Genetic and pathogenic characterization of new infectious bronchitis virus strains in the GVI-1 and GI-19 lineages isolated in central China

Avian infectious bronchitis(IB)is a highly contagious infectious disease caused by infectious bronchitis virus(IBV),which is prevalent in many countries worldwide and causes serious harm to the poultry industry.At present,many commercial IBV vaccines have been used for the prevention and control of IB;however,IB outbreaks occur frequently.In this study,two new strains of IBV,SX/2106 and SX/2204,were isolated from two flocks which were immunized with IBV H120 vaccine in central China.Phylogenetic and recombination analysis indicated that SX/2106,which was clustered into the GI-19 lineage,may be derived from recombination events of the GI-19 and GI-7 strains and the LDT3-A vaccine.Genetic analysis showed that SX/2204 belongs to the GVI-1 lineage,which may have originated from the recombination of the GI-13 and GVI-1 strains and the H120 vaccine.The virus cross-neutralization test showed that the antigenicity of SX/2106 and SX/2204 was different from H120.Animal experiments found that both SX/2106 and SX/2204 could replicate effectively in the lungs and kidneys of chickens and cause disease and death,and H120 immunization could not provide effective protection against the two IBV isolates.It is noteworthy that the pathogenicity of SX/2204 has significantly increased compared to the GVI-1 strains isolated previously,with a mortality rate up to 60%.Considering the continuous mutation and recombination of the IBV genome to produce new variant strains,it is important to continuously monitor epidemic strains and develop new vaccines for the prevention and control of IBV epidemics.

Evolutionary implications of Avian Infectious Bronchitis Virus(AIBV)analysis

For developing efficient vaccines, it is essential to identify which amino acid changes are most important to the survival of the virus. We investigate the amino acid substitution features in the Avian Infectious Bronchitis Virus （AIBV） antigenic domain of a vaccine serotype （DE072） and a virulent viral strain （GA98） to better understand adaptive evolution of AIBV. In addition, the SARS Coronavirus （SARS-CoV） was also analyzed in the same way. It is interesting to find that extreme comparability exists between AIBV and SARS in amino acid substitution pattern. It suggests that amino acid changes that result in overall shift of residue charge and polarity should be paid special attention to during the development of vaccines.

E Protein Prokaryotic Expression of Avian Infectious Bronchitis Virus

The small envelope protein （E） gene of avian infectious bronchitis virus （IBV） M41 strain was cloned, and then it was subcloned into prokaryotic expressing vector pGEX-6P-1. The recombinant plasmid was transformed into E.coli. BL21 and induced by IPTG. SDS-PAGE result showed that when objective protein fused with GST （about 20 ku）, the relative molecular mass of fusion protein was 38 ku. It indicated that objective protein was about 12.4 ku. The result showed that E protein was expressed successfully, it was useful to the subsequent E protein research.

Genotyping and pathotyping of diversified strains of infectious bronchitis viruses circulating in Egypt

