Angiotensin converting enzyme 2 alleviates infectious bronchitis virus-induced cellular inflammation by suppressing IL-6/JAK2/STAT3 signaling pathway / 生物工程学报

The goal of this study was to investigate the regulatory effect of angiotensin converting enzyme 2 (ACE2) on cellular inflammation caused by avian infectious bronchitis virus (IBV) and the underlying mechanism of such effect. Vero and DF-1 cells were used as test target to be exposed to recombinant IBV virus (IBV-3ab-Luc). Four different groups were tested: the control group, the infection group[IBV-3ab-Luc, MOI (multiplicity of infection)=1], the ACE2 overexpression group[IBV-3ab Luc+pcDNA3.1(+)-ACE2], and the ACE2-depleted group (IBV-3ab-Luc+siRNA-ACE2). After the cells in the infection group started to show cytopathic indicators, the overall protein and RNA in cell of each group were extracted. real-time quantitative polymerase chain reaction (RT-qPCR) was used to determine the mRNA expression level of the IBV nucleoprotein (IBV-N), glycoprotein 130 (gp130) and cellular interleukin-6 (IL-6). Enzyme linked immunosorbent assay (ELISA) was used to determine the level of IL-6 in cell supernatant. Western blotting was performed to determine the level of ACE2 phosphorylation of janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). We found that ACE2 was successfully overexpressed and depleted in both Vero and DF-1 cells. Secondly, cytopathic indicators were observed in infected Vero cells including rounding, detaching, clumping, and formation of syncytia. These indicators were alleviated in ACE2 overexpression group but exacerbated when ACE2 was depleted. Thirdly, in the infection group, capering with the control group, the expression level of IBV-N, gp130, IL-6 mRNA and increased significantly (P < 0.05), the IL-6 level was significant or extremely significant elevated in cell supernatant (P < 0.05 or P < 0.01); the expression of ACE2 decreased significantly (P < 0.05); protein phosphorylation level of JAK2 and STAT3 increased significantly (P < 0.05). Fourthly, comparing with the infected group, the level of IBV-N mRNA expression in the ACE2 overexpression group had no notable change (P > 0.05), but the expression of gp130 mRNA, IL-6 level and expression of mRNA were elevated (P < 0.05) and the protein phosphorylation level of JAK2 and STAT3 decreased significantly (P < 0.05). In the ACE2-depleted group, there was no notable change in IBV-N (P > 0.05), but the IL-6 level and expression of mRNA increased significantly (P < 0.05) and the phosphorylation level of JAK2 and STAT3 protein decreased slightly (P > 0.05). The results demonstrated for the first time that ACE2 did not affect the replication of IBV in DF-1 cell, but it did contribute to the prevention of the activation of the IL-6/JAK2/STAT3 signaling pathway, resulting in an alleviation of IBV-induced cellular inflammation in Vero and DF-1 cells.

The potential inhibitory impact of curcumin, epigallocatechin gallate and their combinations on infectious bronchitis virus in vivo

Aims@#Infectious bronchitis virus (IBV) is a highly contagious, acute viral respiratory disease that mostly affects chickens. The poultry sector has suffered enormous losses as a result of IBV. Currently, live attenuated vaccines are routinely used to prevent and control IBV. However, due to the enormous genetic variety, vaccinations are becoming ineffective, with low cross-protection effects among vaccine serotypes. The present study aimed at investigating the possible antiviral effects of curcumin, epigallocatechin gallate (EGCG) and their mixtures against IBV in vivo.@*Methodology and results@#Curcumin, EGCG and their combinations were administered to infected and uninfected chicken groups and viral load titers were determined by real-time PCR. The clinical symptoms of both the negative and positive control groups were also compared. Finally, the trachea tissues of each group were examined histopathologically. According to our findings, the viral titer and the clinical signs dropped significantly during the pretreatment infection procedure. Curcumin, EGCG and their combinations also show significant antiviral activities.@*Conclusion, significance and impact of study@#This study clearly shown that natural compounds and their combinations, such as curcumin or/and ECGC can reduce viral pathogenicity in vivo, suggesting that they might have therapeutic implications in the poultry sector.

