RESUMO
Human parainfluenza virus type 3 (HPIV3) is a major pediatric respiratory pathogen lacking available vaccines or antiviral drugs. We generated live-attenuated HPIV3 vaccine candidates by codon-pair deoptimization (CPD). HPIV3 open reading frames (ORFs) encoding the nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and polymerase (L) were modified singly or in combination to generate 12 viruses designated Min-N, Min-P, Min-M, Min-FHN, Min-L, Min-NP, Min-NPM, Min-NPL, Min-PM, Min-PFHN, Min-MFHN, and Min-PMFHN. CPD of N or L severely reduced growth in vitro and was not further evaluated. CPD of P or M was associated with increased and decreased interferon (IFN) response in vitro, respectively, but had little effect on virus replication. In Vero cells, CPD of F and HN delayed virus replication, but final titers were comparable to wild-type (wt) HPIV3. In human lung epithelial A549 cells, CPD F and HN induced a stronger IFN response, viral titers were reduced 100-fold, and the expression of F and HN proteins was significantly reduced without affecting N or P or the relative packaging of proteins into virions. Following intranasal infection in hamsters, replication in the nasal turbinates and lungs tended to be the most reduced for viruses bearing CPD F and HN, with maximum reductions of approximately 10-fold. Despite decreased in vivo replication (and lower expression of CPD F and HN in vitro), all viruses induced titers of serum HPIV3-neutralizing antibodies similar to wt and provided complete protection against HPIV3 challenge. In summary, CPD of HPIV3 yielded promising vaccine candidates suitable for further development.
Assuntos
Códon , Vírus da Parainfluenza 3 Humana , Vacinas Atenuadas , Replicação Viral , Animais , Vírus da Parainfluenza 3 Humana/imunologia , Vírus da Parainfluenza 3 Humana/genética , Humanos , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/genética , Códon/genética , Cricetinae , Infecções por Respirovirus/imunologia , Infecções por Respirovirus/prevenção & controle , Infecções por Respirovirus/virologia , Chlorocebus aethiops , Células Vero , Fases de Leitura Aberta/genética , Mesocricetus , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Vacinas Virais/imunologia , Vacinas Virais/genética , Proteínas Virais/imunologia , Proteínas Virais/genética , Vacinas contra Parainfluenza/imunologia , Vacinas contra Parainfluenza/genéticaRESUMO
Interferon (IFN) regulatory factors (IRF) are key transcription factors in cellular antiviral responses. IRF7, a virus-inducible IRF, expressed primarily in myeloid cells, is required for transcriptional induction of interferon α and antiviral genes. IRF7 is activated by virus-induced phosphorylation in the cytoplasm, leading to its translocation to the nucleus for transcriptional activity. Here, we revealed a nontranscriptional activity of IRF7 contributing to its antiviral functions. IRF7 interacted with the pro-inflammatory transcription factor NF-κB-p65 and inhibited the induction of inflammatory target genes. Using knockdown, knockout, and overexpression strategies, we demonstrated that IRF7 inhibited NF-κB-dependent inflammatory target genes, induced by virus infection or toll-like receptor stimulation. A mutant IRF7, defective in transcriptional activity, interacted with NF-κB-p65 and suppressed NF-κB-induced gene expression. A single-action IRF7 mutant, active in anti-inflammatory function, but defective in transcriptional activity, efficiently suppressed Sendai virus and murine hepatitis virus replication. We, therefore, uncovered an anti-inflammatory function for IRF7, independent of transcriptional activity, contributing to the antiviral response of IRF7.