AIM: To characterize the circulating infectious bronchitis virus(IBV) strains in Egypt depending on the sequence of the spike-1(S1) gene [hypervariable region-3(HVR-3)] and to study the pathotypic features of these strains.METHODS: In this work, twenty flocks were sampled for IBV detection using RRT-PCR and isolation of IBV in specific pathogen free(SPF) chicks during the period from 2010 to 2015. Partial sequencing and phylogenetic analysis of 400 bp representing the HVR-3 of the S1 gene was conducted. Pathotypic characterization of one selected virus from each group(Egy/Var-Ⅰ, Egy/Var-Ⅱ and classic) was evaluated in one day old SPF chicks. The chicks were divided into 4 groups 10 birds each including the negative control group. Birds were inoculated at one day by intranasal instillation of 105EID50/100 μL of IBV viruses [IBV-EG/1212B-2012(Egy/Var-Ⅱ), IBV/EG/IBV1-2011(Egy/Var-Ⅰ) and IBV-EG/11539F-2011(classic)], while the remaining negative control group was kept uninfected. The birds were observed for clinical signs, gross lesions and virus pathogenicity. The real-time rR TPCR test was performed for virus detection in the tissues. Histopathological examinations were evaluated in both trachea and kidneys.RESULTS: The results revealed that these viruses were separated into two distinct groups; variant(GI-23) and classic(GI-1), where 16 viruses belonged to a variant group, including 2 subdivisions [Egy/Var-Ⅰ(6 isolates) and Egy/Var-Ⅱ(10 isolates)] and 4 viruses clustered to the classic group(Mass-like). IBV isolates in the variant group were grouped with other IBV strains from the Middle East. The variant subgroup(Egy/Var-Ⅰ) was likely resembling the original Egyptian variant strain(Egypt/Beni-Suif/01) and the Israeli strain(IS/1494/2006). The second subgroup(Egy/Var-Ⅱ) included the viruses circulating in the Middle East(Ck/EG/BSU-2 and Ck/EG/BSU-3/2011) and the Israeli strain(IS/885/00). The two variant subgroups(Egy/Var-Ⅰ and Egy/Var-Ⅱ) found to be highly pathogenic to SPF chicks with mortalities up to 50% than those of the classic group which was of low virulence(10% mortality). Pathogenicity indices were 25(Egy/Var-Ⅱ), 24(Egy/Var-Ⅰ) and 8(classic); with clinical scores 3, 2 and 1 respectively.CONCLUSION: These findings indicated that the recent circulating Egyptian IBVs have multiple heterogeneous origins in marked diversifying nature of their spread, with high pathotype in specific pathogen free chicks.

Protection against Infectious Bronchitis Virus, a Corona Virus Infection, Using Ostrich Antibodies

In chickens, infectious bronchitis (IB) is a major respiratory disease. The respiratory system is the primary multiplication site of IB virus (IBV), a coronavirus, after which the virus is distributed to other organs. Poultry farms sustain considerable economic damage due to IB outbreaks in flocks, since IB causes a severe reduction in weight gain in chicks. In the present study, we produced the ostrich IgY against IBV by immunizing female ostriches with the IB viral antigens. The resultant purified IgY showed a strong neutralizing activity against IBV infection of cultured primary chick kidney cells. The infectivity of IBV was markedly inhibited in the trachea of chicks when ostrich IgY was injected intra-muscularly into newly hatched chicks prior to viral inhalation challenge at two weeks of age. Furthermore, the infection was strongly blocked in the tracheae when IgY was injected into chicks at one day and one week of age, with viral inhalation performed at three weeks of age. These findings suggest that the injection of ostrich IgY can help protect young chicks from IBV infections. In south Asian and African countries, broiler chicks are sent to poultry market around 30 days of age, so it is important to prevent IB outbreaks in very young flocks. We strongly believe that ostrich IgY will be a powerful weapon against IB infection in poultry farms on a wide scale and also hope that these findings will aid in the development of antibody vaccines for new type corona viruses, SARS-CoV and MERS-CoV.

Cloning and Sequencing of S Gene of Novel Variant of Infectious Bronchitis Virus ZJ971 Isolates in China

A novel proventriculopathogic variant (isolate ZJ971) of infectious bronchitis virus (IBV) was identified from enlarged proeventriculus of the sick chickens in the study. The S gene cDNA segment with 3.6 kb in length was amplified by RT-PCR with special primers from the ZJ971 viral isolate of (IBV) and cloned into plasmid pBluescript SK( + ). The recombinants containing S gene of IBV-ZJ971 isolate were identified by digestion of restriction enzyme EcoRI, BamHI and PCR amplification. The cloned S gene from isolate IBV-7J971 was composed of 3492 bp in length encoding for a polypeptide of 1080 amino acids. Comparing the nucleotide of S gene of IBV isolate ZJ971 with that of reported IBV strains Beaudette, M41, Ark99 and CuT2, the homology was 97.3%, 97.5%, 88.6% and 85.6%, respectively; and the homology of the deduced amino acids of S protein of IBV isolate ZJ971 was 96%, 96.3%, 86.1% and 83.1% respectively; especially, the mutation of 3241st nucleotide of S gene of IBV isolate ZJ971 from G to T resulted in the translating termination of S protein at 3240th nucleotide site.

