) that can infect a wide range of animal species, other influenza viruses, including influenza B virus (IBV), influenza C virus (ICV), and influenza D virus (IDV) have a limited host range. Swine can be infected with all four different genera of influenza viruses. IAV infection of pigs causes the well-known swine influenza that poses significant threats to human and animal health. However, influenza virus infection of pigs with IBV, ICV, and IDV are not well-characterized. Herein, we compared pathogenicity of IBV and IDV using intratracheal and intranasal infection of pigs, which are IAV seropositive, and commingled naïve pigs with the infected animals to determine their transmissibility. Both viruses caused fever and some lung lesions, replicated in the lungs of infected pigs, but only IDV transmitted to the contact animals. Although IBV and IDV displayed differing levels of replication in the respiratory tract of infected pigs, no significant differences in pathogenicity of both viruses were observed. These results indicate that both IBV and IDV can replicate, and are pathogenic in pigs.

Asunto(s)

Virus de la Influenza B/fisiología , Infecciones por Orthomyxoviridae/transmisión , Infecciones por Orthomyxoviridae/virología , Enfermedades de los Porcinos/transmisión , Enfermedades de los Porcinos/virología , Thogotovirus/fisiología , Animales , Modelos Animales de Enfermedad , Especificidad del Huésped , Virus de la Influenza A , Virus de la Influenza B/patogenicidad , Gammainfluenzavirus , Pulmón/patología , Pulmón/virología , Infecciones por Orthomyxoviridae/patología , Porcinos , Enfermedades de los Porcinos/patología , Thogotovirus/patogenicidad , Estados Unidos , Carga Viral , Virulencia , Replicación Viral

RESUMEN

The Prp8 intein is one of the most widespread eukaryotic inteins, present in important pathogenic fungi, including Cryptococcus and Aspergillus species. Because the processed Prp8 carries out essential and non-redundant cellular functions, a Prp8 intein inhibitor is a mechanistically novel antifungal agent. In this report, we demonstrated that cisplatin, an FDA-approved cancer drug, significantly arrested growth of Prp8 intein-containing fungi C. neoformans and C. gattii, but only poorly inhibited growth of intein-free Candida species. These results suggest that cisplatin arrests fungal growth through specific inhibition of the Prp8 intein. Cisplatin was also found to significantly inhibit growth of C. neoformans in a mouse model. Our results further showed that cisplatin inhibited Prp8 intein splicing in vitro in a dose-dependent manner by direct binding to the Prp8 intein. Crystal structures of the apo- and cisplatin-bound Prp8 inteins revealed that two degenerate cisplatin molecules bind at the intein active site. Mutation of the splicing-site residues led to loss of cisplatin binding, as well as impairment of intein splicing. Finally, we found that overexpression of the Prp8 intein in cryptococcal species conferred cisplatin resistance. Overall, these results indicate that the Prp8 intein is a novel antifungal target worth further investigation.

Asunto(s)

Antifúngicos/administración & dosificación , Cisplatino/administración & dosificación , Criptococosis/microbiología , Cryptococcus neoformans/efectos de los fármacos , Proteínas Fúngicas/genética , Inteínas , Proteínas de Unión al ARN/genética , Secuencia de Aminoácidos , Animales , Antifúngicos/química , Cisplatino/química , Cryptococcus neoformans/genética , Cryptococcus neoformans/crecimiento & desarrollo , Cryptococcus neoformans/metabolismo , Femenino , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Humanos , Ratones , Ratones Endogámicos BALB C , Modelos Moleculares , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/metabolismo , Alineación de Secuencia

RESUMEN

Influenza is a common and contagious respiratory disease caused by influenza A, B, C, and D viruses (IAV, IBV, ICV, and IDV). A multiplex real-time RT-PCR assay was developed for simultaneous detection of IAV, IBV, ICV, and IDV. The assay was designed to target unique sequences in the matrix gene of IBV and ICV, the RNA polymerase subunit PB1 of IDV, and combined with USDA and CDC IAV assays, both target the matrix gene. The host 18S rRNA gene was included as an internal control. In silico analyses indicated high strain coverages: 97.9% for IBV, 99.5% for ICV, and 100% for IDV. Transcribed RNA, viral isolates and clinical samples were used for validation. The assay specifically detected target viruses without cross-reactivity, nor detection of other common pathogens. The limit of detection was approximately 30 copies for each viral RNA template, which was equivalent to a threshold cycle value of ~37.

