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.

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.

Isolation and genetic stability of an infectious bronchitis virus strain (IBV) / Isolamento e estabilidade genética de uma cepa do vírus da bronquite infecciosa (IBV)

O vírus da bronquite infecciosa (IBV) é um importante patógeno respiratório presente na avicultura comercial e tem provocado grandes perdas econômicas em todo o mundo. A vacinação é realizada pela indústria produtora de aves, mas continuam surgindo novos sorotipos e variações antigênicas, dificultando o controle de IBV. Nós realizamos uma caracterização molecular de uma cepa de IBV obtida diretamente de tecidos e comparamos com a mesma cepa que havia sido passada três vezes em ovo embrionado. Nós mostramos uma variação significante na sequência viral depois de ter sido isolada em ovo embrionado.(AU)

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).

Sequencing and Serologic Identification of S1 Genes of Infectious Bronchitis Viruses Isolated during 2012-2013 in Guangxi Province, China / 病毒学报

We wished to ascertain the prevalence as well as the genetic and antigenic variation of infectious bronchitis viruses (IBVs) circulating in the Guangxi Province of China in recent years. The S1 gene of 15 IBV field isolates during 2012-2013 underwent analyses in terms of the similarity of amino-acid sequences, creation of phylogenetic trees, recombination, and serologic identification. Similarities in amino-acid sequences among the 15 isolates of the S1 gene were 54.3%-99.6%, and 43.3%-99.3% among 15 isolates and reference strains. Compared with the vaccine strain H120, except for GX-YL130025, the other 14 isolates showed a lower similarity of amino-acid sequences of the S1 gene (65.1-81.4%). Phylogenetic analyses of the S1 gene suggested that 15 IBV isolates were classified into eight genotypes, with the predominant genotype being new-type II. Recombination analyses demonstrated that the S1 gene of the GX-NN130048 isolate originated from recombination events between vaccine strain 4/91 and a LX4-like isolate. Serotyping results suggested that seven serotypes prevailed during 2012-2013 in Guangxi Province, and that only one isolate was consistent with the vaccine strain H120 in serotype (which has been used widely in recent years). The serotype of recombinant isolate GX-NN130048 was different from those of its parent strains. These results suggested that not only the genotype, but also the serotype of IBV field isolates in Guangxi Province had distinct variations, and that increasing numbers of genotypes and serotypes are in circulation. We showed that recombination events can lead to the emergence of new serotypes. Our study provides new evidence for understanding of the molecular mechanisms of IBV variations, and the development of new vaccines against IBVs.

The Isolation and Identification of Infectious Bronchitis Virus PTFY Strain in Muscovy Ducks / 病毒学报

In July 2009, some farms of breeding Muscovy ducks on the peak of egg laying suffered the decrease of hatching rate and the quality of the eggs showing low mortality and no evident respiratory symptoms. The swelling and congestive ovary was visible after autopsy. This study was brought out for the diagnosis of these cases. The virus was isolated and identified by the methods of virus culture in chicken embryo, physical and chemical properties test, hemagglutinin test, NDV (Newcastle diseases Virus) interference test, electron microscope observation, pathogenicity test and the gene sequence analysis. The results indicated the virus showed the characters of inducing dwarf embryo after inocubation, the sensibility to lipid solvent and the hemagglutination capacity after pancreatic enzyme treatment, the typical morphology of coronavirus, the interference to NDV replication and the homology among 84.7% - 99% of the particial N gene sequences to the reference IBV (Avian infectious bronchitis virus) strains. The strain was identified as IBV isolate and this study confirmed the pathogenicity of IBV to Muscovy ducks.

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.

Comportamiento del virus de la bronquitis infecciosa aviar en aves con sintomatología respiratoria provenientes de granjas de producción del Departamento de Cundinamarca / Avian Infectious Bronchitis virus behavior in birds from commercial farms with a history of respiratory disease, located in the department of Cundinamarca

Objetivo. Evaluar la dinámica serológica contra el virus de bronquitis infecciosa aviar y su relación con la presentación y/o antecedentes de signos clínicos y hallazgos patológicos, bajo condiciones de campo. Materiales y métodos. Se realizó un muestreo al azar en dos fases, en pollo de engorde y reproductoras de granjas del Departamento de Cundinamarca. En la primera fase se tomó muestra de sangre a un total de 224 aves, distribuidas en 7 granjas. En la segunda fase, realizada 20 días posteriores al primer muestreo, se tomó una segunda muestra al mismo número de aves empleadas inicialmente. Las muestras de los sueros obtenidos se emplearon para la realización del inmunoensayo ligado a enzima (ELISA), diseñado para detectar anticuerpos frente al virus de la bronquitis infecciosa aviar en suero sanguíneo. Resultados. Se obtuvo que del total de las granjas analizadas el 85.72% mostró reactividad serológica al virus de la Bronquitis Infecciosa aviar (VBIA), con correlación ante la presencia de los signos clínicos o antecedentes respiratorios en granja.

