Percepción del riesgo en el proceso de producción de vacunas aviares / Risk perception in the avian vaccine production process

La importancia que tienen para la avicultura cubana el virus de la enfermedad infecciosa de la bolsa (Gumboro) y el virus de la viruela aviar, así como la producción de vacunas que permitan controlar las enfermedades producidas por estos agentes biológicos, justifican la necesidad del establecimiento de una buena gestión de la bioseguridad, ya que el desconocimiento de los peligros y riesgos del personal que labora en estas vacunas puede provocar accidentes de consecuencias indeseables para el producto, escapes de estos microorganismos durante sus procesos productivos y la consecuente contaminación del medio ambiente. El objetivo de la presente investigación fue realizar un análisis de la percepción de riesgo existente en el personal responsable del proceso de producción de dos vacunas aviares. Para ello se utilizó el software RISKPERCEP en una instalación de producción de vacunas aviares; su aplicación mostró variables que demostraron subestimación del riesgo por el personal expuesto y variables con tendencia a la sobrestimación, asociadas fundamentalmente al incorrecto diseño de la instalación. Se concluye que existe la necesidad de una buena capacitación y que se impartan cursos de actualización de bioseguridad donde se tengan en cuenta todos los aspectos del diseño del laboratorio que puedan solucionarse(AU)

The importance of infectious bursal disease virus and fowl pox virus for Cuban poultry farming, as well as the production of vaccines to control the diseases caused by these biological agents, justifies the need for establishment of a good Biosafety management; since the ignorance of the dangers and risks on the part of the personnel that works in them can cause accidents with undesirable consequences for the product, escapes of these microorganisms during their production processes and the consequent contamination of the environment. The objective of the research was to carry out an analysis of the perception of risk in the personnel responsible for the production process of two avian vaccines. The RISKPERCEP software was used in an avian vaccine production facility; its application showed variables that demonstrated underestimation of the risk by the exposed personnel and variables with a tendency to overestimate; fundamentally associated with the incorrect design of the facility. Finally, it is proposed that biosafety update courses be given and that all aspects of the laboratory design that can be solved are taken into account(AU)

Bourse aiguë de l'enfant en chirurgie pédiatrique du chu Gabriel touré / Acute bursa of the child in pediatric surgery at the teaching hospital Gabriel Touré

La bourse aiguë est une urgence médico-chirurgicale de part ses nombreuses étiologies menaçant le pronostic fonctionnel des testicules et leurs annexes. Objectifs : Identifier les causes des bourses aiguës de l'enfant et décrire leurs aspects cliniques et thérapeutiques. Matériels et méthode : Il s'agissait d'une étude descriptive retroprospective allant du 1er janvier 2010 au 31 Décembre 2015 portant sur tous les enfants âgés de 0 à 15 ans reçus et traités pour bourse aiguë dans le service de Chirurgie Pédiatrique du CHU Gabriel Touré. Résultats: En 6 ans, nous avons enregistré 42 patients soit une fréquence de 1,4% des urgences chirurgicales. L'âge moyen était de 2,98 ans (24jours-14 ans). La prématurité a représenté 11,9 % des cas. La tuméfaction scrotale douloureuse était le principal motif de consultation (76,2%), Les principales étiologies étaient la HISE (90,5%), le traumatisme scrotal (4,7%), l'orchiépididymite (2,4%) et la torsion testiculaire (2,4%). Le traitement était chirurgical dans 97,6% des cas. L'évolution après 3 mois était simple dans 97,6% des cas. Conclusion: La bourse aigue de l'enfant est une pathologie peu fréquente touchant surtout les nourrissons. La hernie inguino-scrotale étranglée était la principale étiologie. Le diagnostic doit être précoce et le traitement adéquat afin de reduire la morbi-mortalité

Acute bursa is a medico-surgical emergency because of its many etiologies threatening the functional prognosis of the testes and their appendages. Objectives: Identify the causes of acute bursaries in the child and describe their clinical and therapeutic aspects. Materials and method: This were a retrospective descriptive study from January 1, 2010, to December 31, 2015, on all children aged 0 to 15 years received and treated for acute scholarship in the Pediatric Surgery department at the teaching hospital Gabriel Touré. Results: In 6 years, we registered 42 patients, ie a frequency of 1.4% of surgical emergencies. The mean age was 2.98 years (24 days-14 years). Prematurity represented 11.9% of cases. Painful scrotal tumefaction was the main reason for consultation (76.2%), The main a etiologies were HISE (90.5%), scrotal trauma (4.7%), orchi epididymitis (2.4%) and testicular torsion (2.4%). The treatment was surgical in 97.6% of cases. The course after 3 months was simple in 97.6% of cases. Conclusion: Acute bursa in children is an uncommon condition, especially affecting infants. Strangulated inguino-scrotal hernia was the main aetiology. The diagnosis must be early and the treatment adequate in order to reduce morbidity and mortality