Diferencias entre el SARS-CoV-2 y el coronavirus de la Bronquitis Infecciosa Aviar (IBV) / Differences between SARS-CoV-2 and Avian Infectious Bronchitis (IBV) coronavirus / Diferenças entre o coronavírus SARS-CoV-2 e Bronquite Infecciosa Aviária (IBV)

Resumen La enfermedad por el nuevo coronavirus 2019 (COVID-19) es causada por el virus denominado SARS-CoV-2, no obstante, en los pollos de corral los coronavirus causan Bronquitis Infecciosa Aviar. En la actualidad, se ha logrado analizar la secuencia genómica del virus SARS-CoV-2, el cual indica que éste emergió de un reservorio animal, incluso se ha considerado que un virus aislado de un murciélago, idéntico a SARS-CoV, sea el progenitor del nuevo coronavirus. En otros estudios, se ha evidenciado que la glicoproteína de la espícula viral tiene un alto grado de emparentamiento entre virus que infectan mamíferos y aves, que es la que permite el contacto con el huésped. Mientras que en el caso del IBV, al inhalarse, el virus se ligará a los receptores de glucoproteína que contienen ácido siálico en las células epiteliales ciliadas del tejido respiratorio, entonces la replicación viral dará como resultado la pérdida de la función ciliar, acopiamiento de moco, necrosis y descamación, provocando dificultad para respirar y asfixia. El IBV afecta tráquea, riñones y tracto reproductivo de muchas aves. En el caso de las gallinas, el IBV virémico causa lesiones en el magnum y en el útero. Esta revisión dilucida algunos puntos clave en las diferencias en el nuevo coronavirus y el virus de la bronquitis infecciosa. Es muy poco probable que SARS-CoV-2 infecte o cause enfermedades en las aves de corral.

Abstract The new coronavirus 2019 disease (COVID-19) is caused by the virus called SARS-CoV-2, however, in free-range chicken's coronaviruses cause Avian Infectious Bronchitis. Currently, it has been possible to analyze the genomic sequence of the SARS-CoV-2 virus, which indicates that it emerged from an animal reservoir, it has even been considered that a virus isolated from a bat, identical to SARS-CoV, is the parent of the new coronavirus. In other studies, it has been shown that the glycoprotein of the viral spicule has a high degree of relationship between viruses that infect mammals and birds, which is the one that allows contact with the host. Whereas in the case of IBV, when inhaled, the virus will bind to sialic acid-containing glycoprotein receptors in hair epithelial cells of respiratory tissue, then viral replication will result in loss of ciliary function, mucus clearance, necrosis, and peeling, causing shortness of breath and suffocation. IBV affects the trachea, kidneys, and reproductive tract of many birds. In chickens, viremic IBV causes lesions in the magnum and the uterus. This review elucidates some key points in the differences between the novel coronavirus and the infectious bronchitis virus. SARS-CoV-2 is highly unlikely to infect or cause disease in poultry.

Resumo A nova doença coronavírus 2019 (COVID-19) é causada pelo vírus denominado SARS-CoV-2, no entanto, em galinhas caipiras, os coronavírus causam bronquite infecciosa aviária. Atualmente, foi possível analisar a sequência genômica do vírus SARS-CoV-2, o que indica que ele emergiu de um reservatório animal, inclusive se considerou que um vírus isolado de um morcego, idêntico ao SARS-CoV, é o progenitor do novo coronavírus. Em outros estudos, foi demonstrado que a glicoproteína da espícula viral possui alto grau de parentesco entre os vírus que infectam mamíferos e aves, o que permite o contato com o hospedeiro. Enquanto no caso do IBV, quando inalado, o vírus se liga aos receptores de glicoproteína contendo ácido siálico nas células epiteliais ciliadas do tecido respiratório, a replicação viral resultará em perda da função ciliar, acúmulo de muco , necrose e descamação, causando falta de ar e sufocação. O IBV afeta a traqueia, os rins e o trato reprodutivo de muitas aves. No caso das galinhas, o IBV virêmico causa lesões no magno e no útero. Esta revisão elucida alguns pontos-chave nas diferenças entre o novo coronavírus e o vírus da bronquite infecciosa. É altamente improvável que o SARS-CoV-2 infecte ou cause doenças em aves.