Objective. Evaluate the serological dynamics against avian infectious bronchitis virus and its relationship with the presentation and / or a history of clinical signs and pathological findings, under field conditions. Materials and methods. A random sampling was conducted in two phases, in broiler and breeder farms located in the Department of Cundinamarca. In the first phase blood sample were taken from a total of 224 birds, distributed over the 7 farms. In the second phase, carried out 20 days after the first, a second sample was collected from the same number of birds used in the first phase. The serum samples were used to carry out the enzyme linked immunoassay (ELISA) intended to detect antibodies against avian infectious bronchitis virus in blood serum. Results. As a result it was found that from the total farms analyzed the 85.72% showed serologic reactivity against Avian Infectious Bronchitis Virus (AIBV) that correlated to the presence of clinical signs or previous history of respiratory disease in the farm.

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.

Antigenic variations of infectious bronchitis virus from broiler flocks in Al-Beheira governorate

Seventeen avian infectious bronchitis virus [IBV] isolates were isolated from broiler chickens showing respiratory and renal lesions. The isolated strains were characterized by real time reverse transcriptase polymerase chain reaction used for N gene, and then RT-PCR and sequence analysis of the hypervariable region 3 of the S1 spike glycoprotein gene of six isolates. Six isolates showed 87.15% to 89.71% and 87.27% to 90.82% amino acid sequence identity and 87.61% to 89.19% and 87.91% to 89.72% nucleotide sequence identity to the Egyptian variant 1 and the IS/885 strains, respectively. The six isolates formed a distinct phylogenetic group with the Ck/Eg/BSU-2/2011 and Ck/Eg/ BSU-3/2011 [Var 2]. Amino acid and nucleotide identities between the six Egyptian isolates and variant 2 [Ck/Eg/BSU-2/2011 and Ck/Eg/BSU-3/2011] ranged from 97.27% to 100% and 97.88% to 99.38%, respectively. The results indicate that the six isolates IBV/CK/Beh/101/013/S1, IBV/CK/Beh/204/013/S1, IBV/CK/Beh/105/013/S1, IBV /CK/Beh/1011/013/S1, IBV/CK/Beh/1017/013/S1, IBV/CK/Beh/2020/013/S1 can be considered a variant 2 as Ck/Eg/BSU-2/2011 and Ck/Eg/BSU-3/2011. This study demonstrates a constant evolution of IBV in Egypt that necessitates continuous monitoring to control the spread of infections, and the development and use of vaccines based on indigenous viruses

Genotypes and serotypes of avian infectious bronchitis viruses isolated during 2009-2011 in Guangxi, China / 病毒学报

In order to investigate the prevalence and track genetic and antigenic evolutions of infectious bronchitis virus (IBV) and their prevalence in Guangxi, China since 1985, gene amplification and sequencing and virus neutralization (VN) test on chicken embryo tracheal organ cultures were used in genotyping and serotyping of 28 IBV isolates during 2009-2011 in Guangxi. The results of N gene sequencing and comparison showed that the 28 isolates and reference strains were classified into three groups, and most isolates belonged to group Ill, while the isolates in 1985-2008 belonged to groups IV and II. The data of VN test indicated that the 28 isolates belonged to 6 serotypes; among them, 71. 4% belonged to serotypes 1, 2, and 3, and 11 (39.3%) shared the same serotype with the current vaccine strains. Given the data of our previous study, it is found that prevalent serotypes and their proportions varied in different areas of Guangxi and during different periods. These data lay a good foundation for developing an oil-emulsified inactivated polyvalent vaccine containing local dominant serotypes for the effective prevention and control of infectious bronchitis.

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.