Co-expression, purification and bioassay of three avian viral antigens / 生物工程学报

To achieve uniform soluble expression of multiple proteins in the same Escherichia coli strain, and simplify the process steps of antigen production in genetic engineering subunit multivalent vaccine, we co-expressed three avian virus proteins including the fowl adenovirus serotype 4 (FAdV-4) Fiber-2 protein, infectious bursal disease virus (IBDV) VP2 protein and egg-drop syndrome virus (EDSV) Fiber protein in E. coli BL21(DE3) cells after optimization of gene codon, promoter, and tandem expression order. The purified proteins were analyzed by Western blotting and agar gel precipitation (AGP). The content of the three proteins were well-proportioned after co-expression and the purity of the purified proteins were more than 80%. Western blotting analysis and AGP experiment results show that all the three co-expression proteins had immunoreactivity and antigenicity. It is the first time to achieve the three different avian virus antigens co-expression and co-purification, which simplified the process of antigen production and laid a foundation for the development of genetic engineering subunit multivalent vaccine.

Exploratory serosurvey for antibodies to avian pathogens in backyard chickens from a satellite community of Jalapa City, Guatemala

An exploratory serosurvey was conducted to determine the presence of circulating antibodies to avian patho-gens in backyard chickens from Los Achiotes (LAC), a satellite community of Jalapa City, located in eastern Guatemala. Blood samples from 51 adult chickens belonging to 51 households were taken and investigated for the presence of antibodies to Avian Influenza (AI), Newcastle Disease (ND), Infectious Bronchitis (IB), Infectious Bursal Disease (IBD), Mycoplasma gallisepticum (MG) and M. synoviae (MS). Antibodies for AI, ND, were investigated by Hemagglutination Inhibition, for IB and IBD by ELISA (BioChek®) and for MG and MS by a rapid serum plate agglutination test. The cut-off point for positive titers was 1:4 for AI and ND and a 0.2 S/P ratio for IB and IBD. All sampled chickens were positive for concomitant antibodies to various pathogens. Over half of the chickens were positive reactors to antibodies to all six tested pathogens; about a third carried antibodies to five and the rest to four or three. The frequencies of positive reactors were: AI = 27 (53%); ND = 49 (96.1%); IB = 50 (98%); IBD = 51 (100%); MG = 45 (88%) and MS = 48 (94%). The results show that the dynamic population of backyard chickens in LAC could be a potential threat to backyard poultry, farm poultry, wild birds and human population. The need to develop interventions and policies following the One Health approach (animal health to achieve human health) is stressed.

Se realizó un estudio serológico exploratorio buscando anticuerpos contra patógenos aviares en gallinas de traspatio de la comunidad Los Achiotes –una comunidad satélite de la Ciudad de Jalapa, en el oriente de Guatemala−. Se tomaron muestras de sangre de 51 gallinas provenientes de sendas casas. Se buscaron anticuerpos contra influenza aviar (IA), enfermedad de Newcastle (ENC), bronquitis infecciosa (BI), enfermedad de Gumboro (EG), Mycoplasma gallisepticum (MG) y M. synoviae (MS). Para investigar la presencia de anticuerpos contra IA y ENC se utilizó la prueba de inhibición de hemoaglutinación; para los anticuerpos contra BI la prueba de ELISA BioChek® y para los anticuerpos contra MG y MS la prueba rápida en placa. El punto de corte para títulos positivos fue de 1:4 para IA y ENC y de una razón S/P de 0.2 para BI y EG. Todas las gallinas muestreadas portaban concomitantemente anticuerpos contra varios patógenos aviares. Más de la mitad de las gallinas portaban anticuerpos contra los seis patógenos estudiados. Las frecuencias de reactores positivos a anticuerpos fueron: IA = 27 (53%); ENC = 49 (96.1%); BI = 50 (98%); EG = 51 (100%); MG = 45 (88%) y MS = 48 (94%). Se concluye que la población dinámica de gallinas de traspatio de Los Achiotes podría ser una potencial amenaza para la avicultura artesanal, la avicultura tecnificada, las aves silvestres y la población humana. Se señala la necesidad de generar intervenciones y políticas desde la corriente denominada Una salud (salud animal para lograr la salud humana).