Cross-species surveillance and risk factors associated with Avian Coronavirus in North-Central and South West Regions of Nigeria

@#Infectious bronchitis viral (IBV) (Avian coronavirus) diseases is among the major reproductive diseases affecting the avian production in Africa. There is scanty information on its current status and vaccination compliance among captive wild birds (CWB) and indigenous chickens (LC) in Nigeria. This study aimed to assess the exposure and the risk factors associated with IBV in CWB and LC from North-central and South west regions of Nigeria. Sera samples from 218 LC and 43 CWB were examined for IBV IgG using enzyme linked immunosorbent assay. Also, owners of LC and managers of CWB were interviewed using a pre-tested structured checklist. An overall IBV prevalence of 42.9% (112/261) was obtained. Captive wild birds and indigenous chickens had 11.6% (5/43) and 49.1% (107/218) prevalence respectively with a significant difference (p< 0.0001, OR= 7.3, 95% CI= 2.8-19.3). Also, geo-location indicated significant difference in IBV exposure among birds (p<0.034). Furthermore, the study showed that there had never been laboratory screening on all acquired wild birds for exposure to infectious agents in the study location while none of these birds (LB/CWB) had history of vaccination. Since IBV is endemic in Nigeria, the use of vaccine for prophylactic measure should be advocated among LC and CWB owners in order to avoid unnecessary losses. Also, the essence of screening for infectious agents in newly acquired wild birds should be considered crucial for health sustenance and public safety.

Construction and characterization of a recombinant vaccine encoding the nucleocapsid protein gene of avian infectious bronchitis virus / Construção e caracterização de uma vacina recombinante codificando o gene da proteina do nucleocapsideo do virus da bronquite infecciosa das galinhas

A vacinação é a forma mais utilizada para prevenir a bronquite infecciosa causada pelo vírus da bronquite infecciosa das galinhas (IBV). Contudo, as vacinas convencionais são incapazes de diferenciar aves infectadas de vacinadas. No presente trabalho foi construído, caracterizado, e avaliado como candidato vacinal, um adenovírus recombinante expressando o gene N do IBV. O gene N foi clonado em um adenovírus humano tipo 5 defectivo e transfectado para as células HEK-293A para gerar rAd5_N. Após o vetor ser obtido como esperado e a confirmação da expressão da proteína N em HEK-293ª, foi realizada inoculação pela via oculo-nasal na dose de 10 7 TCID 50 /0,1mL para imunização de galinhas livres de patógenos específicos (SPF). A resposta imunológica do Ad5_N e a proteção contra o desafio ao IBV foram avaliadas e comparadas com uma vacina viva comercial. Não foram detectados anticorpos anti-IBV em aves vacinadas com o Ad5_N. A vacina comercial induziu anticorpos detectáveis a partir do 7º dia pós-vacinal. Em aves vacinadas com o Ad5_N não houve aumento na expressão de IFNγ. Neste estudo, o rAd5_N obtido não conferiu proteção contra desafio com IBV-M41. Os resultados indicam a necessidade de avaliar adenovírus recombinantes expressando outros genes do IBV.(AU)

Genomic characteristics of natural recombinant infectious bronchitis viruses isolated in Korea / 대한수의학회지

Two infectious bronchitis virus (IBV) K046-12 and K047-12 strains were isolated and the nearly complete genomes of them were sequenced. Sequence comparisons showed that the K046-12 genome was most similar to Korean IBV strains, and the K047-12 genome was most similar to QX-like IBV strains. Phylogenetic analysis showed that nearly all K046-12 and most K046-12 genes were placed in the same cluster as Korean IBV isolates, but the S1 region was placed in the same cluster as Mass-type IBVs. For K047-12, nearly all K047-12 and most K047-12 genes were located in the same cluster as QX-like IBVs, but the M region was located in the same cluster as Korean IBV isolates with K047-12. Recombination analysis confirmed that K046-12 is a recombinant strain with the primary parental sequence derived from Korean IBVs and minor parental sequence derived from Mass-type IBV, and K047-12 is a recombinant strain with the major parental sequence derived from QX-IBV and minor parental sequence derived from Korean IBVs. This study showed that new IBV recombinants are constantly generated among various IBVs, including those used for vaccination. Therefore, genetic analysis of new virus isolates should be performed for effective infectious bronchitis control and appropriate vaccine development.

Serologic monitoring of animal welfare-oriented laying-hen farms in South Korea / 대한수의학회지

As animal welfare issue becomes important, the European Union bans conventional cages for laying hens from 2012. So the alternative housing systems like floor pens, aviaries or free range systems have been suggested. From 2011 to 2014, we monitored 20 welfare-oriented laying hen farms in South Korea to figure out serological status of major viral diseases. During this period, total 3,219 blood samples were collected from the randomly selected chickens to test and evaluate the hemagglutination inhibition titers for low pathogenic avian influenza, Newcastle disease and egg drop syndrome '76. A total of 2,926 blood samples were tested through enzyme linked immunosorbent assay (ELISA) to assess the serological status of infectious bronchitis (IB). The distribution of ELISA titers for IB was various from almost 0 to 20,000 through the all weeks of age. Also, the antibody coefficient of variation for most of the diseases in this study was higher than those of typical cage layers. As this study was the first surveillance for major avian viral diseases of the animal welfare-oriented farms in South Korea, the results obtained from this study will help to determine what information and resources are needed to maintain better biosecurity and to improve the health and welfare of laying hen flocks.

