Pathobiology of cryptosporidiosis (C. baileyi) in broiler chickens.

Pathologic and clinicopathologic changes were examined in broiler chickens inoculated with Cryptosporidium baileyi (Cb) alone or in combination with infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV) or Escherichia coli (Ec). Concurrent infections with Cb and either IBV or Ec resulted in a greater respiratory inflammatory response than either agent given alone. Concurrent Cb, IBV or Ec infections resulted in a decreased density of respiratory cryptosporidial stages. No interactions between Cb and IBDV were observed. Clinicopathologic results in broiler chicks exhibiting signs of respiratory cryptosporidiosis indicated that pO2 decreased, pCO2 increased, HCO3 increased and CO2 increased. Changes in blood gases and serum electrolyte values correlated with signs of acute respiratory disease. Blood gases and serum electrolyte values were unchanged in birds with bursal and cloacal infections only. Results of these studies clarified pathogenetic events associated with avian respiratory cryptosporidiosis, and demonstrated that cryptosporidiosis may enhance the severity of respiratory disease caused by other avian pathogens.     

Complete genome sequences of two chinese virulent avian coronavirus infectious bronchitis virus variants

Avian coronavirus infectious bronchitis virus (IBV) is variable, which causes many serotypes. Here we reported the complete genome sequences of two virulent IBV variants from China, GX-YL5 and GX-YL9, belonging to different serotypes. Differences between GX-YL5 and GX-YL9 were found mainly in stem-loop structure I in the predicted RNA secondary structure of open reading frame (ORF) 1b and the S protein gene fusion region, which will help us understand the molecular evolutionary mechanism of IBV and the disconcordance between the genotypes and serotypes of coronavirus.     

Development and characterization of a recombinant infectious bronchitis virus expressing the ectodomain region of S1 gene of H120 strain

Infectious bronchitis (IB), caused by infectious bronchitis virus (IBV), is a highly contagious chicken disease, and can lead to serious economic losses in poultry enterprises. The continual introduction of new IBV serotypes requires alternative strategies for the production of timely and safe vaccines against the emergence of variants. Modification of the IBV genome using reverse genetics is one way to generate recombinant IBVs as the candidates of new IBV vaccines. In this study, the recombinant IBV is developed by replacing the ectodomain region of the S1 gene of the IBV Beaudette strain with the corresponding fragment from H120 strain, designated as rBeau-H120(S1e). In Vero cells, the virus proliferates as its parental virus and can cause syncytium formation. The peak titer would reach 10^5.9 50 % (median) tissue culture infective dose/mL at 24 h post-infection. After inoculation of chickens with the recombinant virus, it demonstrated that rBeau-H120(S1e) remained nonpathogenic and was restricted in its replication in vivo. Protection studies showed that vaccination with rBeau-H120 (S1e) at 7-day after hatch provided 80 % rate of immune protection against challenge with 10³ 50 % embryos infection dose of the virulent IBV M41 strain. These results indicate that rBeau-H120 (S1e) has the potential to be an alternative vaccine against IBV based on excellent propagation property and immunogenicity. This finding might help in providing further information that replacement of the ectodomain fragment of the IBV Beaudette S1 gene with that from a present field strain is promising for IBV vaccine development.     

The Replicase Gene of Avian Coronavirus Infectious Bronchitis Virus Is a Determinant of Pathogenicity

We have previously demonstrated that the replacement of the S gene from an avirulent strain (Beaudette) of infectious bronchitis virus (IBV) with an S gene from a virulent strain (M41) resulted in a recombinant virus (BeauR-M41(S)) with the in vitro cell tropism of the virulent virus but that was still avirulent. In order to investigate whether any of the other structural or accessory genes played a role in pathogenicity we have now replaced these from the Beaudette strain with those from M41. The recombinant IBV was in effect a chimaeric virus with the replicase gene derived from Beaudette and the rest of the genome from M41. This demonstrated that it is possible to exchange a large region of the IBV genome, approximately 8.4 kb, using our transient dominant selection method. Recovery of a viable recombinant IBV also demonstrated that it is possible to interchange a complete replicase gene as we had in effect replaced the M41 replicase gene with the Beaudette derived gene. Analysis of the chimaeric virus showed that it was avirulent indicating that none of the structural or accessory genes derived from a virulent isolate of IBV were able to restore virulence and that therefore, the loss of virulence associated with the Beaudette strain resides in the replicase gene.     

Morphogenesis of Avian Infectious Bronchitis Virus and a Related Human Virus (Strain 229E)

Avian infectious bronchitis virus (IBV) and strain 229E, a virus recently recovered from patients with colds, have been shown to possess a similar distinctive morphology in negatively stained preparations. An electron microscopic study of the morphogenesis of IBV in the chorioallantoic membrane and of strain 229E in WI-38 cells was performed. In infected cells, round electron-dense particles 82 mmu in diameter were observed to form by a process of budding from membranes of the endoplasmic reticulum and cytoplasmic vesicles. The particles in IBV-infected cells were similar in size and shape to those in strain 229E-infected cells but showed certain differences in internal structure. The evidence that the particles represent virions and the implications of these findings in the classification of this virus group are discussed.     