Development and Application of the Reverse Genetic Technologies for Infectious Bursal Disease Virus / 病毒学报

Infectious bursal disease virus (IBDV) is an important member of the Birnaviridae family. IBUV mainly targets the bursa of Fabricius, the central immune organ of chicken, resulting in chicken infectious bursal disease (IBD). IBD represents one of the great challenges for ongoing development of the poultry industry. Reverse genetics for IBDV emerged over twenty years ago. Since then, the technologies behind virus rescue have continually improved leading to a deep understanding of IBDV gene function and tailored vaccine development. Our lab has also been instrumental in the field of IBDV research. Here we review studies on the pathogenic mechanism and the effective prevention and control of IBD.

Immune response elicited by the oral administration of an intermediate strain of IBDV in chickens

The immune response elicited by the oral inoculation of an intermediate strain of infectious bursal disease virus was studied in chickens. A strong over expression of IL-6, IL-8, IFNα and IFNγ was observed in bursa at 3 days post inoculation together with an increase in splenic NO2 release. An influx of T-lymphocytes was also detected.

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.

Immunomodulatory effects of Pimpinella anisum L. (Aniseed) in Broiler Chicks against Newcastle disease and infectious bursal disease viruses / Efecto inmunomodulador de Pimpinella anisum L. (anís) en pollos de engorde contra la enfermedad de Newcastley la enfermedad viral de la bursitis infecciosa

Pimpinella anisum L. (Aniseed) is mostly used as an immune stimulant, growth promoter, antifungal, antibacterial in many countries for centuries. The aim of this study was to determine the immunomodulatory effect of aniseed against Newcastle Disease (ND) and infectious bursal disease (IBD) viruses. The immunomodulatory effect of aniseed against ND and IBD viruses were determined by modifying splenic cell migration inhibition assay and differential leukocyte count for cellular immunity. Haemagglutination inhibition and indirect haemagglutination were used for measurement of humoral immune response against ND and IBD viruses, respectively. The present study suggests that the aniseed addition to basal diet at the rate of 0.5 g/kg and 1 g/kg of feed had best immunomodulatory activity both for humoral and cellular immune responses. However, at higher doses aniseed had adverse effects. Aniseed possesses significant immunomodulatory activity when it is added at lower doses i.e., 0.5 g/kg and 1 g/kg.

Pimpinella anisum L. (Anís) se utiliza principalmente como un estimulante inmunológico, promotor del crecimiento, antifúngico, y antibacteriano, en muchos países durante siglos. El objetivo de este estudio fue determinar el efecto inmunomodulador de anís contra la enfermedad de Newcastle (ND) y la enfermedad de la bursitis infecciosa (IBD). El efecto inmunomodulador de anís contra los virus ND y e IBD se determinaron mediante la modificación del ensayo de inhibición de la migración de células del bazo y recuento diferencial de leucocitos de la inmunidad celular. La inhibición de la hemaglutinación y hemaglutinación indirecta se utilizaron para la medición de la respuesta inmune humoral contra el virus de ND e IBD, respectivamente. El presente estudio sugiere que la adición de anís a la dieta basal a la tasa de 0,5 g/kg y 1 g/kg de alimentación tuvo una mejor actividad inmunomoduladora tanto para las respuestas inmunes humorales como celulares. Sin embargo, a dosis más altas de anís tuvo efectos adversos. El anís posee una importante actividad inmunomoduladora cuando se añade en dosis más bajas, es decir, 0,5 g/kg y 1 g/kg.