Assuntos
Fator Regulador 7 de Interferon , NF-kappa B , Animais , Humanos , Camundongos , Células HEK293 , Inflamação/genética , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/imunologia , NF-kappa B/genética , NF-kappa B/imunologia , Vírus Sendai/fisiologia , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/imunologia , Replicação Viral , Mutação , Regulação da Expressão Gênica/genética , Vírus da Hepatite Murina/fisiologia , Infecções por Coronavirus/imunologia , Infecções por Respirovirus/imunologiaRESUMO
Respiratory diseases constitute a major health problem for ruminants, resulting in considerable economic losses throughout the world. Parainfluenza type 3 virus (PIV3) is one of the most important respiratory pathogens of ruminants. The pathogenicity and phylogenetic analyses of PIV3 virus have been reported in sheep and goats. However, there are no recent studies of the vaccination of sheep or goats against PIV3. Here, we developed a purified inactivated ovine parainfluenza virus type 3 (OPIV3) vaccine candidate. In addition, we immunized sheep with the inactivated OPIV3 vaccine and evaluated the immune response and pathological outcomes associated with OPIV3 TX01 infection. The vaccinated sheep demonstrated no obvious symptoms of respiratory tract infection, and there were no gross lesions or pathological changes in the lungs. The average body weight gain significantly differed between the vaccinated group and the control group (P < 0.01). The serum neutralization antibody levels rapidly increased in sheep post-vaccination and post-challenge with OPIV3. Furthermore, viral shedding in nasal swabs and viral loads in the lungs were reduced. The results of this study suggest that vaccination with this candidate vaccine induces the production of neutralizing antibodies and provides significant protection against OPIV3 infection. These results may be helpful for further studies on prevention and control strategies for OPIV3 infections.
Assuntos
Infecções por Respirovirus , Doenças dos Ovinos , Vacinas de Produtos Inativados , Vacinas Virais , Animais , Ovinos , Infecções por Respirovirus/veterinária , Infecções por Respirovirus/prevenção & controle , Infecções por Respirovirus/virologia , Infecções por Respirovirus/imunologia , Vacinas de Produtos Inativados/imunologia , Doenças dos Ovinos/prevenção & controle , Doenças dos Ovinos/virologia , Doenças dos Ovinos/imunologia , Vacinas Virais/imunologia , Respirovirus/imunologia , Imunogenicidade da Vacina , Vacinação/veterináriaRESUMO
Objective: This study compared clinical and immunological responses to coinfection challenge of beef calves mucosally primed and differentially boosted with commercial combination vaccines containing antigens against bovine coronavirus (BCoV), bovine parainfluenza virus Type 3 (BPIV3), and bovine respiratory syncytial virus (BRSV). Animals: Nineteen commercial beef heifers. Procedure: At birth, calves were mucosally (IN) primed with modified-live virus (MLV) vaccines, differentially boosted by injection of either combination MLV (IN-MLV) or inactivated virus (IN-KV) vaccines at a mean age of 44 d, and then challenged by coinfection with BCoV, BPIV3, and BRSV at weaning. Results: Both groups were similarly protected from clinical disease and had anamnestic neutralizing antibody responses to all 3 viruses. The IN-KV group shed more BCoV, and less BPIV3 and BRSV, than the IN-MLV group. Conclusion: These data indicated similar clinical and immunological protection between IN-MLV and IN-KV; however, shed of virus varied. Clinical relevance: Whereas boosting with KV or MLV appeared to have similar efficacy, viral shed differences may affect disease control.
Efficacité comparative des vaccins vivants modifiés et inactivés pour stimuler les réponses au virus respiratoire syncytial bovin, au virus parainfluenza bovin de type 3 et au coronavirus bovin après amorçage via la muqueuse de veaux de boucherie nouveau-nés. Objectif: Cette étude a comparé les réponses cliniques et immunologiques à une co-infection de veaux de boucherie amorcés par voie muqueuse et différentiellement stimulés avec des vaccins combinés commerciaux contenant des antigènes contre le coronavirus bovin (BCoV), le virus parainfluenza bovin de type 3 (BPIV3) et le virus respiratoire syncytial bovin (BRSV). Animaux: Dix-neuf génisses de boucherie commerciales. Procédure: À la naissance, les veaux ont été vaccinés au niveau des muqueuses (IN) avec des vaccins à virus vivants modifiés (MLV), stimulés de manière différentielle par l'injection de vaccins combinés MLV (IN-MLV) ou de virus inactivés (IN-KV) à un âge moyen de 44 jours. puis provoqué par une co-infection avec BCoV, BPIV3 et BRSV au sevrage. Résultats: Les deux groupes étaient protégés de la même manière contre la maladie clinique et présentaient des réponses anamnestiques en anticorps neutralisants contre les 3 virus. Le groupe IN-KV a excrété plus de BCoV et moins de BPIV3 et de BRSV que le groupe IN-MLV. Conclusion: Ces données indiquent une protection clinique et immunologique similaire entre IN-MLV et IN-KV; cependant, l'excrétion du virus variait. Pertinence clinique: Alors que le rappel avec KV ou MLV semble avoir une efficacité similaire, les différences d'excrétion virale peuvent affecter la limitation de la maladie.(Traduit par Dr Serge Messier).
