Efficient assembly of full-length infectious clone of Brazilian IBDV isolate by homologous recombination in yeast

The Infectious Bursal Disease Virus (IBDV) causes immunosuppression in young chickens. Advances in molecular virology and vaccines for IBDV have been achieved by viral reverse genetics (VRG). VRG for IBDV has undergone changes over time, however all strategies used to generate particles of IBDV involves multiple rounds of amplification and need of in vitro ligation and restriction sites. The aim of this research was to build the world's first VRG for IBDV by yeast-based homologous recombination; a more efficient, robust and simple process than cloning by in vitro ligation. The wild type IBDV (Wt-IBDV-Br) was isolated in Brazil and had its genome cloned in pJG-CMV-HDR vector by yeast-based homologous recombination. The clones were transfected into chicken embryo fibroblasts and the recovered virus (IC-IBDV-Br) showed genetic stability and similar phenotype to Wt-IBDV-Br, which were observed by nucleotide sequence, focus size/morphology and replication kinetics, respectively. Thus, IBDV reverse genetics by yeast-based homologous recombination provides tools to IBDV understanding and vaccines/viral vectors development.

Partial VP1 sequencing of Brazilian infectious bursal disease virus strains

Infectious bursal disease virus (IBDV) is classified according to the antigenicity and virulence into classical virulent (cv), very virulent (vv), and antigenic variant strains. The molecular basis for the IBDV antigenic variation is well established and is associated to the capsid protein, VP2 (gene VP2 of segment A), whereas both VP2 and the RNA-dependent RNA polymerase, VP1 (gene VP1 of segment B), have been correlated with the virulence. In this study, seventeen Brazilian IBDV samples previously characterized by the VP2 gene as cv (three) and vv (fourteen) strains were genetically and molecularly analyzed for their VP1 gene. All of the strains kept with the same cv or vv classification except one sample, Br/03/DR. This sample was classified as vv by its VP2 gene, but it was most closely related to the cv strains by its VP1 partial sequence and phylogeny. Studies on the phylogeny of VP1 have suggested a possible reassortment event that originated the vvVP1. In this case, the sample carrying vvVP2 and cvVP1 could be a descendant of IBDV ancestors prior to the reassortment of vvVP1; alternatively, it could be the result of a genetic exchange between the segments of different strains or with a live attenuated vaccine. Nevertheless, this is the first report of natural genetic reassortment of IBDV in Brazil.

Desenvolvimento de sistemas de genética reversa para os vírus da doença de gumboro e influenza aviária através do uso de recombinação homóloga em levedura / Development of reverse genetics systems for infectious bursal disease virus and avian influenza by yeast-based homologous recombination

A Doença de Gumboro (DG) é uma doença imunossupressora comum em aves jovens infectadas pelo Vírus da Doença de Gumboro (Infectious Bursal Disease Vírus, IBDV), sendo responsável por perdas econômicas no setor avícola. O vírus influenza apresenta-se com um alto nível de mutação, o que resulta no surgimento de vírus imunologicamente distintos capazes de causar pandemias ou epidemias. Entende-se por sistema de genética reversa viral (SGRV) a geração/recuperação de vírus por meio da transfecção celular do cDNA viral clonado ou seu RNA viral transcrito in vitro. SGRV pode ser usado na elucidação dos mecanismos de replicação do influenza e IBDV, e aplicações biotecnológicas como desenvolvimento de vacinas. Diante desse levantado, objetivou-se a construção de dois SGRVs por recombinação homóloga em levedura (RHL): um para IBDV e outro para influenza aviária (IA). Para o SGRV do IBDV, IBDV foi isolado no Brasil, teve seu genoma amplificado e clonado por RHL no vetor pJG-CMV-HDR. Os clones foram transfectados em fibroblasto de embrião de galinha (FEG) e o vírus gerado (IC-IBDVBr) mostrou estabilidade gênica e fenótipo similar ao vírus parental. A geração e crescimento do IC-IBDVBr não foram possíveis em células Vero. Para o SGRV do IA, IA foi isolado no Brasil, seu genoma foi amplificado e clonado em pDrive/pGEM-T Easy e depois subclonado por RHL no vetor pJGCh2008. Os clones em pJG-Ch2008 responsáveis pela codificação das proteínas do complexo polimerase viral (CPV) foram transfectados simultaneamente em células Human Embryonic Kidney 293T com plasmídeos contendo o gene repórter red fluorescent protein ou Gaussia luciferase, ambos flanqueados pela untransleated region do influenza. A funcionalidade do CPV do IA foi verificada pela expressão de RFP e GLuc. A recuperação do IA em FEG pelos clones em pJG-Ch2008 não foi possível. A funcionalidade do CPV mais a integridade dos clones indicam que a recuperação do IA não foi possível provavelmente devido à eficiência da transfecção celular. A construção do SGRV para IBDV, o primeiro do mundo feito por RHL e o primeiro desenvolvido no Brasil, junto com os passos iniciais para a construção do primeiro SGRV para influenza feito por RHL e a consequente construção do CPV por essa tecnologia, disponibilizam ao país ferramentas capazes de contribuir no esclarecimento do ciclo replicativo de ambos os vírus, além de criar bases para o futuro desenvolvimento de vacinas e vetores virais.