Preparation and immunogenicity of silk fibroin/chitosan microspheres for DNA vaccine delivery against infectious bursal disease virus / 生物工程学报

To evaluate the immunities of biodegradable microsphere as a release delivery system for DNA vaccine against Infectious Bursal Disease Virus, in our study, silk fibroin/chitosan microsphere adjuvant was prepared with a precipitation/coacervation method. Both glutaraldehyde and Na2SO4 solution were used in cross-linking. No immune chicken were intramuscularly inoculated at 14 day-old and boosted 2 weeks later. The results show that glutaraldehyde destroyed the DNA activity of the vaccine whereas Na2SO4 solution did not. Factors of the chitosan concentration 0.5% (pH 5.0), silk fibroin concentration 0.6%, plasmid DNA (500 microg/mL) dissolved in 2% Na2SO4 solution were optimized to produce microsphere, with a loading capacity of 89.14%. The average particle size of SF-CS/pCI-VP2/4/3 microsphere is 1.98 microm, and it can protect the loading DNA vaccine from DNase I digestion. Data from anti IBDV ELISA antibodies in the serum show that immunization activity of the microsphere groups were generally higher than plasmid vaccine group (P < 0.05), and the SF/CS compound microspheres group was better than that of sole CS microsphere group. The developed SF/CS microspheres are a very promising vaccine delivery system.

Preparation and identification of monoclonal antibodies against infectious bursal disease virus (IBDV) VP4 / 生物工程学报

Infectious bursal disease virus (IBDV) VP4 plays an important role in immunosuppression of host. In order to develop monoclonal antibodies (McAbs) against VP4, we vaccinated BALB/c mice with His-VP4, screened and subcloned positive clones. We established 4 hybridoma cell lines that stably secreted McAbs against VP4 and named these cell lines 3B3, 3H11, 4C8 and 4G6, respectively. We tested the dissociation constant (Kd) of these McAbs, and found that their K(d)s were 4.61 x 10(-11), 1.71 x 10(-10), 4.26 x 10(-11), 5.02 x 10(-11), respectively. The isotypes of these McAbs were determined to be IgG1, IgG1, IgG2b and IgG1. These McAbs specifically bound to VP4 in IBDV infected DF-1 cells as demonstrated by Western blotting analysis and fluorescence antibody assay. These McAbs would help to detect IBDV infection and to analyze the biological activities of IBDV VP4.

Immunogenicity of recombinant Lactobacillus casei expressing VP2 protein of infectious bursal disease virus in chickens / 生物工程学报

In order to determine immunogenicity and protective effect in chickens, we used the IBDV (Infectious bursal disease virus)-Vp2/Lactobacillus casei as antigen transfer system. First, the immunized and control chickens were challenged by IBDV/DQ at lethal dose to determine the protective ratio. Second, chickens were orallyand intranasally vaccinated twice with 10(9) CFU/mL pLA-VP2/L. casei, pLA/L. casei and PBS as negativecontrol and commercial vaccine as positive control. The bursa injury and the lesion score wererecorded post challenge. The level of specific IgG and sIgA in pLA-VP2/L. casei and positive control groups was significantly higher than that in negativecontrol groups. The protection efficacy in pLA-VP2/L. casei oral group was higher than that inintranasal group. The SI. of pLA-VP2/L. casei oral group was significant higher than other groups. The lesion score indicated the pLA-VP2/L. casei was safer than commercial vaccine for bursa. Collectively, the pLA-VP2/L. casei could be a vaccine candidate for IBDV.

Canarypox virus expressing infectious bursal disease VP2 protein as immunogen for chickens

Canarypox viruses (CNPV) carrying the coding sequence of VP2 protein from infectious bursal disease virus (IBDV) were obtained. These viruses were able to express VP2 protein in vitro and to induce IBDV-neutralizing antibodies when inoculated in specific pathogen-free chickens demonstrating that CNPV platform is usefulness to develop immunogens for chickens.

Cloning and expression of gumboro VP2 antigen in Aspergillus niger

Infectious Bursal Disease Virus [IBDV] causes a highly immunosuppressive disease in chickens and is a pathogen of major economic importance to the poultry industry worldwide. The VP2 protein is the major host-protective immunogen of IBDV and has been considered as a potential subunit vaccine against the disease. VP2 coding sequence was cloned in an inducible fungal vector and the protein was expressed in Aspergillus niger [A. niger]. Aiming at a high level of expression, a multicopy AMA1-pyrG-based episomal construct driven by a strong inducible promoter, glaA, was prepared and used in transformation of A. niger pyrG[-] protoplasts. SDS-PAGE and western blot analysis was carried out to confirm the expression of the protein. A number of pyrG[+] positive transformants were isolated and the presence of expression cassette was confirmed. Western blot analysis of one of these recombinant strains using monospecific anti-VP2 antibodies demonstrated the successful expression of the protein. The recombinant protein was also detected by serum obtained from immunized chicken. In the present study, we have generated a recombinant A. niger strain expressing VP2 protein intracellulary. This recombinant strain of A. niger may have potential applications in oral vaccination against IBDV in poultry industry

