RESUMO
Marek's disease virus (MDV), an alpha herpes virus, causes a lymphoproliferative state in chickens known as Marek's disease (MD), resulting in severe monetary losses to the poultry industry. Because lymphocytes of bursa of Fabricius and spleen are prime targets of MDV replication during the early cytolytic phase of infection, the immune response in bursa and spleen should be the foundation of late immunity induced by MDV. However, the mechanism of the MDV-mediated host immune response in lymphocytes in the early stage is poorly understood. The present study is primarily aimed at identifying the crucial genes and significant pathways involved in the immune response of chickens infected with MDV CVI988 and the very virulent RB1B (vvRB1B) strains. Using the RNA sequencing approach, we analyzed the generated transcriptomes from lymphocytes isolated from chicken bursa and spleen. Our findings validated the expression of previously characterized genes; however, they also revealed the expression of novel genes during the MDV-mediated immune response. The results showed that after challenge with CVI988 or vvRB1B strains, 634 and 313 differentially expressed genes (DEGs) were identified in splenic lymphocytes, respectively. However, 58 and 47 DEGs were observed in bursal lymphocytes infected with CVI988 and vvRB1B strains, respectively. Following MDV CVI988 or vvRB1B challenge, the bursal lymphocytes displayed changes in IL-6 and IL-4 gene expression. Surprisingly, splenic lymphocytes exhibited an overwhelming alteration in the expression of cytokines and cytokine receptors involved in immune response signaling. On the other hand, there was no distinct trend between infection with CVI988 and vvRB1B and the expression of cytokines and chemokines, such as IL-10, IFN-γ, STAT1, IRF1, CCL19, and CCL26. However, the expression profiles of IL-1ß, IL-6, IL8L1, CCL4 (GGCL1), and CCL5 were significantly upregulated in splenic lymphocytes from chickens infected with CVI988 compared with those of chickens infected with vvRB1B. Because these cytokines and chemokines are considered to be associated with B cell activation and antigenic signal transduction to T cells, they may indicate differences of immune responses initiated by vaccinal and virulent strains during the early phase of infection. Collectively, our study provides valuable data on the transcriptional landscape using high-throughput sequencing to understand the different mechanism between vaccine-mediated protection and pathogenesis of virulent MDV in vivo.
Assuntos
Herpesvirus Galináceo 2/fisiologia , Imunidade/genética , Linfócitos/metabolismo , Linfócitos/virologia , Doença de Marek/genética , Doença de Marek/virologia , Transcriptoma , Animais , Linfócitos B/metabolismo , Linfócitos B/virologia , Biomarcadores , Galinhas , Biologia Computacional/métodos , Citocinas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Redes Reguladoras de Genes , Doença de Marek/imunologia , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Baço/imunologia , Baço/metabolismo , Baço/virologia , Replicação ViralRESUMO
The Bombyx mori nucleopolyhedrovirus (BmNPV) baculovirus expression system (BES) is a eukaryotic expression system. It possesses great capability for post-translation modification in expression of foreign proteins. With the counterselection cassette rpsL-neo and phage λ-Red recombinase, the defective-rescue BmNPV BES reBmBac can be employed for efficient heterologous multigene coexpression at different gene sites in one baculovirus genome. In the present study, a recombinant baculovirus, reBm-Cαγ, carrying two types of chicken interferon (IFN) genes (chIFN-α and chIFN-γ) was constructed using the reBmBac system. The chIFN-α and chIFN-γ genes were inserted into the same baculovirus genome at the polyhedron and p10 gene sites, respectively. The recombinant baculovirus was capable of coexpressing both chIFN-α and chIFN-γ. The expression levels of the two types of IFN in the coexpression product were exponentially high, at approximately 1.7 and 2.5 times higher, respectively, than those in the corresponding single-expression products. The increase in expression level corresponds to replacement of the nonessential p10 gene in the reBm-Cαγ recombinant baculovirus. This coexpression of recombinant chicken IFNs showed superior antiviral activity.
Assuntos
Baculoviridae/genética , Bombyx/genética , Expressão Gênica , Vetores Genéticos/genética , Interferon Tipo I/genética , Interferon gama/genética , Proteínas Recombinantes/genética , Animais , Embrião de Galinha , Galinhas , Fibroblastos/metabolismo , LarvaRESUMO
We characterized 55 influenza A(H9N2) viruses isolated in Pakistan during 2014-2016 and found that the hemagglutinin gene is of the G1 lineage and that internal genes have differentiated into a variety of novel genotypes. Some isolates had up to 4-fold reduction in hemagglutination inhibition titers compared with older viruses. Viruses with hemagglutinin A180T/V substitutions conveyed this antigenic diversity and also caused up to 3,500-fold greater binding to avian-like and >20-fold greater binding to human-like sialic acid receptor analogs. This enhanced binding avidity led to reduced virus replication in primary and continuous cell culture. We confirmed that altered receptor-binding avidity of H9N2 viruses, including enhanced binding to human-like receptors, results in antigenic variation in avian influenza viruses. Consequently, current vaccine formulations might not induce adequate protective immunity in poultry, and emergence of isolates with marked avidity for human-like receptors increases the zoonotic risk.