Complete genomic analysis of a novel infectious bronchitis virus isolate / 病毒学报

The genome of CK/CH/SD09/005, an isolate of infectious bronchitis virus (IBV), was characterized to enable the further understanding of the epidemiology and evolution of IBV in China. Twenty-five pairs of primers were designed to amplify the full-length genome of CK/CH/SD09/005. The nucleotide sequence of CK/CH/SD09/005 was compared with reference IBV strains retrieved from GenBank. The phylogenic relationship between CK/CH/SD09/005 and the reference strains was analyzed based on S1 gene sequences. The complete genome of CK/CH/SD09/005 consisted of 27691 nucleotides (nt), excluding the 5' cap and 3' poly A tail. The whole-genome of CK/CH/SD09/005 shared 97 - 99% nucleotide sequence homology with the GX-NN09032 strain, which was the only complete genome that was closely related to CK/CH/SD09/005. When compared with all reference strains except GX-NN09032, CK/CH/SD09/005 showed the highest similarity to ck/CH/LDL/091022 and SDIB821/2012 (QX-like) in the replicase gene (Gene 1) and 3'UTR, with a sequence identity rate of 97% and 98%, respectively. However, CK/CH/SD09/005 exhibited lower levels of similarity with ck/CH/LDL/091022 and SDIB821/2012 in S-3a-3b-3c/ E-M-5a-5b-N with a sequence identity of 72% - 90%. CK/CH/SD09/005 showed the highest level of nucleotide identity with Korean strain 1011, and Chinese strains CK/CH/LXJ/02I, DK/CH/HN/ZZ2004 and YX10, in ORF 3c/E (97%), 5a (96%), 5b (99%) and N (96%), respectively. ORFs 3a, 3b and M of CK/CH/SD09/005 exhibited no more than 90% homology with the reference strains, excluding GX-NN09032. The phylogenic analysis based on the S1 gene revealed that CK/CH/SD09/005 and 39 published strains were classified into seven clades (genotypes). CK/CH/SD09/005 was distributed in clade IV with several isolates collected between 2007 and 2012. CK/CH/SD09/005 showed 66% - 69% and 72% - 81% nucleotide identities with the IBV strains of other six clades in the S1 and S2 subunits, respectively. More over, multiple substitutions were found throughout the entire S gene of CK/CH/SD09/005, while insertions and deletions were located within the S1 gene. These results indicated that CK/CH/SD09/005 is a novel variant that may be derived from the QX-like strains that are prevalent in China. Multiple genetic mechanisms, including recombinations, mutations, insertions and deletions, are likely to have contributed to the emergence of this IBV strain.

Dynamic distribution of the avian infectious bronchitis virus isolate strain Jin-13 in SPF chickens / 病毒学报

This study aimed to understand the dynamic distribution of infectious bronchitis virus (IBV) Jin-13 strain in SPF chickens. Ninety-day-old SPF chickens were inoculated with Jin-13, a virulent strain, and dissected at day 1, 4, 7, 10, 14, 21, 28 or 35 post-inoculation (dpi). Samples of heart, liver, spleen, lung, trachea, kidney and duodenum were collected and the N gene was detected by Sybr Green I real-time quantitative RT-PCR assays. The established method had a good linear correlation from 7.77 x 10(8) to 10(0) copies/microL. SPF chickens developed typical clinical signs of IBV at the 4th dpi, and the IBV viral concentration of tissues and organs gradually increased with a peak of up to 7.13 x 10(4) copies/microL. The viral concentration of most organs decreased by the 10th dpi, but those of the kidney, trachea and lung remained positive for IBV at 28 dpi and the heart was still positive for IBV at > 35 dpi. The results of this study, showed that the Jin-13 strain can cause prolonged virus excertion in chickens with severe renal damage.

Rescue of the recombinant infectious bronchitis virus with the ectodomain region of H120 spike glycoprotein / 病毒学报

To explore the expression potential of heterogeneous genes using the backbone of infectious bronchitis virus (IBV) Beaudette strain, the ectodomain region of the Spike gene (1,302 bp) of IBV H120 strain was amplified by RT-PCR and replaced into the corresponding location of the IBV Beaudette strain full-length cDNA. This recombinant was designated as BeauR-H120(S1). BeauR-H120(S1) was directly used as the DNA template for the transcription of viral genomic RNA in vitro. Then, the transcription product was transfected into Vero cells by electroporation. At 48 h post-transfection, the transfected Vero cells were harvested, and passaging continued. A syncytium was not observed until the recombinant virus had passed through four passages. The presence of rBeau-H120(S1) was verified by the detection of the replaced ectodomain region of the H120 Spike gene using RT-PCR. Western blot analysis of rBeau-H120 (S1)-infected Vero cell lysates demonstrated that the nucleocapsid (N) protein was expressed, which implied that rBeau-H120(S1) could propagate in Vero cells. The TCIDs0 and EIDs0 data demonstrated that the titer levels of rBeau-H120(S1) reached 10(590+/-0.22)TCID50/mL and 10(6.13+/-0.23)EID50/mL in Vero cells and 9-day-old SPF chicken embryos, respectively. Protection studies showed that the percentage of antibody-positive chickens, which were vaccinated with rBeau-H120(S1) at 7 days after hatching, rose to 90% at 21 days post-inoculation. Inoculation provided an 85% rate of immune protection against a challenge of the virulent IBV M41 strain (103EID50/chicken). This recombinant virus constructed using reverse genetic techniques could be further developed as a novel genetic engineering vaccine against infectious bronchitis.

