RESUMO
The influenza A virus (IAV) ribonucleoprotein (vRNP) complex consists of polymerase subunits, nucleoprotein (NP) and viral RNA and is responsible for RNA transcription and replication. Interactions between the vRNP complex and host factors play important roles in virus replication, pathogenicity and species tropism. In this study, Strep-tag affinity purification coupled with mass spectrometry was used to identify host factors that interact with IAV vRNP complex in infected human cells. We purified vRNP complex from HEK 293T cells infected with a recombinant mouse-adapted IAV (A/Chicken/Hubei/489/2004) containing a Strep-tag PB2 subunit and identified Y-box-binding protein 3 (YBX3) as a negative regulator of IAV replication. Overexpression of YBX3 inhibited the virus replication, viral protein expression and vRNA synthesis. Conversely, RNAi knockdown of YBX3 resulted in significantly increased virus growth rate. Furthermore, knockdown of YBX3 augmented the nuclear accumulation of NP and viral primary transcription in infected cells. Our results suggest that YBX3 restricts IAV replication by interacting with vRNP complex and subsequently imparing its function.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas de Choque Térmico/metabolismo , Vírus da Influenza A/genética , Vírus da Influenza A/metabolismo , Proteínas Nucleares/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas do Core Viral/metabolismo , Replicação Viral , Células A549 , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Núcleo Celular/metabolismo , Citoplasma/metabolismo , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , Cães , Células HEK293 , Proteínas de Choque Térmico/genética , Interações entre Hospedeiro e Microrganismos , Humanos , Vírus da Influenza A/enzimologia , Vírus da Influenza A/crescimento & desenvolvimento , Células Madin Darby de Rim Canino , Espectrometria de Massas , Camundongos , Ligação Proteica , RNA Interferente Pequeno , RNA Viral/metabolismo , Transcrição Gênica , Regulação para Cima , Proteínas do Core Viral/genética , Replicação Viral/fisiologiaRESUMO
Newcastle disease virus (NDV) has been used as a vector in the development of vaccines and gene delivery. In the present study, we generated the thermostable recombinant NDV (rNDV) expressing the different forms of hemagglutinin (HA) of highly pathogenic avian influenza virus (HPAIV) H5N1 based on the full-length cDNA clone of thermostable TS09-C strain. The recombinant thermostable Newcastle disease viruses, rTS-HA, rTS-HA1 and rTS-tPAs/HA1, expressed the HA, HA1 or modified HA1 protein with the tissue plasminogen activator signal sequence (tPAs), respectively. The rNDVs displayed similar thermostability, growth kinetics and pathogenicity compared with the parental TS09-C virus. The tPAs facilitated the expression and secretion of HA1 protein in cells infected with rNDV. Animal studies demonstrated that immunization with rNDVs elicited effective H5N1- and NDV-specific antibody responses and conferred immune protection against lethal H5N1 and NDV challenges in chickens and mice. Importantly, vaccination of rTS-tPAs/HA1 resulted in enhanced protective immunity in chickens and mice. Our study thus provides a novel thermostable NDV-vectored vaccine candidate expressing a soluble form of a heterologous viral protein, which will greatly aid the poultry industry in developing countries.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Vacinas contra Influenza , Influenza Aviária , Doença de Newcastle , Animais , Anticorpos Antivirais/sangue , Galinhas/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Virus da Influenza A Subtipo H5N1 , Vacinas contra Influenza/genética , Influenza Aviária/prevenção & controle , Camundongos , Doença de Newcastle/prevenção & controle , Vírus da Doença de Newcastle , Ativador de Plasminogênio Tecidual/genética , Vacinas Combinadas/genéticaRESUMO
To study the typical vaginal bacterial flora of giant pandas (Ailuropoda melanoleuca), we took vaginal swabs for the sake of bacterial isolation, from 24 healthy female giant pandas. A total of 203 isolates were identified, representing a total of 17 bacterial species. The most common bacteria isolated were Lactobacillus spp. (54.2%, 13/24), followed by Staphylococcus epidermidis (41.7%, 10/24) and Escherichia coli (33.3%, 8/24). Some opportunistic pathogenic bacteria, such as Peptostreptococcus spp., Klebsiella pneumoniae , and Proteus mirabilis , were also isolated but showed no pathology. Antimicrobial susceptibility testing of aerobic bacterial isolates was performed with the disk diffusion method. Of the 152 isolates, resistance was most frequently observed with chloramphenicol (17.8%), followed by tetracycline (14.5%), ciprofloxacin (12.5%), streptomycin (11.8%), and florfenicol (11.8%), whereas 7.2% were multidrug resistant. This is the first report of the normal culturable vaginal bacterial flora of giant pandas and the antimicrobial susceptibility patterns of the isolates.
Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Farmacorresistência Bacteriana , Ursidae/microbiologia , Vagina/microbiologia , Animais , FemininoRESUMO
In order to study the typical vaginal bacterial flora of giant pandas (Ailuropoda melanoleuca), we took vaginal swabs for the sake of bacterial isolation, from 24 healthy female giant pandas. A total of 203 isolates were identified, representing a total of 17 bacterial species. The most common bacteria isolated were Lactobacillus spp. (54.2%, 13 of 24), followed by Staphylococcus epidermidis (41.7%, 10 of 24) and Escherichia coli (33.3%, 8 of 24). Some opportunistic pathogenic bacteria, such as Peptostreptococcus spp., Klebsiella pneumoniae, and Proteus mirabilis, were also isolated but showed no pathology. Antimicrobial susceptibility testing of aerobic bacterial isolates was performed with disk diffusion method. Of the 152 isolates, resistance was most frequently observed with chloramphenicol (17.8%), followed by tetracycline (14.5%), ciprofloxacin (12.5%), streptomycin (11.8%), and florfenicol (11.8%), while 7.2% were multidrug resistant. This is the first report of the normal vaginal culturable bacterial flora of giant pandas, followed by the antimicrobial susceptibility patterns of the isolates.
