RESUMO
There is an urgent need to develop new vaccines against highly pathogenic PRRS virus (HP-PRRSV) variant in China. The actual use of each codon pairs is more or less frequent than that of the statistical prediction and codon pair bias (CPB) usage affects gene translation. We "shuffled" the existing codons in HP-PRRSV genes GP5, M, nsp2 and nsp9, so that the CPB of these genes could be more negative. De-optimization of nsp9, the RNA-dependent RNA polymerase, significantly decreased PRRSV replication in porcine alveolar macrophages (PAMs). In vitro study showed that HV-nsp9(min) and HV-nsp29(min) were remarkably attenuated in PAMs, and inoculation of pigs with 2 mlâ10(5.0) TCID50/ml of HV-nsp9(min) or HV-nsp29(min) did not cause PRRS. Importantly, pigs immunized with HV-nsp29(min) were fully protected against different HP-PRRSV strains׳ lethal challenges. Our results imply that the CPB de-optimized HV-nsp29(min) has the potential to be used as a live vaccine candidate against HP-PRRSV.
Assuntos
Códon/química , Macrófagos Alveolares/efeitos dos fármacos , Síndrome Respiratória e Reprodutiva Suína/prevenção & controle , Vírus da Síndrome Respiratória e Reprodutiva Suína/efeitos dos fármacos , RNA Polimerase Dependente de RNA/imunologia , Proteínas Virais/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Códon/imunologia , Engenharia Genética , Imunização , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/virologia , Síndrome Respiratória e Reprodutiva Suína/imunologia , Síndrome Respiratória e Reprodutiva Suína/mortalidade , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Biossíntese de Proteínas , RNA Polimerase Dependente de RNA/administração & dosagem , RNA Polimerase Dependente de RNA/genética , Análise de Sobrevida , Suínos , Vacinas Atenuadas , Carga Viral , Proteínas Virais/administração & dosagem , Proteínas Virais/genética , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Replicação Viral/efeitos dos fármacosRESUMO
Large-scale production of long dsRNA is needed if antiviral applications of RNAi are to succeed in shrimp farm operations. A novel hairpin-RNA expression vector was developed based on the RNA-dependent RNA polymerase (RdRp) gene of yellow head virus (YHV), the cause of a lethal shrimp disease. Using transformed RNase-deficient Escherichia coli, large amounts (approximately 5 mg dsRNA from 130 ml bacterial culture) of long dsRNA (>300 nt) were produced. Large-scale in vivo dsRNA production was approximately one-fourth the cost of production of a commercial in vitro transcription kit. The hairpin-RNA consisted of the target RdRp sequence ("forward") and a 100-base shortened version of its inverted repeat ("reverse") to introduce a loop and bypass the difficulty of including a small "loop" connector into the "carrier" vector. A test group of whiteleg shrimp Penaeus (Litopenaeus) vannamei (approximately 10-15 g) was injected with 25 microg of this dsRNA 1-day prior to YHV challenge while control groups were injected with NaCl solution or similarly prepared dsGFP-RNA. The group injected with YHV-specific dsRNA did not develop yellow head disease during 14-day of observation after YHV challenge, whereas the control groups injected with NaCl and dsGFP-RNA developed gross signs of yellow head disease and died within 7-10 days after challenge. Quantitative RT-PCR and immunohistochemistry revealed that both viral mRNA and viral proteins were suppressed in the protected shrimp.
Assuntos
Biotecnologia , Penaeidae/imunologia , Penaeidae/virologia , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , RNA Polimerase Dependente de RNA , Roniviridae/patogenicidade , Animais , Biotecnologia/economia , Biotecnologia/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Imuno-Histoquímica , Penaeidae/enzimologia , Penaeidae/genética , Interferência de RNA/imunologia , RNA de Cadeia Dupla/administração & dosagem , RNA de Cadeia Dupla/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Polimerase Dependente de RNA/administração & dosagem , RNA Polimerase Dependente de RNA/genética , RNA Polimerase Dependente de RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Roniviridae/enzimologia , Roniviridae/genética , Proteínas Virais/genética , Proteínas Virais/metabolismo , Replicação ViralRESUMO
We have previously demonstrated that vaccination of mice with plasmid DNA vectors expressing immunodominant mycobacterial genes induced cellular immune responses and significant protection against challenge with Mycobacterium tuberculosis. We demonstrate here, using in vitro-synthesized RNA, that vaccination with DNA or RNA constructs expressing the M. tuberculosis MPT83 antigen are capable of inducing specific humoral and T-cell immune responses and confer modest but significant protection against M. tuberculosis challenge in mice. This is the first report of protective immunity conferred against intracellular bacteria by an RNA vaccine. This novel approach avoids some of the drawbacks of DNA vaccines and illustrates the potential for developing new antimycobacterial immunization strategies.