Protection of chickens against infectious bronchitis virus with a multivalent DNA vaccine and boosting with an inactivated vaccine

The protective efficacy of DNA plasmids encoding avian infectious bronchitis virus (IBV) S1, N, or M protein was investigated in chickens. Chickens were inoculated monovalently (with plasmid pVAX1-16S1, pVAX1-16M, or pVAX1-16N alone) or multivalently (combination of the three different plasmids, pVAX1-16S1/M/N). A prime-boost immunization protocol against IBV was developed. Chickens were immunized with the multivalent DNA vaccine twice and then boosted with an inactivated vaccine once. Antibody titers of the chickens immunized with pVAX1-16S1/M/N were much higher than those of the monovalent groups (p < 0.01). A protective rate up to 90% was observed in the pVAX1-16S1/M/N group. The serum antibody titers in the prime-boost birds were significantly higher than those of the multivalent DNA vaccine group (p < 0.01) but not significantly different compared to the inactivated vaccine group at 49 days of age. Additionally, the prime-boost group also showed the highest level of IBV-specific cellular proliferation compared to the monovalent groups (p < 0.01) but no significant difference was found compared to the multivalent DNA vaccine group, and the prime-boost group completely protected from followed viral challenge.

Detection of infectious bronchitis virus and Avian metapneumovirus by a duplex semi-nested reversetranscription–polymerase chain reaction assay / Detecção do vírus da bronquite infecciosa das galinhas e do metapneumovírus aviário utilizando uma reação de transcrição reversa com reação em cadeia pela polimerase duplex

Duplex RT-PCR assay is reported for the simultaneous detection of avian infectious bronchitis virus (IBV) and avian metapneumovirus (aMPV), the causative agents of major diseases in poultry. The duplex RT-PCR assay optimized showed a detection limit of 10-3 (101 EID50/50m L for IBV and 100.5 EID50/50m L for aMPV, respectively when two viruses were mixed and 10-1 for each one separated (103 EID50/50m L for IBV and 102.5 EID50/50m L for aMPV, respectively. It was specific, sensitive and applicable for the rapid detection of these viruses in clinical samples.

Descreve-se um ensaio de duplex RT-PCR assay para a detecção simultânea do vírus da bronquite infecciosa das galinhas (IBV) e do metapneumovirus aviário (aMPV), agentes etiológicos de doenças de elevada importância em avicultura. A duplex RT-PCR otimizada mostrou um limiar de detecção de 10-3 (101 EID50/50m L para IBV e 100.5 EID50/50m L para aMPV, respectivamente, quando da combinação dos dois vírus e 10-1 para cada um dos vírus em separado(103 EID50/50m L para IBV e 102.5 EID50/50m L para aMPV, respectivamente. O ensaio foi demonstrado como específico, sensível e aplicável à rápida detecção destes vírus em amostras clínicas.

Evaluation of antigenic relationship of Guangxi isolates of infectious bronchitis virus / 病毒学报

Monovalent antisera of 3 vaccine strains and 7 representative field isolates were prepared based on the comparison of genetic diversity of the hypervariable region I of S1 gene (HVR I from 3 infectious bronchitis (IB) vaccine strains (H120, Ma5 and 4/91) ,one reference strain M41 and 26 IB field isolates. These 30 strains were classified in 7 different genotypes, respectively. Virus-neutralizing test on tracheal organ cultures (TOC) with chicken embryo were used to evaluate relatedness values of the antigenicity based on the antibody titer, to analyze the antigenic relationships between the isolates and vaccine strains, as well as to determine the serotypes of 26 IB viruses isolated from the field in Guangxi between 1985 and 2008. The results showed 30 strains were classified into 7 distinct serotypes and there were two predominant serotypes within the 26 isolates, serotypes 1 (totally 13 isolates) and serotype 2 (totally 5 isolates), respectively. In addition, there were some differences observed between the results of serotyping and the genotyping (including the S1, N, M and 3'UTR). The results of the study demonstrated that there were different predominant serotypes and multiple serotypes of IBV circulated in Guangxi in recent years, antigenic variation existed between Guangxi field isolates and vaccine strains.