Asunto(s)

Enfermedades de los Bovinos/diagnóstico , Infecciones por Orthomyxoviridae/veterinaria , Orthomyxoviridae/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/normas , Enfermedades de los Porcinos/diagnóstico , Animales , Bovinos , Enfermedades de los Bovinos/virología , Diagnóstico Diferencial , Genes Virales/genética , Orthomyxoviridae/clasificación , Infecciones por Orthomyxoviridae/virología , ARN Viral/genética , Sensibilidad y Especificidad , Porcinos , Enfermedades de los Porcinos/virología

RESUMEN

We performed swine influenza virus (SIV) surveillance in Midwest USA and isolated 100 SIVs including endemic and reassortant H1 and H3 viruses with 2009 pandemic H1N1 genes. To determine virus evolution when different genotypes and subtypes of influenza A viruses circulating in the same swine herd, a virus survival experiment was conducted in pigs mimicking field situations. Five different SIVs were used to infect five pigs individually, then two groups of sentinel pigs were introduced to investigate virus transmission. Results showed that each virus replicated efficiently in lungs of each infected pig, but only reassortant H3N2 and H1N2v viruses transmitted to the primary contact pigs. Interestingly, the parental H1N2v was the majority of virus detected in the second group of sentinel pigs. These data indicate that the H1N2v seems to be more viable in swine herds than other SIV genotypes, and reassortment can enhance viral fitness and transmission.

Asunto(s)

Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N2 del Virus de la Influenza A/genética , Subtipo H3N2 del Virus de la Influenza A/genética , Infecciones por Orthomyxoviridae/veterinaria , Virus Reordenados/genética , Enfermedades de los Porcinos/epidemiología , Animales , Monitoreo Epidemiológico , Aptitud Genética , Genotipo , Subtipo H1N1 del Virus de la Influenza A/clasificación , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Subtipo H1N2 del Virus de la Influenza A/clasificación , Subtipo H1N2 del Virus de la Influenza A/patogenicidad , Subtipo H3N2 del Virus de la Influenza A/clasificación , Subtipo H3N2 del Virus de la Influenza A/patogenicidad , Medio Oeste de Estados Unidos/epidemiología , Infecciones por Orthomyxoviridae/epidemiología , Infecciones por Orthomyxoviridae/transmisión , Infecciones por Orthomyxoviridae/virología , Filogenia , Virus Reordenados/clasificación , Virus Reordenados/patogenicidad , Porcinos , Enfermedades de los Porcinos/transmisión , Enfermedades de los Porcinos/virología , Replicación Viral

RESUMEN

Rift Valley fever virus (RVFV) is the causative agent of Rift Valley fever (RVF) that affects both livestock and humans. There are neither fully licensed RVF vaccines available for human or animal use, nor effective antiviral drugs approved for human use in the U.S. To identify antiviral compounds effective for RVF, we developed and employed a cell-based high-throughput assay using a recombinant RVFV MP-12 strain, which expresses Renilla luciferase in place of the NSs protein, to screen 727 small compounds purchased from the National Institutes of Health. Twenty-three compounds were initially identified using the screening assay. Two compounds, 6-azauridine and mitoxantrone, also inhibited the replication of the parental MP-12 strain encoding the NSs gene, with limited cytotoxic effects. The respective 50% inhibitory concentrations were 29.07 µM and 79.85 µM when tested with the parental MP-12 strain at a multiplicity of infection of 2. The compounds were further evaluated using the STAT-1 KO mouse model. At one hour post intranasal inoculation of MP-12 strain, mice were intranasally treated with each indicated compound twice daily. Mice treated with either placebo or 6-azauridine displayed severe weight loss and reached the threshold for euthanasia with obvious neurologic symptoms. Onset of disease was, however, delayed in mice treated with either ribavirin or mitoxantrone. The results indicated that mitoxantrone can reduce the severity of diseases in RVFV-infected mice. Our studies build the foundation for the initial screening and efficacy studies of RVF antivirals in a BSL-2 environment, avoiding the higher risks of BSL-3 exposure with wild-type virus.