Assuntos
Animais Recém-Nascidos , Doenças dos Bovinos , Coronavirus Bovino , Vírus da Parainfluenza 3 Bovina , Infecções por Vírus Respiratório Sincicial , Vírus Sincicial Respiratório Bovino , Vacinas de Produtos Inativados , Vacinas Virais , Animais , Bovinos , Coronavirus Bovino/imunologia , Vírus da Parainfluenza 3 Bovina/imunologia , Vírus Sincicial Respiratório Bovino/imunologia , Doenças dos Bovinos/prevenção & controle , Doenças dos Bovinos/virologia , Doenças dos Bovinos/imunologia , Feminino , Vacinas de Produtos Inativados/imunologia , Vacinas de Produtos Inativados/administração & dosagem , Infecções por Vírus Respiratório Sincicial/veterinária , Infecções por Vírus Respiratório Sincicial/prevenção & controle , Infecções por Vírus Respiratório Sincicial/imunologia , Vacinas Virais/imunologia , Vacinas Virais/administração & dosagem , Animais Recém-Nascidos/imunologia , Infecções por Coronavirus/veterinária , Infecções por Coronavirus/prevenção & controle , Infecções por Coronavirus/imunologia , Infecções por Coronavirus/virologia , Anticorpos Antivirais/sangue , Vacinas Atenuadas/imunologia , Vacinas Atenuadas/administração & dosagem , Infecções por Respirovirus/veterinária , Infecções por Respirovirus/prevenção & controle , Infecções por Respirovirus/imunologia , Imunização Secundária/veterináriaRESUMO
Viral infectivity depends on multiple factors. Recent studies showed that the interaction between viral RNAs and endogenous microRNAs (miRNAs) regulates viral infectivity; viral RNAs function as a sponge of endogenous miRNAs and result in upregulation of its original target genes, while endogenous miRNAs target viral RNAs directly and result in repression of viral gene expression. In this study, we analyzed the possible interaction between parainfluenza virus RNA and endogenous miRNAs in human and mouse lungs. We showed that the parainfluenza virus can form base pairs with human miRNAs abundantly than mouse miRNAs. Furthermore, we analyzed that the sponge effect of endogenous miRNAs on viral RNAs may induce the upregulation of transcription regulatory factors. Then, we performed RNA-sequence analysis and observed the upregulation of transcription regulatory factors in the early stages of parainfluenza virus infection. Our studies showed how the differential expression of endogenous miRNAs in lungs could contribute to respiratory virus infection and species- or tissue-specific mechanisms and common mechanisms could be conserved in humans and mice and regulated by miRNAs during viral infection.