Effects of Traditional Chinese Herbal Medicine on Immune Organ Indexes and Macrophages Phagocytic Indexes in Chickens Infected by Infectious Bronchitis Virus

[ Objective] To study the therapeutic effects of traditional Chinese medicine prescriptions on infectious bronchitis （IB） and find a novel avenue for prevention and treatment of viral diseases in poultry. [Method] A total of 160 cockerels at the age of 15 d were divided into four groups randomly, including traditional Chinese medicine group, moroxydine control group, challenge control group and healthy control group. Except the healthy control group, other groups were challenged with infectious bronchitis virus （IBV） on Day 15. After 48 h post challenge, the traditional Chinese medicine groupand moroxydine control group were respectively administrated with Chinese herbal medicine prescription and moroxydine, continuously for 5 d. The immune organ indexes and macrephage phagocytic indexes were detected on Day 18, 24 and 30, respectively. [ Result] The immune organ indexes and macrophage phagocytic indexes were not significantly different between traditional Chinese medicine group and moroxydine control group on Day 18. But all the indexes of the traditional Chinese medicine groups were increased significantly （ P 〈 0.05） on Day 24 and 30, and showed extremely significant difference （ P 〈 0.01 ） with those of challenge control group on Day 30. [ Conclusion] The traditional Chinese herbal medicine can enhance macrophage phagocytic indexes and immune organs indexes of chickens infected by IBV.

Molecular Characteristics of S1 Gene of Infectious Bronchitis Virus Isolated from Chicken Proventriculus

Infectious bronchitis virus was isolated from swollen proventriculi of clinically ill chicken. The suspected virus samples (2/97, 3/97, 1/98) were adapted in SPF chicken embryos for virus isolation and identification. All the virus isolates were able to agglutinate chicken erythrocytes after treatment with trypsin, and interfer with the reproduction of Newcastle disease virus in chicken embryos, and have low antigenic relat-edness values with reference positive IBV. The isolates 2/97, 3/97, 1/98 RNAs extracted from the allantoic fluid of inoculated embryonated eggs were converted to cDNA by reverse transcription with 3'-primer of S1 gene of (IBV). Polymerase chain reaction (PCR) was performed with two primers which span the S1 gene. Amplified product of 1. 93 kb was subjected to EcoR I and BamH I digestion and the fragments obtained were the same as expected size. The PCR product was ligated to pBlueScript-SK ( + ) vector, and its nucleotide sequence was determined by the dideoxy-mediated chain termination method. Nucleotide sequence analysis showed 73. 6 - 99. 7% homology between the isolated IBV and the IBV strains in GenBank. The homology of amino acid was 71. 4 - 99.4%.

Isolation and Identification of Avian Infectious Bronchitis Virus

[ Objective] The aim was to isolate and identify avian infectious bronchitis virus （IBV） from diseased chickens. [ Method] IBVs were iso- lated from the diseased chickens in a chicken farm in Anhui Province with blind passage method to observe virus pathogenicity. Then animal regres- sion test was made to replicate symptoms of bronchial congestion in SPF chickens and S1 gene segments were amplified and isolated, followed by comparison with IBV vaccine strains. [ Result] Detection of Hemagglutinating activity （HA） showed that allantoic fluid had no concerning effect on erythrocyte, suggesting that NDV and AIV were not included in the isolated viruses. However, the erythrocyte could be agglutinated with allantoic fluid treated with 1% of pancreatin, which is in consistent with biological characters of IBV. After SPF chickens were inoculated with the 6^th SPF al- lantoic fluid, bronchial congestion was replicated, proving that the isolated virus was avian IBV, named IBV XZ strain. [ Conclusion] This study pro- vides a theoretical basis for prevention of avian infectious bronchitis.