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).

Correlations in the results of virus neutralization test, hemagglutination inhibition test, and enzyme-linked immunosorbent assay to determine infectious bronchitis virus vaccine potency

The virus neutralization (VN) test was used to determine potency of the infectious bronchitis (IB) vaccine. The results of VN, hemagglutination inhibition (HI), and enzyme-linked immunosorbent assay (ELISA) were compared with those of the IBV M41. The r² values between VN and HI titers and the ELISA antibody titer were 0.8782 and 0.0336, respectively, indicating a high correlation between VN and HI, but not VN and ELISA. The Cohen's kappa coefficient between the VN titer of 2 log₁₀ and HI titer of 5 log₂ was 0.909. Our results showed that VN could be replaced with HI for testing the potency of IBV M41.

Increased expression of interleukin-6 related to nephritis in chickens challenged with an avian infectious bronchitis virus variant / Aumento da expressão de interleucina-6 relacionada com nefrite in galinhas desafiadas com uma variante do vírus da bronquite infecciosa

A Brazilian field isolate (IBV/Brazil/PR05) of avian infectious bronchitis virus (IBV), associated with development of nephritis in chickens, was previously genotyped as IBV variant after S1 gene sequencing. The aim of this study was to evaluate the levels of IL-6 in kidneys and trachea of birds vaccinated and challenged with IBV/Brazil/PR05 strain, correlating these results with scores of microscopic lesions, specific IBV antigen detection and viral load. The up-regulation of IL-6 and the increased levels of viral load on renal and tracheal samples were significantly correlated with scores of microscopic lesions. Reduced levels of viral load were detected in kidneys of birds previously vaccinated and challenged, compared to non-vaccinated challenged group, although markedly microscopic lesions were observed for both groups. The expression of IL-6, present both in the kidney and in the tracheas, was dependent on the load of the virus present in the tissue, and the development of lesions was related with IL-6 present in the tissues. These data suggest that variant IBV/Brazil/PR05 can induce the expression of proinflammatory cytokines in a manner correlated with viral load and increased IL-6 is involved in the tissue with the influx of inflammatory cells and subsequent nephritis. This may contribute with a model to the development of immunosuppressive agents of IL-6 to prevent acute inflammatory processes against infection with IBV and perhaps other coronaviruses, as well as contribute to the understanding of the immunopathogenesis of IBV nephropatogenic strains.

Uma estirpe variante do vírus da bronquite infecciosa (VBI) associada com o desenvolvimento de nefrite em galinhas, foi isolado e identificado como variante por análise do gene S1. A estirpe IBV/Brazil/PR05 foi testada quanto à sua capacidade de induzir a expressão de interleucina-6 (IL-6) nos tecidos renais e traqueais. Galinhas vacinadas com a estirpe Massachusetts H120 e não vacinadas foram desafiadas com a estirpe IBV/Brazil/PR05. Cinco dias após a infecção, traquéias e rins foram coletados para análise por RT-qPCR, imunohistoquímica e histopatologia. Foi determinada a expressão relativa de IL-6 e da carga viral. A expressão de IL-6 e carga viral foram correlacionadas com o desenvolvimento de nefrite e lesão traqueal. A expressão de IL-6 foi maior quando houve aumento da carga viral na traqueia e nos rins. A carga viral presente nos rins foi inferior quando as aves foram vacinadas, entretanto foi observada nefrite acentuada. Houve alta correlação entre o desenvolvimento de nefrite e o nível de expressão de IL-6, bem como a expressão de IL-6 e a carga viral. A expressão de IL-6, presente tanto nos rins e nas traqueias, foi relacionada a carga viral presente nestes tecidos, e o desenvolvimento das lesões foi relacionado com a expressão de IL-6. Estes dados sugerem que a variante IBV/Brazil/PR05 pode induzir a expressão de citocinas pró-inflamatórias de forma correlacionada com a carga viral, e o aumento de IL-6 está envolvido com o influxo de células inflamatórias no tecido, o que evolui para o desenvolvimento de nefrite. Isto pode contribuir como um modelo para o desenvolvimento de agentes imunossupressores da IL-6 para evitar processos inflamatórios agudos contra infecção com o VBI e talvez outros coronavírus, bem como contribuir para o entendimento da imunopatogênese das estirpes nefropatogênicas deste vírus.