Asunto(s)

Antivirales/farmacología , Fiebre del Valle del Rift/tratamiento farmacológico , Virus de la Fiebre del Valle del Rift/efectos de los fármacos , Animales , Antivirales/aislamiento & purificación , Azauridina/farmacología , Línea Celular , Modelos Animales de Enfermedad , Descubrimiento de Drogas , Femenino , Ensayos Analíticos de Alto Rendimiento , Concentración 50 Inhibidora , Ratones , Mitoxantrona/farmacología , Virus de la Fiebre del Valle del Rift/fisiología , Bibliotecas de Moléculas Pequeñas/farmacología , Replicación Viral/efectos de los fármacos

RESUMEN

[This corrects the article DOI: 10.1038/s41541-017-0034-4.].

RESUMEN

Since December 2014, Eurasian-origin, highly pathogenic avian influenza H5 viruses including H5N1, H5N2, and H5N8 subtypes (called H5Nx viruses), which belong to the H5 clade 2.3.4.4, have been detected in U.S. wild birds. Subsequently, highly pathogenic H5N2 and H5N8 viruses have caused outbreaks in U.S. domestic poultry. Vaccination is one of the most effective ways to control influenza outbreaks and protect animal and public health. Newcastle disease virus (NDV)-based influenza vaccines have been demonstrated to be efficacious and safe in poultry. Herein, we developed an NDV-based H5 vaccine (NDV-H5) that expresses a codon-optimized ectodomain of the hemagglutinin from the A/chicken/Iowa/04-20/2015 (H5N2) virus and evaluated its efficacy in chickens. Results showed that both live and inactivated NDV-H5 vaccines induced hemagglutinin inhibition antibody titers against the H5N2 virus in immunized chickens after prime and booster, and both NDV-H5 vaccines completely protected chickens from lethal challenge with the highly pathogenic H5N2 A/turkey/Minnesota/9845-4/2015 virus. No clinical signs and only minimal virus shedding was observed in both vaccinated groups. In contrast, all mock-vaccinated, H5N2-infected chickens shed virus and died within 5 days post challenge. Furthermore, one dose of the live NDV-H5 vaccine also provided protection of 90% chickens immunized by coarse spraying; after exposure to H5N2 challenge, sera from vaccinated surviving chickens neutralized both highly pathogenic H5N1 and H5N8 viruses. Taken together, our results suggest that the NDV-based H5 vaccine is able to protect chickens against intercontinental highly pathogenic H5Nx viruses and can be used by mass application to protect the poultry industry.

RESUMEN

Although several studies have investigated the functions of influenza PA-X, the impact of different expressions of PA-X protein including full-length, truncated or PA-X deficient forms on virus replication, pathogenicity and host response remains unclear. Herein, we generated two mutated viruses expressing a full-length or deficient PA-X protein based on the A/California/04/2009 (H1N1) virus that expresses a truncated PA-X to understand three different expressions of PA-X protein on virus replication, pathogenicity and host immune responses. The results showed that expression of either full-length or truncated PA-X protein enhanced viral replication and pathogenicity as well as reduced host innate immune response in mice by host shutoff activity when compared to the virus expressing the deficient PA-X form. Furthermore, the full-length PA-X expression exhibited a greater effect on virus pathogenicity than the truncated PA-X form. Our results provide novel insights of PA-X on viral replication, pathogenicity and host immune responses.