Prokaryotic Expression of Infectious Bronchitis Virus S1 Gene and Analysis of Biological Activity of Recombinant Protein

[Objective] To study the prokaryotic expression and antigenicity identification of S1 gene from avian infectious bronchitis virus （IBV）. [Method] The S1 gene was cloned into a pMD18-T vector to yield a recombinant plasmids pMD18-T-IBV-S1. Then S1 gene was inserted into the multiple cloning site of a prokaryotic expression vector pET-32a （ ＋ ）. The recombinant plasmid was transformed into E. coil BL21. The recombinant protein was induced by IPTG and measured by SDS-PAGE and western-blotting. [Result] The S1 gene was successfully expressed in E. coil BL21, the fusion proteins were about 66.0 kDa in a form of inclusion body. Western-blotting test showed that the recombinant proteins could be identified by IBV polyclonal antibody. [ Conclusion] The recombinant proteins of S1 gene have the antigenicity, which lays a good foundation for further research on new generation vaccine of IBV.

The Establishment of Fluorescent PCR Detection Method for Avian Infectious Bronchitis Virus

[ Objective] The aim was to to establish a kind of peculiar, sensitive and quick fluorescent PCR detection method. [Method] A peculiar, sensitive and quick method of fluorescent PCR detection for avian infectious bronchitis virus was established, the standard curve was built, specific primers, susceptibility and repeatability was detected. [ Result] This method diagnosed avian infectious bronchitis virus peculiarly, sensitively and quickly, simple and easy to use, time short, suitable for clinical testing. [ Conclusion] This research laid the foundation to diagnose avian infectious bronchitis virus.

Isolation and Identification of Avian Nephropathogenic Infectious Bronchitis Virus

In November 2009, a respiratory disease with rapid transmission, rapid onset and mortality of about 8% appeared many times in a large chicken farm in Jiangsu Province of China. Necropsy revealed tracheal bleeding, kidney enlargement and white-spotted kidney. An isolate from the farm was identified as an avian infectious bronchitis virus （IBV） by chicken embryo inoculation, hemagglutination assay, virus interference assay, animal regression and tracheal rings culture. The complete ,S1 gene was amplified by RT-PCR, and its homology to that of the vaccine strains com- monly used in China was analyzed with DNAStar software. Therefore, the IBV isolate was initially classified into nephropathogenic IBV and named IBV JS09 strain.

Development of a Microplate Lectin-Capture RT-PCR (MLC-RT-PCR) for the Detection of Avian Infectious Bronchitis Virus

Rapid, sensitive and specific methods are necessary to confirm the diagnosis of outbreaks of avian infectious bronchitis virus (IBV) infection. The amplification of IBV genome by reverse transcription followed by polymerase chain reaction (RT-PCR) has been one of the most used methods for the detection of this virus in clinical samples. To reduce the time and the number of steps in the molecular diagnosis of IBV, we developed a sensitive and rapid detection method based on viral capture by a lectin (Concanavalin A—Con A) in the microplate wells, followed by RT-PCR to amplify the S1 gene. The detection limit of IBV was 103 EID50/ml for the amplification of 5’part of the S1 gene, and 104 EID50/ml for the amplification of full S1 gene. This technique was specific for IBV detection, and no amplified products were detected for other avian viral pathogens (bursal infectious disease virus, avian metapneumovirus and Newcastle disease virus). The MLC-RT-PCR was as sensitive as conventional RT-PCR, and virus isolation method for the detection of IBV in tissue samples collected from experimentally infected birds. The MLC-RT-PCR technique demonstrated a great potential for the rapid and specific diagnosis of IBV.

Molecular Detection and Sequencing for S1 Glycoprotein Gene of Bronchitis Virus of 2016 Epidemic from Sindh and Punjab