Elevated level of renal xanthine oxidase mRNA transcription after nephropathogenic infectious bronchitis virus infection in growing layers

To assess relationships between xanthine oxidase (XOD) and nephropathogenic infectious bronchitis virus (NIBV) infection, 240 growing layers (35 days old) were randomly divided into two groups (infected and control) of 120 chickens each. Each chicken in the control and infected group was intranasally inoculated with 0.2 mL sterile physiological saline and virus, respectively, after which serum antioxidant parameters and renal XOD mRNA expression in growing layers were evaluated at 8, 15 and 22 days post-inoculation (dpi). The results showed that serum glutathione peroxidase and superoxide dismutase activities in the infected group were significantly lower than in the control group at 8 and 15 dpi (p < 0.01), while serum malondialdehyde concentrations were significantly higher (p < 0.01). The serum uric acid was significantly higher than that of the control group at 15 dpi (p < 0.01). In addition, the kidney mRNA transcript level and serum activity of XOD in the infected group was significantly higher than that of the control group at 8, 15 and 22 dpi (p < 0.05). The results indicated that NIBV infection could cause the increases of renal XOD gene transcription and serum XOD activity, leading to hyperuricemia and reduction of antioxidants in the body.

Production and immunogenicity of chimeric virus-like particles containing the spike glycoprotein of infectious bronchitis virus

Infectious bronchitis virus (IBV) poses a severe threat to the poultry industry and causes heavy economic losses worldwide. Vaccination is the most effective method of preventing infection and controlling the spread of IBV, but currently available inactivated and attenuated virus vaccines have some disadvantages. We developed a chimeric virus-like particle (VLP)-based candidate vaccine for IBV protection. The chimeric VLP was composed of matrix 1 protein from avian influenza H5N1 virus and a fusion protein neuraminidase (NA)/spike 1 (S1) that was generated by fusing IBV S1 protein to the cytoplasmic and transmembrane domains of NA protein of avian influenza H5N1 virus. The chimeric VLPs elicited significantly higher S1-specific antibody responses in intramuscularly immunized mice and chickens than inactivated IBV viruses. Furthermore, the chimeric VLPs induced significantly higher neutralization antibody levels than inactivated H120 virus in SPF chickens. Finally, the chimeric VLPs induced significantly higher IL-4 production in mice. These results demonstrate that chimeric VLPs have the potential for use in vaccines against IBV infection.

Detecção molecular de vírus da bronquite infecciosa em plantéis de avós, matrizes e frangos de corte no Rio Grande do Sul e Mato Grosso / Molecular detection of infectious bronchitis virus in flocks of grandmothers, mothers and broiler chicken in Rio Grande do Sul and Mato Grosso

O vírus da Bronquite Infecciosa das galinhas (VBI) pertence ao grupo 3 da família Coronaviridae e é o causador de desordens respiratórias e renais em frangos de corte. A vacinação com vacinas vivas é praticada em matrizes e avós e muitas vezes também nos plantéis destinados ao abate. As vacinas utilizadas no Brasil são usualmente do sorogrupo Massachusetts e baseadas nas amostras H120 e H52. É comum que após a vacinação o vírus vacinal seja detectado por isolamento em ovos embrionados ou por métodos moleculares por até 4 semanas. Após essa data, normalmente, não há detecção de vírus e o VBI, quando encontrado, pode representar recirculação do vírus vacinal no plantel ou a introdução de uma nova cepa do vírus. No presente estudo, para avaliar a circulação do vírus em plantéis de frangos e reprodutoras nos estados do Rio Grande do Sul e Mato Grosso do Sul, foram coletadas 240 traqueias e rins de aves de 48 plantéis, sendo (20 exemplares/4 plantéis) de avós, (80 exemplares/16 plantéis) de matrizes e (140 exemplares/28 plantéis) de frangos de corte, as quais foram analisadas em misturas de cinco amostras. Todos os animais eram vacinados e as amostras foram coletadas ao redor de 2 a 48 semanas após a vacinação. A presença de VBI foi determinada com auxílio de uma reação em cadeia da polimerase tipo nested, direcionada ao gene da proteína S1, padronizada neste estudo. Das 48 amostras testadas, 14 resultaram positivas: cinco foram oriundas de aves vacinadas há menos de quatro semanas na data da coleta e nove eram de amostras de aves vacinadas há mais de quatro semanas, o que pode ser devido à recirculação do vírus vacinal ou mesmo introdução de vírus selvagem nos plantéis.