Asunto(s)

Regulación Viral de la Expresión Génica/genética , Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Infecciones por Orthomyxoviridae/virología , Proteínas Represoras/genética , Proteínas no Estructurales Virales/genética , Células A549 , Animales , Línea Celular , Línea Celular Tumoral , Perros , Femenino , Células HEK293 , Humanos , Inflamación/genética , Inflamación/inmunología , Pulmón/inmunología , Pulmón/patología , Pulmón/virología , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos BALB C , Mutación/genética , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/patología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Represoras/metabolismo , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/genética

RESUMEN

In our previous studies, the reassortant virus containing only the PR8 H1N1 matrix (M) gene in the background of the modified bat influenza Bat09 : mH1mN1 virus could be generated. However, whether M genes from other origins can be rescued in the background of the Bat09 : mH1mN1 virus and whether the resulting novel reassortant virus is virulent remain unknown. Herein, two reassortant viruses were generated in the background of the Bat09 : mH1mN1 virus containing either a North American or a Eurasian swine influenza virus M gene. These two reassortant viruses and the reassortant virus with PR8 M as well as the control Bat09 : mH1mN1 virus replicated efficiently in cultured cells, while the reassortant virus with PR8 M grew to a higher titre than the other three viruses in tested cells. Mouse studies showed that reassortant viruses with either North American or Eurasian swine influenza virus M gene did not enhance virulence, whereas the reassortant virus with PR8 M gene displayed higher pathogenicity when compared to the Bat09 : mH1mN1 virus. This is most likely due to the fact that the PR8 H1N1 virus is a mouse-adapted virus. Furthermore, reassortment potential between the Bat09 : mH1mN1 virus and an H3N2 swine influenza virus (A/swine/Texas/4199-2/1998) was investigated using co-infection of Madin-Darby canine kidney cells, but no reassortant viruses were detected. Taken together, our results indicate that the modified bat influenza virus is most likely incapable of reassortment with influenza A viruses with in vitro co-infection experiments, although reassortant viruses with different M genes can be generated by reverse genetics.

Asunto(s)

Variación Genética , Subtipo H1N1 del Virus de la Influenza A/genética , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Infecciones por Orthomyxoviridae/veterinaria , Virus Reordenados/genética , Virus Reordenados/aislamiento & purificación , Proteínas de la Matriz Viral/genética , Animales , Quirópteros , Modelos Animales de Enfermedad , Subtipo H1N1 del Virus de la Influenza A/clasificación , Subtipo H1N1 del Virus de la Influenza A/aislamiento & purificación , Ratones , Infecciones por Orthomyxoviridae/patología , Infecciones por Orthomyxoviridae/virología , Porcinos , Carga Viral , Virulencia , Replicación Viral

RESUMEN

Although several studies have exploited the effects of PB1-F2 in swine influenza viruses, its contribution to the pathogenicity of swine influenza viruses remains unclear. Herein, we investigated the effects of PB1-F2 on the pathogenicity of influenza virus using a virulent H1N1 A/swine/Kansas/77778/2007 (KS07) virus, which expresses a full-length PB1-F2, in mice and pigs. Using reverse genetics, we generated the wild-type KS07 (KS07_WT), a PB1-F2 knockout mutant (KS07_K/O) and its N66S variant (KS07_N66S). KS07_K/O showed similar pathogenicity in mice to the KS07_WT, whereas KS07_N66S displayed enhanced virulence when compared to the other two viruses. KS07_WT exhibited more efficient replication in lungs and nasal shedding in infected pigs than the other two viruses. Pigs infected with the KS07_WT had higher pulmonary levels of granulocyte-macrophage colony-stimulating factor, IFN-γ, IL-6 and IL-8 at 3 and 5 days post-infection, as well as lower levels of IL-2, IL-4 and IL-12 at 1 day post-infection compared to those infected with the KS07_K/O. These results indicate that PB1-F2 modulates KS07 H1N1 virus replication, pathogenicity and innate immune responses in pigs and the single substitution at position 66 (N/S) in the PB1-F2 plays a critical role in virulence in mice. Taken together, our results provide new insights into the effects of PB1-F2 on the virulence of influenza virus in swine and support PB1-F2 as a virulence factor of influenza A virus in a strain- and host-dependent manner.