Effects of Dangguibuxue Tang, a Chinese herbal medicine, on growth performance and immune responses in broiler chicks

The effects of Dangguibuxue Tang (DBT) on growth performance and immunity response in immunosuppressed broiler chicks were investigated in this study. 240 one-d-old broiler chicks (DaHeng S01) were randomly divided into 4 groups, 2.0% DBT-treatment (A), 0.5% DBT-treatment (B), cyclophosphamide-control (C), and control group (D). From 4 d to 7 d of age, chicks in group A, B and C were given cyclophosphamide (CY) at a dosage of 100mg/kg body weight (BW) daily by intraperitoneal injection to induce immunosuppression. Chicks in group D were given an equal volume of physiological saline daily by intraperitoneal injection and considered normal chicks. Groups A and B were supplemented with 2.0% or 0.5% of DBT in the drinking water from 8 d to 42 d of age. Groups C and D did not receive any additional medication. The results revealed that chicks from group B had lower feed:gain rate (FGR), lower total mortality, higher immunity organ indexes, higher levels of Newcastle disease (ND) antibody and infectious bursal disease (IBD) antibody, higher interleukin-2 and interleukin-6 levels, and greater lymphocyte proliferative responses to concanavalin A (ConA) during the experiment than those from group C. However, no significant difference in the immunity status in the two levels of DBT-treatment was observed. These results indicate that supplementation of 0.5% of DBT can improve both cellular immunity and humoral immunity in immunosuppressed broiler chicks.

Distribution and Quantification of Lymphocytes in the Major Lymphoid Organs of Naturally Gumboro Infected Broilers / Distribución y Cuantificación de Linfocitos en los Órganos Linfoides Principales de Pollos de Engorda Infectados Naturalmente con Gumboro

Gumboro disease is caused by the infectious bursal disease virus (IBDV) which rapidly destroys immature B-lymphocytes of bursa of Fabricious, and causes immune suppression and high mortality in commercial broiler farms in Bangladesh. To investigate the possible effect of IBDV on lymphocytes and its distribution in the major lymphoid organs, bursa of Fabricious including spleen and thymus of naturally Gumboro-infected broilers, a research was conducted in the Department of Anatomy and Histology, collaboration with the Department of Pathology, Bangladesh Agricultural University, Bangladesh. Bursa of Fabricious, spleen and thymus of 21-days-old Gumboro-infected and non-infected broilers of same age (control) were routinely processed and stained by hematoxylin and eosin to examine the distribution of lymphocytes in the major lymphatic organs as well as quantified the number of lymphocytes under high power magnification field and compared with those of control. The number of lymphocytes in bursa of Fabricious, spleen and thymus of Gumboro-infected broilers were 27.20 ± 1.53, 66.50 ± 2.70 and 79.30 ± 3.92 whereas 121 ± 3.82, 89.90 ± 2.09 and 106.30 ± 4.07 were in non-infected control respectively. The numbers of lymphocytes were significantly (p < 0.05) lower in all lymphatic organs of Gumboro-infected broilers than those of non-infected control. The significant numbers of lymphocytes decrease in spleen and thymus suggest that IBVD not only destroy lymphocytes in bursa of Fabricious, but also in spleen and thymus and thus may severely suppress the immune response of IBVD affected broilers.