Infectious bronchitis virus (IBV, Avian Coronavirus) from chickens belongs to group 3 of the family Coronaviridae and causes respiratory and renal disorders in broilers. Vaccination using live vaccines is generally performed in mothers and grandmothers, as well as often in flocks for slaughter. The vaccines used in Brazil are usually from serogroup Massachusetts and based on standard samples of the virus at passages H120 and H52. It is common that after vaccination the vaccine virus is detected by isolation in embryonated eggs or by molecular methods for up to four weeks. After, there is usually no virus detection and any IBV found may represent recirculation of the vaccine virus in the flock or the introduction of a new strain. In this study, to evaluate the circulation of the virus in poultry flocks and breeders in the state of Rio Grande do Sul and Mato Grosso do Sul, 240 samples were collected from tracheas and kidneys of birds from 48 flocks, and (20 biological samples / 4 flocks) from grandmothers (80 samples/16 flocks) and mothers (140 samples/28 flocks) from broilers, which were analyzed in pools of five samples. All animals were vaccinated and samples were collected around 2-48 weeks after vaccination. The presence of IBV was determined with the aid of a polymerase chain reaction "nested" gene-directed protein S1, standardized in this study. From the 48 samples tested, 14 were positive: 5 were from birds vaccinated after less than 4 weeks and 9 were from birds vaccinated more than four weeks should be wild viruses or represent the recirculation of the vaccine virus.

Development of an attenuated vaccine strain from a korean respiratory type infectious bronchitis virus / 대한수의학회지

An attenuated vaccine strain AVR1/08 of Korean respiratory type of infectious bronchitis virus (IBV) was developed by 89th passages of IBV D85/06 strain in chicken eggs. The AVR1/08 strain had higher virus titer at least 20 times (10(1.3)) than the parent virus D85/06 by egg inoculation method. The AVR1/08 strain had a single point mutation (S to Y) at position 56 of spike protein of IBV compared to parent virus IBV D85/06 strain. The mutation was observed consistently at viruses after 47th passage in chicken eggs. The AVR1/08 strain showed no virulence even after 6 passages in chickens and all chickens inoculated induced anti-IBV antibody 14 days after vaccination. The AVR1/08 strain had broad protective efficacy against QX type Korean nephropathogenic virus (Q43/06 strain), KM91 type Korean nephropathogenic virus (KM91 strain) and Korean respiratory virus (D85/06 strain). In contrast, Massachusetts (Mass) type attenuated vaccine strain H120 showed protection of 37.5 to 50% against these three viruses. Our results indicate that the AVR1/08 strain has potential as an attenuated vaccine effective in controlling IBVs circulating in Korea.

Construção de uma biblioteca de fragmentos de anticorpos monoclonais de galinhas com cadeia única (scFv) por phage display com reatividade cruzada para estirpes heterólogas do vírus da bronquite infecciosa aviária / Construction of an avian single chain monoclonal antibodies (scFv) library by phage display that cross-reacted with heterologous avian infectious bronchitis virus strains

Anticorpos monoclonais constituem a base de vários testes usados na detecção e na identificação de antígenos. Nesse contexto, tais imuno-reagentes têm sido extensivamente empregados na identificação de estirpes virais envolvidas na etiologia de surtos de bronquite infecciosa a campo, permitindo o aperfeiçoamento das técnicas de detecção e caracterização antigênica do vírus da bronquite infecciosa das galinhas (VBI). No presente estudo, uma biblioteca de fragmentos de anticorpos de galinha originalmente preparada por phage display contra a estirpe vacinal (H120) do VBI foi usada para a seleção de fragmentos de anticorpos recombinantes com reatividade cruzada para as estirpes heterólogas IBVPR01 e IBVPR05, isoladas de surtos a campo no Brasil e a estirpe SE-17, isolada nos Estados Unidos. Após três ciclos de panning, foi identificado, pelo ELISA, um conjunto de 15 anticorpos scFv expressos em fagos e com reatividade cruzada para essas mesmas estirpes do VBI. A análise por Western-blotting revelou que dois desses clones apresentavam fagos expressando fragmentos de anticorpos monoclonais com reatividade cruzada para a nucleoproteína N das três estirpes do VBI e também para a forma recombinante dessa nucleoproteína derivada da estirpe M41. Concluindo, os fragmentos de anticorpos monoclonais recombinantes scFv-N produzidos em fagos interagem com um epítopo mais conservado da proteína N do VBI e apresentam um grande potencial para utilização na detecção e no diagnóstico direto desse vírus.