Asunto(s)

Subtipo H1N1 del Virus de la Influenza A/inmunología , Subtipo H1N1 del Virus de la Influenza A/patogenicidad , Infecciones por Orthomyxoviridae/veterinaria , Proteínas Virales/genética , Animales , Línea Celular , Femenino , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Subtipo H1N1 del Virus de la Influenza A/genética , Ratones , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/virología , Porcinos/inmunología , Porcinos/virología , Enfermedades de los Porcinos/virología , Virulencia/genética , Factores de Virulencia/genética , Replicación Viral/genética

RESUMEN

Rift Valley fever virus (RVFV), a Category A pathogen and select agent, is the causative agent of Rift Valley fever. To date, no fully licensed vaccine is available in the U.S. for human or animal use and effective antiviral drugs have not been identified. The RVFV MP12 strain is conditionally licensed for use for veterinary purposes in the U.S. which was excluded from the select agent rule of Health and Human Services and the U.S. Department of Agriculture. The MP12 vaccine strain is commonly used in BSL-2 laboratories that is generally not virulent in mice. To establish a small animal model that can be used in a BSL-2 facility for antiviral drug development, we investigated susceptibility of six mouse strains (129S6/SvEv, STAT-1 KO, 129S1/SvlmJ, C57BL/6J, NZW/LacJ, BALB/c) to the MP12 virus infection via an intranasal inoculation route. Severe weight loss, obvious clinical and neurologic signs, and 50% mortality was observed in the STAT-1 KO mice, whereas the other 5 mouse strains did not display obvious and/or severe disease. Virus replication and histopathological lesions were detected in brain and liver of MP12-infected STAT-1 KO mice that developed the acute-onset hepatitis and delayed-onset encephalitis. In conclusion, the STAT-1 KO mouse strain is susceptible to MP12 virus infection, indicating that it can be used to investigate RVFV antivirals in a BSL-2 environment.

Asunto(s)

Modelos Animales de Enfermedad , Fiebre del Valle del Rift/virología , Virus de la Fiebre del Valle del Rift/clasificación , Animales , Encéfalo/virología , Hígado/virología , Ratones , Ratones Endogámicos , Replicación Viral/fisiología

RESUMEN

In order to produce an efficient poultry H9 avian influenza vaccine that provides cross-protection against multiple H9 lineages, two Newcastle disease virus (NDV) LaSota vaccine strain recombinant viruses were generated using reverse genetics. The recombinant NDV-H9Con virus expresses a consensus-H9 hemagglutinin (HA) that is designed based on available H9N2 sequences from Chinese and Middle Eastern isolates. The recombinant NDV-H9Chi virus expresses a chimeric-H9 HA in which the H9 ectodomain of A/Guinea Fowl/Hong Kong/WF10/99 was fused with the cytoplasmic and transmembrane domain of the fusion protein (F) of NDV. Both recombinant viruses expressed the inserted HA stably and grew to high titers. An efficacy study in chickens showed that both recombinant viruses were able to provide protection against challenge with a heterologous H9N2 virus. In contrast to the NDV-H9Chi virus, the NDV-H9Con virus induced a higher hemagglutination inhibition titer against both NDV and H9 viruses in immunized birds, and efficiently inhibited virus shedding through the respiratory route. Moreover, sera collected from birds immunized with either NDV-H9Con or NDV-H9Chi were able to cross-neutralize two different lineages of H9N2 viruses, indicating that NDV-H9Con and NDV-H9Chi are promising vaccine candidates that could provide cross-protection among different H9N2 lineage viruses.

Asunto(s)

Protección Cruzada , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Vacunas contra la Influenza/inmunología , Gripe Aviar/prevención & control , Virus de la Enfermedad de Newcastle/genética , Animales , Anticuerpos Antivirales/sangre , Pollos/inmunología , Pollos/virología , Pruebas de Inhibición de Hemaglutinación , Subtipo H9N2 del Virus de la Influenza A , Pruebas de Neutralización , Virus de la Enfermedad de Newcastle/inmunología , Esparcimiento de Virus