La enfermedad de Gumboro es causada por el virus de la bursitis infecciosa (VBI), que destruye rápidamente los linfocitos B inmaduros de la bolsa de Fabricio, y causa supresión inmune y la elevada mortalidad en las granjas comerciales de pollos de engorde en Bangladesh. Para investigar el posible efecto del VBI en los linfocitos y su distribución en los órganos linfoides principales, la bolsa de Fabricio, incluyendo el bazo y el timo de pollos de engorde naturalmente infectados con Gumboro, se realizó una investigación en el Departamento de Anatomía e Histología, y el Departamento de Patología, Universidad Agrícola de Bangladesh, Bangladesh. Tanto la bolsa de Fabricio, bazo y el timo de pollos de engorde con 21 días de edad infectados con Gumboro y no infectados de la misma edad (control) se procesaron de forma rutinaria y se tiñeron con H & E para examinar la distribución de los linfocitos en los órganos linfáticos principales, así cuantificar el número de linfocitos bajo campo de alta magnificación y compararlos con los de control. El número de linfocitos en la bolsa de Fabricio, bazo y timo de pollos infectados con Gumboro fue 27,20 ± 1,53, 66,50 ± 2,70 y 79,30 ± 3,92, respectivamente, mientras que en los controles no infectados fue 121 ± 3,82, 89,90 ± 2,09 y 106,30 ± 4,07 respectivamente. El número de linfocitos fue significativamente (p < 0,05) más bajo en todos los órganos linfáticos de pollos de engorde infectados con Gumboro que los no infectados. La disminuición significativa de linfocitos en el bazo y timo, sugiere que el VBI no sólo destruye linfocitos en la bolsa de Fabricio, sino también en el bazo y el timo y, por tanto, puede suprimir severamente la respuesta inmune de pollos de engorde afectados por VBI.

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.

Evaluation of modified vaccinia virus Ankara expressing VP2 protein of infectious bursal disease virus as an immunogen in chickens

A recombinant modified vaccinia Ankara (MVA) virus expressing mature viral protein 2 (VP2) of the infectious bursal disease virus (IBDV) was constructed to develop MVA-based vaccines for poultry. We demonstrated that this recombinant virus was able to induce a specific immune response by observing the production of anti-IBDV-seroneutralizing antibodies in specific pathogen-free chickens. Besides, as the epitopes of VP2 responsible to induce IBDV-neutralizing antibodies are discontinuous, our results suggest that VP2 protein expressed from MVA-VP2 maintained the correct conformational structure. To our knowledge, this is the first report on the usefulness of MVA-based vectors for developing recombinant vaccines for poultry.

The development and application of a SYBR Green I real-time PCR assay for detection of infectious bursal disease virus / 病毒学报

To meet the needs of detection of infectious bursal disease virus (IBDV) under high efficient culture, a SYBR Green I real-time RT-PCR (qRT-PCR) was developed using a pair of primers specific to the conserved region of VP4 gene of IBDV and compared with TCID50 method by monitoring the proliferation dynamics of IBDV in DF-1 cell line adherent to micro carrier in tubular reactor. The results showed that the RT-PCRassay was linear in the range of 4. 03 X 10(1)-10(9) copies/microL. The IBDV RNA detection limit was 40 copies/microL, which was 1 000 times more sensitive than conventional PCR. No cross-reactions with other viruses was observed. The intra-assay coefficient of variation was less than 0.05%. There was a parallel correlation of IBDV proliferation dynamics in DF-1 cell under Micro carrier suspension and static adherent culture by the qRT-PCR assay and TCID50 method. The detection results of the IBDV samples from tubular and flask culture showed the differences of the micro carrier and adherent culture by both methods. In conclusion, the qRT-PCR assay is more rapid and sensitive than the TCID50 method, which is more appropriate for the real time detection of IBDV.

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.

Molecular characterization of two Bangladeshi infectious bursal disease virus isolates using the hypervariable sequence of VP2 as a genetic marker

Two Bangladeshi infectious bursal disease virus (IBDV) isolates collected in 2007, termed GB1 and GB3, were subjected to comparative sequencing and phylogenetic analyses. Sequence analysis of a 474-bp hypervariable region in the VP2 gene revealed that among four major amino acid substitutions observed in the strains, two were unique to GB1 and GB3 (Ser217Leu and Ala270Thr) while one substitution was only found in GB1 (Asn299Ser). Among IBDVs from Bangladesh including GB1 and GB3, the rate of identity and homology was around 97~99%. The amino acid sequences of GB1 and GB3 differ from those of previous Bangladeshi IBDV isolates and contain amino acid substitutions Pro222Ala and Asn299Ser (in GB3 only). Phylogenetic analysis revealed that GB1 and GB3 are grouped with other very virulent IBDVs of European and American origin in contrast to two previously isolated Bangladeshi IBDV strains (GenBank accession Nos. AF362776 and AF260317), which belong to the Asian group. It was concluded that GB1 and GB3 belong to a very virulent group of IBDVs. However, amino acid sequences of GB1 and GB3 differ from those of the other Bangladeshi IBDVs by one or two amino acids encoded in the hypervariable region of the VP2 gene.