Infectious Bronchitis (IB) is highly contagious disease of commercial poultry causing substantial economic loses by producing poor quality meat in broilers and effecting production in breeder birds. The causative agent has been reported as most hazardous pathogen among other infectious agent even after being immunized with multi-variant strain vaccine. Currently, different strain such as H-120, 4/91 and D274 have been used extensively for immunoprophylaxis against velogenic strain across Pakistan with minimal protection reported. In current study PCR analysis was used to investigate the molecular nature of IB isolates from Punjab and Sind province of Pakistan in 2016 epidemics. Total of 100 tracheal samples were considered for virus inoculation in 10 days old chicken embryonated eggs. The IBV infected amniotic fluid was neutralized with monoclonal antisera of H-120, 4/91 and D274 strains. The IBV screened samples were subjected for RNA extraction and subsequent to PCR using type specific primer of each strain. The amplified product of 840 bp was sequenced through Sanger sequencing. On the basis of PCR results, four similar amplified products from both regions were obtained showing similarities in agarose gel electrophoresis, but they differ from each other on the basis of nucleotides sequence. Phylogenetic analysis revealed that nucleotide sequences of isolates from Karachi were similar to the IBV H-120, Mass-41 and Connecticut 46 reference strains. Whereas, isolates from the Punjab province are analogous to the Mans-2, Mans-3, 9/41(UK) but did not show significant similarity with other reference strain. Therefore, it is recommended that use of M-41 and H-120 in vaccine production could be effective measure against velogenic infectious agent in Sindh particularly in Karachi, whereas, it would be better to incorporate either of the variant GQ281656.1, AY279533.1 in vaccine because of their highest level of resemblance with genetically sequenced isolates from Lahore and its surroundings.

黄芩苷体外抗IBV QX株的作用研究

为研究黄芩苷体外抗禽传染性支气管炎病毒(Avian infectious bronchitis virus,IBV)QX株的效果,试验对IBV进行复壮与扩繁,制备原代鸡胚肾(CEK)细胞,并测定IBV对CEK细胞的TCID50和黄芩苷对CEK细胞的最大无毒浓度(MNTC);在MNTC基础上采用CCK法测定不同浓度黄芩苷对IBV的有效抑制率,观察不同浓度黄芩苷对IBV导致的CEK细胞的细胞病变效应(CPE)的抑制情况,并采用qPCR法测定不同浓度黄芩苷对IBV N基因相对表达量的影响。结果表明:成功复壮并扩繁了IBV,其未被其他病毒污染,对CEK细胞的TCID50为1×10^(-4.75)/0.1 mL;黄芩苷对CEK细胞的MNTC为9.75 mg/L;该浓度黄芩苷对IBV的有效抑制率最高,为64.03%,能有效减轻IBV对CEK细胞的CPE;各浓度黄芩苷均可以极显著降低CEK细胞中IBV N基因相对表达量(P<0.01)。说明黄芩苷对CEK细胞的MNTC为9.75 mg/L,该浓度黄芩苷具有较好的体外抗IBV QX株的作用。

Identification of one peptide which inhibited infectivity of avian infectious bronchitis virus in vitro

Purified avian infectious bronchitis virus (IBV) was used to screen a random phage dis-play peptide library. After the fourth panning, 10 positive phages were sequenced and characterized. The phages specifically inhibited IBV infectivity in HeLa cells and blocked IBV haemagglutination. One linear peptide “GSH HRH VHS PFV” from the positive phages with the highest neutralization titer was synthesized and this peptide inhibited IBV infection in HeLa as well. The results may contribute to development of antiviral therapeutics for IBV and studying the determinants for viral and cell interac-tion.

Responses of the Toll-like receptor and melanoma differentiation-associated protein 5 signaling pathways to avian infectious bronchitis virus infection in chicks

Avian infectious bronchitis virus(IBV) is a Gammacoronavirus in the family Coronaviridae and causes highly contagious respiratory disease in chickens. Innate immunity plays significant roles in host defense against IBV. Here, we explored the interaction between IBV and the host innate immune system. Severe histopathological lesions were observed in the tracheal mucosa at 3–5days post inoculation(dpi) and in the kidney at 8 dpi, with heavy viral loads at 1–11 and 1–28 dpi,respectively. The expression of m RNAs encoding Toll-like receptor(TLR) 3 and TLR7 were upregulated at 3–8 dpi, and that of TIR-domain-containing adapter-inducing interferon(IFN) β(TRIF) was upregulated at 21 dpi in the trachea and kidney. Myeloid differentiation primary response protein 88(My D88) was upregulated in the trachea during early infection. Tumor necrosis factor receptor-associated factor(TRAF) 3 and TRAF6 were upregulated expression in both tissues.Moreover, melanoma differentiation-associated protein 5(MDA5), laboratory of genetics and physiology 2(LGP2), stimulator of IFN genes(STING), and mitochondrial antiviral signaling protein(MAVS), as well as TANK binding kinase 1(TBK1), inhibitor of kappa B kinase(IKK) ?, IKKα, IKKβ,IFN regulatory factor(IRF) 7, nuclear factor of kappa B(NF-κB), IFN-α, IFN-β, various interleukins(ILs), and macrophage inflammatory protein-1β(MIP-1β) were significantly upregulated in the trachea and downregulated in the kidney. These results suggested that the TLR and MDA5 signaling pathways and innate immune cytokine were induced after IBV infection. Additionally,consistent responses to IBV infection were observed during early infection, with differential and complicated responses in the kidney.