Assuntos
Pulmão , MicroRNAs , Animais , MicroRNAs/genética , Camundongos , Humanos , Pulmão/virologia , Pulmão/imunologia , Pulmão/metabolismo , RNA Viral/genética , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Regulação da Expressão Gênica , Infecções Respiratórias/imunologia , Infecções Respiratórias/virologia , Infecções Respiratórias/genética , Infecções por Respirovirus/imunologiaRESUMO
Background: It remains unclear whether BPIV3 infection leads to stress granules formation and whether G3BP1 plays a role in this process and in viral replication. This study aims to clarify the association between BPIV3 and stress granules, explore the effect of G3BP1 on BPIV3 replication, and provide significant insights into the mechanisms by which BPIV3 evades the host's antiviral immunity to support its own survival. Methods: Here, we use Immunofluorescence staining to observe the effect of BPIV3 infection on the assembly of stress granules. Meanwhile, the expression changes of eIF2α and G3BP1 were determined. Overexpression or siRNA silencing of intracellular G3BP1 levels was examined for its regulatory control of BPIV3 replication. Results: We identify that the BPIV3 infection elicited phosphorylation of the eIF2α protein. However, it did not induce the assembly of stress granules; rather, it inhibited the formation of stress granules and downregulated the expression of G3BP1. G3BP1 overexpression facilitated the formation of stress granules within cells and hindered viral replication, while G3BP1 knockdown enhanced BPIV3 expression. Conclusion: This study suggest that G3BP1 plays a crucial role in BPIV3 suppressing stress granule formation and viral replication.
Assuntos
DNA Helicases , Proteínas de Ligação a Poli-ADP-Ribose , RNA Helicases , Proteínas com Motivo de Reconhecimento de RNA , Grânulos de Estresse , Replicação Viral , Animais , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas com Motivo de Reconhecimento de RNA/metabolismo , DNA Helicases/metabolismo , DNA Helicases/genética , RNA Helicases/metabolismo , RNA Helicases/genética , Grânulos de Estresse/metabolismo , Bovinos , Fator de Iniciação 2 em Eucariotos/metabolismo , Infecções por Respirovirus/imunologia , Infecções por Respirovirus/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Fosforilação , Linhagem Celular , Grânulos Citoplasmáticos/metabolismoRESUMO
Human parainfluenza virus type 3 (hPIV3) is a respiratory pathogen that can cause severe disease in older people and infants. Currently, vaccines against hPIV3 are in clinical trials but none have been approved yet. The haemagglutinin-neuraminidase (HN) and fusion (F) surface glycoproteins of hPIV3 are major antigenic determinants. Here we describe naturally occurring potently neutralizing human antibodies directed against both surface glycoproteins of hPIV3. We isolated seven neutralizing HN-reactive antibodies and a pre-fusion conformation F-reactive antibody from human memory B cells. One HN-binding monoclonal antibody (mAb), designated PIV3-23, exhibited functional attributes including haemagglutination and neuraminidase inhibition. We also delineated the structural basis of neutralization for two HN and one F mAbs. MAbs that neutralized hPIV3 in vitro protected against infection and disease in vivo in a cotton rat model of hPIV3 infection, suggesting correlates of protection for hPIV3 and the potential clinical utility of these mAbs.
Assuntos
Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Proteína HN , Vírus da Parainfluenza 3 Humana , Infecções por Respirovirus , Sigmodontinae , Proteínas Virais de Fusão , Animais , Vírus da Parainfluenza 3 Humana/imunologia , Vírus da Parainfluenza 3 Humana/genética , Humanos , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/química , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/química , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/química , Proteínas Virais de Fusão/imunologia , Proteínas Virais de Fusão/química , Proteína HN/imunologia , Proteína HN/química , Proteína HN/genética , Infecções por Respirovirus/imunologia , Infecções por Respirovirus/virologia , Modelos Animais de Doenças , Testes de Neutralização , Linfócitos B/imunologia , Modelos MolecularesRESUMO
Un ensayo inmunoenzimático comercial Abboutt-RSV-EIA fue evaluado comprarándolo con la inmunofluorescencia indirecta. Aspirados nasofaríngeos de 95 niños con infección respiratoria aguda baja fueron procesados por inmunofluorescencia y por enzimoinmunoensayo para revelar la presencia de antígenos del virus respiratorio sincicial. De los 60 materiales positivos por inmunofluorescencia, 46 también lo fueron por enzimoinmunoensayo (sensibilidad 78,7%); de 35 negativos, 34 fueron también negativos por el Abbout-RSV-EIA (especificidad 97,1%). Según los resultados presentados, la evaluada es aceptable como una alternativa para el diagnóstico rápido de VRS en lugares donde no se cuente con otros recursos