Monoclonal antibodies are the basis of various techniques used for antigen detection or characterization, and their use is specially recommended for the identification of viral strains involved in the etiology of infectious bronchitis outbreaks. These antibodies are homogeneous, highly specific and fully characterizable, allowing the improvement of immunological techniques detection and antigenic characterization of avian infectious bronchitis virus strains (IBV). A phage display library was used, which was prepared previously against the IBV vaccine strain (H120) for the selection of new scFv antibody fragments specific for heterologous IBV strains isolated from outbreaks in Brazil (IBVPR01, IBVPR05) and USA (SE-17). After three cycles of panning, a set of 15 scFv antibodies were expressed in phages and cross-reacted in ELISA with these three viral strains. Western-blotting analysis showed that two of the clones were expressing scFv specific for the nucleoprotein of these IBV strains, as well as to the recombinant form of this protein derived from M41. In conclusion, the recombinant fragments of monoclonal antibodies expressed in phage have a great potential for future use in immunodiagnostic techniques and to study the evolution of infectious bronchitis virus.

Clonagem, expressão e caracterização da nucleoproteína recombinante do vírus da bronquite infecciosa em Escherichia coli e em Pichia pastoris / Cloning, expression, and characterization of the nucleocapsid protein of infectious bronchitis virus in Escherichia coli and Pichia pastoris

O gene da proteína de nucleocapsídeo (1.230 pb) da estirpe M41 do vírus da bronquite infecciosa (VBI) foi amplificado pelas reações de transcrição reversa e em cadeia da polimerase (RT-PCR) e clonado, em seguida, em dois sistemas; pET28a - Escherichia coli e pFLD -Pichia pastoris. Os produtos recombinantes construídos para expressão (pET28a-N ou pFLD-N) foram identificados por análises de PCR e de sequenciamento de nucleotídeos. Os clones transformantes da linhagem BL21 de E. coli e da linhagem GS115 de P. pastoris foram submetidos aos protocolos apropriados de indução. A expressão da proteína N de fusão com etiqueta de poli-histidina e com massa molecular de 54 kDa foi determinada pelas técnicas de SDS-PAGE e de Western blotting, confirmando-se que ambas proteínas N recombinantes apresentaram tamanhos e antigenicidade compatíveis com a proteína N nativa do próprio VBI. O sistema E. coli expressou uma quantidade relevante da proteína N recombinante, enquanto que o sistema P. pastoris produziu uma baixa recuperação dessa proteína recombinante. A proteína N recombinante gerada pelo sistema bacteriano foi purificada em resina de níquel-sepharose. O conjunto de resultados indica que o sistema de expressão constituído por pET28a ­ E. coli é mais efetivo para produzir a proteína N recombinante do VBI destinada ao uso como antígeno para detectar anticorpos anti-virais específicos em ensaios de imunodiagnóstico para essa infecção viral.

The nucleocapsid protein (N) gene (1,230 bp) of the M41 strain of infectious bronchitis virus (IBV) was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR), and cloned in two systems; pET28a Escherichia coli and pFLD Pichia pastoris. The recombinant expression constructs (pET28a-N or pFLD-N) were identified by PCR and sequencing analysis. The transformant clones of BL21 strain of E. coli or GS115 of P. pastoris were submitted to appropriate inducting protocols. Expression of histidine-tagged fusion N proteins with a molecular mass of 54 kDa was determined by SDS-PAGE and Western blotting analysis, confirming that both recombinant N proteins were comparable in size and antigenicity to native IBV N protein. The E. coli system overexpressed the recombinant N protein, while the P. pastoris system produced a low yield of this recombinant protein. The bacteria expressed N protein was purified by chromatography on nickel-sepharose resin. These results indicated that the pET28a E. coli expression system is more effective to generate N recombinant protein for using as an antigen to detect anti-IBV antibodies in immuno-assays for this viral infection.

Bronquite infecciosa das galinhas: conhecimentos atuais, cepas e vacinas no Brasil / Infectious bronchitis of chickens: current knowledge, strains and vaccines in Brazil

A bronquite infecciosa das galinhas (IB) é uma doença viral aguda e altamente contagiosa que provoca grandes perdas econômicas à indústria avícola em todo o mundo. Considerando que surtos têm ocorrido no Brasil com emergência de novas variantes de IBV, desafiando as estratégias de vacinação atuais, este trabalho objetiva revisar os conhecimentos sobre IB e IBV, a sua distribuição, as cepas e as vacinas utilizadas no Brasil.