The Efficacy of a Live Attenuated TW I-Type Infectious Bronchitis Virus Vaccine Candidate

Infectious bronchitis(IB) is a highly contagious avian disease caused by infection with infectious bronchitis virus(IBV),which seriously affects the development of the global poultry industry. The distribution of TW I-type IBV in China has increased in recent years, becoming a widespread genotype. We previously isolated a TW I-type IBV strain termed CK/CH/GD/GZ14 in 2014, but its pathogenicity and possibility for vaccine development were not explored. Therefore, this research aimed to develop a live-attenuated virus vaccine based on the CK/CH/GD/GZ14 strain. The wild type IBV CK/CH/GD/GZ14 strain was serially passaged in SPF embryos for 145 generations. The morbidity and mortality rate of wildtype strain in 14 day-old chickens is 100% and 80% respectively, while the morbidity rate in the attenuated strain was 20%in the 95 th and 105 th generations and there was no death. Histopathological observations showed that the pathogenicity of the 95th and 105th generations in chickens was significantly weakened. Further challenge experiments confirmed that the attenuated CK/CH/GD/GZ14 strain in the 95th and 105 th generations could resist CK/CH/GD/GZ14(5th generation)infection and the protection rate was 80%. Tracheal cilia stagnation, virus shedding, and viral load experiments confirmed that the 95 th and 105th generations provide good immune protection in chickens, and the immunogenicity of the 105th generation is better than that of the 95th generation. These data suggest that the attenuated CK/CH/GD/GZ14 strain in the105th generation may be applied as a vaccine candidate against TW I-type IBV.

Genome sequencing and characterization analysis of a Beijing isolate of chicken coronavirus infectious bronchitis virus

Avian infectious bronchitis virus (AIBV) is classified as a member of the genus coronavirus in the family coronaviridae. The enveloped virus has a positive-sense, sin-gle-stranded RNA genome of approximately 28 kilo-bases, which has a 5′ cap structure and 3′ polyadenylation tract. The complete genome sequence of infectious bronchitis virus (IBV), Beijing isolate, was determined by cloning sequencing and primer walking. The whole genome is 27733 nucleotides in length, has ten open reading frames: 5′-orf1a-orf1ab-s-3a- 3b-e-m- 6a-6b-n-3′. Alignments of the genome sequence of IBV Beijing isolate with those of two AIBV strains and one SARS coronavirus were performed respectively. The genome sequence of IBV Beijing isolate compared with that of the IBV strain LX4 (uncompleted, 19440 bp in size) was 91.2% similarity. However, the full-length genome sequence of IBV Beijing isolate was 85.2% identity to that of IBV Strain Beaudette, and was only 50.8% homology to that of SARS coronavirus. The results showed that the genome of IBV has remarkable variation. And IBV Beijing isolate is not closely related to SARS coronavirus. Phylogenetic analyses based on the whole genome sequence, S protein, M protein and N pro-tein, also showed that AIBV Beijing isolate is lone virus in group Ⅲ and is distant from SARS coronavirus. In conclu-sion, this study will contribute to the studies of diagnosis and diseases control on IBV in China.

Phylogenetic analysis of avian infectious bronchitis virus isolates from Morocco:a retrospective study(1983 to 2014)

Dear Editor,Infectious bronchitis(IB),one of the most common and difficult poultry diseases,is caused by a gammacoronavirus named infectious bronchitis virus(IBV).IBV frequently causes respiratory and/or renal diseases in chickens and egg production losses in hens.IB has