Sequencing and phylogenetic analysis of the infectious bursal disease virus isolates from outbreak in layer flocks in the state of Minas Gerais / Sequenciamento e análise filogenética de isolados do vírus da doença infecciosa bursal de surtos ocorridos em lotes de poedeiras no estado de Minas Gerais

Sequencing and phylogenetic analysis based on the nucleotide sequence of the gene encoding VP2 protein was carried out in order to characterize the agent of two outbreaks of infectious bursal disease in layer flocks in the state of Minas Gerais in 2004. The results indicate the outbreaks could be related to the vaccinal virus.

O sequenciamento e a análise filogenética a partir da seqüência nucleotídica do gene que codifica a proteína VP2 foram realizados com o intuito de caracterizar os agentes causadores de dois surtos da doença infecciosa bursal em lotes de poedeiras do estado Minas Gerais, em 2004. Os resultados indicam que os surtos analisados podem estar relacionados com o vírus de origem vacinal.

Protection of chicken against very virulent IBDV provided by in ovo priming with DNA vaccine and boosting with killed vaccine and the adjuvant effects of plasmid-encoded chicken interleukin-2 and interferon-gamma

The aim of this study was to examine the efficacy of in ovo prime-boost vaccination against infectious bursal disease virus (IBDV) using a DNA vaccine to prime in ovo followed by a killed-vaccine boost post hatching. In addition, the adjuvant effects of plasmid-encoded chicken interleukin-2 and chicken interferon-gamma were tested in conjunction with the vaccine. A plasmid DNA vaccine (pcDNA-VP243) encoding the VP2, VP4, and VP3 proteins of the very virulent IBDV (vvIBDV) SH/92 strain was injected into the amniotic sac alone or in combination with a plasmid encoding chicken IL-2 (ChIL-2) or chicken IFN-gamma (ChIFN-gamma) at embryonation day 18, followed by an intramuscular injection of a commercial killed IBD vaccine at 1 week of age. The chickens were orally challenged with the vvIBDV SH/92 strain at 3 weeks of age and observed for 10 days. In ovo DNA immunization followed by a killed-vaccine boost provided significantly better immunity than the other options. No mortality was observed in this group after a challenge with the vvIBDV. The prime-boost strategy was moderately effective against bursal damage, which was measured by the bursa weight/body weight ratio, the presence of IBDV RNA, and the bursal lesion score. In ovo DNA vaccination with no boost did not provide sufficient immunity, and the addition of ChIL-2 or ChIFN-gamma did not enhance protective immunity. In the ConA-induced lymphocyte proliferation assay of peripheral blood lymphocyte collected 10 days post-challenge, there was greater proliferation responses in the DNA vaccine plus boost and DNA vaccine with ChIL-2 plus boost groups compared to the other groups. These findings suggest that priming with DNA vaccine and boosting with killed vaccine is an effective strategy for protecting chickens against vvIBDV.

Efficacy of VP2 protein expressed in E. coli for protection against highly virulent infectious bursal disease virus

The ability of a heat-inactivated whole virus from a highly virulent infectious bursal disease virus (hvIBDV) and VP2 protein from hvIBDV expressed in E. coli provided protection against a hvIBDV challenge in specificpathogen- free (SPF) chickens. Six out of seven chickens that were injected three times with crude VP2 protein developed significant antibody titer against IBDV. However, only four out of the seven chickens survived the hvIBDV challenge. Despite showing low antibody titer profiles, all chickens immunized with the heat-inactivated whole virus also survived the challenged with hvIBDV. However, all of these chickens had bursal atrophy and mild to moderate depletion of lymphocytes. Thus, antibodies raised against IBDV VP2 protein expressed in E. coli and denatured IBDV proteins induced some degree of protection against mortality but not against bursal damage following challenge with hvIBDV.