Infectious bronchitis (IB) is an acute, highly contagious disease of chickens, caused by infectious bronchitis virus (IBV), which results in great economic losses to the poultry industry worldwide, despite the routine use of vaccines. Several outbreaks do occur periodically in densely populated poultry regions in Brazil and there are constant emergence of new variants. The aim of this paper is to review the current knowledge about IBV and IB, the distribution, strains and vaccines in Brazil.

Anticorpos contra vírus de galinha em cracídeos / Detection of antibodies against commercial poultry viruses in cracids

Os cracídeos são Galliformes silvestres das Américas. Com o objetivo de investigar a presença de anticorpos contra vírus de galinhas em cracídeos, foram coletadas 51 amostras de soro de 10 diferentes espécies dessas aves. Esses animais eram mantidos em criatórios conservacionistas e zoológicos nos Municípios de Santa Maria, Soledade, Passo Fundo, Sapucaia, Gravataí, Viamão e Três Coroas, Estado do Rio Grande do Sul, Brasil. Anticorpos neutralizantes foram detectados em 5,9 por cento (3/51) do total de amostras testadas contra o vírus da bronquite infecciosa das galinhas, 15,7 por cento (8/51) contra o reovírus aviário e 35,3 por cento (18/51) contra o vírus da doença infecciosa da bolsa. Todas as amostras foram negativas para o vírus da bouba aviária no teste de IDGA. A detecção de anticorpos para vírus de aves comerciais sugere que os cracídeos podem ser susceptíveis à infecção por esses vírus.

The cracids are wild Galliformes native from the Americas. Fifty one serum samples were collected from individuals of 10 different species of cracids in order to obtain information regarding to the antibody status of different viruses. These birds were kept in shelters and zoos localized in Santa Maria, Soledade, Passo Fundo, Sapucaia, Gravataí, Viamão and Três Coroas counties, in the Rio Grande do Sul State, Brazil. Neutralizing antibodies were detected in the individuals serum from different species specific referring to infectious bronchitis virus in 5.9 percent (3/51) of the samples, to avian reovirus in 15.7 percent (8/51) and, to infectious bursal disease virus in 35.3 percent (18/51). All samples were negative for fowlpox virus, as measured by IDGA test. The detection of commercial poultry viruses antibodies suggests that cracids could be susceptible to infection by those viruses.

Pathogenicity and antigenicity of a new variant of Korean nephropathogenic infectious bronchitis virus

Despite the existence of an active vaccination program, recently emerged strains of nephropathogenic infectious bronchitis virus (IBV) in Korea have caused significant economic losses in the poultry industry. In this study, we assessed the pathogenic and antigenic characteristics of a K-IIb type field strain of IBV that emerged in Korea since 2003, such as Kr/Q43/06. Specific pathogen free 1-week-old chickens exhibited severe respiratory symptoms (dyspnea) and nephropathogenic lesions (swollen kidneys with nephritis and urate deposits) following challenge with the recent IBV field strain. The antigenic relatedness (R value), based on a calculated virus neutralization index, of the K-IIb type field strain and K-IIa type strain KM91 (isolated in 1991) was 30%, which indicated that the recent strain, Kr/Q43/06, is a new variant that is antigenically distinct from strain KM91. This report is the first to document the emergence of a new antigenic variant of nephropathogenic IBV in chicken from Korea.

Detection and molecular characterization of infectious bronchitis virus isolated from recent outbreaks in broiler flocks in Thailand

Thirteen field isolates of infectious bronchitis virus (IBV) were isolated from broiler flocks in Thailand between January and June 2008. The 878-bp of the S1 gene covering a hypervariable region was amplified and sequenced. Phylogenetic analysis based on that region revealed that these viruses were separated into two groups (I and II). IBV isolates in group I were not related to other IBV strains published in the GenBank database. Group 1 nucleotide sequence identities were less than 85% and amino acid sequence identities less than 84% in common with IBVs published in the GenBank database. This group likely represents the strains indigenous to Thailand. The isolates in group II showed a close relationship with Chinese IBVs. They had nucleotide sequence identities of 97-98% and amino acid sequence identities 96-98% in common with Chinese IBVs (strain A2, SH and QXIBV). This finding indicated that the recent Thai IBVs evolved separately and at least two groups of viruses are circulating in Thailand.