Assembly and immunogenicity of baculovirus-derived infectious bronchitis virus–like particles carrying membrane,envelope and the recombinant spike proteins

Objective

To assemble infectious bronchitis virus (IBV)-like particles bearing the recombinant spike protein and investigate the humoral immune responses in chickens.

Results

IBV virus-like particles (VLPs) were generated through the co-infection with three recombinant baculoviruses separately encoding M, E or the recombinant S genes. The recombinant S protein was sufficiently flexible to retain the ability to self-assemble into VLPs. The size and morphology of the VLPs were similar to authentic IBV particles. In addition, the immunogenicity of IBV VLPs had been investigated. The results demonstrated that the efficiency of the newly generated VLPs was comparable to that of the inactivated M41 viruses in eliciting IBV-specific antibodies and neutralizing antibodies in chickens via subcutaneous inoculation.

Conclusions

This work provides basic information for the mechanism of IBV VLP formation and develops a platform for further designing IBV VLP-based vaccines against IBV or other viruses.

     

Immunosuppressive effect of Cryptosporidium baileyi infection on vaccination against avian infectious bronchitis in chicks

Two-day-old commercial chicks were inoculated orally with 2 × 10⁶ oocysts of Cryptosporidium baileyi and vaccinated with 10^3.5 EID₅₀/head of a commercially available avian infectious bronchitis (IB) live virus vaccine at 4 and 14 days following inoculation. Chicks infected with C. baileyi were shown to have an immunosuppressive effect on IB virus. It is concluded that infection with the protozoon in early life may increase their susceptibility to IB.     

Proteomic analysis of chicken embryonic trachea and kidney tissues after infection <Emphasis Type="Italic">in ovo</Emphasis> by avian infectious bronchitis coronavirus

Background  

Avian infectious bronchitis (IB) is one of the most serious diseases of economic importance in chickens; it is caused by the avian infectious coronavirus (IBV). Information remains limited about the comparative protein expression profiles of chicken embryonic tissues in response to IBV infection in ovo. In this study, we analyzed the changes of protein expression in trachea and kidney tissues from chicken embryos, following IBV infection in ovo, using two-dimensional gel electrophoresis (2-DE) coupled with matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS).     

Structural View and Substrate Specificity of Papain-like Protease from Avian Infectious Bronchitis Virus

Papain-like protease (PLpro) of coronaviruses (CoVs) carries out proteolytic maturation of non-structural proteins that play a role in replication of the virus and performs deubiquitination of host cell factors to scuttle antiviral responses. Avian infectious bronchitis virus (IBV), the causative agent of bronchitis in chicken that results in huge economic losses every year in the poultry industry globally, encodes a PLpro. The substrate specificities of this PLpro are not clearly understood. Here, we show that IBV PLpro can degrade Lys⁴⁸- and Lys⁶³-linked polyubiquitin chains to monoubiquitin but not linear polyubiquitin. To explain the substrate specificities, we have solved the crystal structure of PLpro from IBV at 2.15-Å resolution. The overall structure is reminiscent of the structure of severe acute respiratory syndrome CoV PLpro. However, unlike the severe acute respiratory syndrome CoV PLpro that lacks blocking loop (BL) 1 of deubiquitinating enzymes, the IBV PLpro has a short BL1-like loop. Access to a conserved catalytic triad consisting of Cys¹⁰¹, His²⁶⁴, and Asp²⁷⁵ is regulated by the flexible BL2. A model of ubiquitin-bound IBV CoV PLpro brings out key differences in substrate binding sites of PLpros. In particular, P3 and P4 subsites as well as residues interacting with the β-barrel of ubiquitin are different, suggesting different catalytic efficiencies and substrate specificities. We show that IBV PLpro cleaves peptide substrates KKAG-7-amino-4-methylcoumarin and LRGG-7-amino-4-methylcoumarin with different catalytic efficiencies. These results demonstrate that substrate specificities of IBV PLpro are different from other PLpros and that IBV PLpro might target different ubiquitinated host factors to aid the propagation of the virus.     

Induction of Caspase-Dependent Apoptosis in Cultured Cells by the Avian Coronavirus Infectious Bronchitis Virus

Avian coronavirus infectious bronchitis virus (IBV) is the causative agent of chicken infectious bronchitis, an acute, highly contagious viral respiratory disease. Replication of IBV in Vero cells causes extensive cytopathic effects (CPE), leading to destruction of the entire monolayer and the death of infected cells. In this study, we investigated the cell death processes during acute IBV infection and the underlying mechanisms. The results show that both necrosis and apoptosis may contribute to the death of infected cells in lytic IBV infection. Caspase-dependent apoptosis, as characterized by chromosomal condensation, DNA fragmentation, caspase-3 activation, and poly(ADP-ribose) polymerase degradation, was detected in IBV-infected Vero cells. Addition of the general caspase inhibitor z-VAD-FMK to the culture media showed inhibition of the hallmarks of apoptosis and increase of the release of virus to the culture media at 16 h postinfection. However, neither the necrotic process nor the productive replication of IBV in Vero cells was severely affected by the inhibition of apoptosis. Screening of 11 IBV-encoded proteins suggested that a 58-kDa mature cleavage product could induce apoptotic changes in cells transiently expressing the protein. This study adds one more example to the growing list of animal viruses that induce apoptosis during their replication cycles.     

Immunological effect of Moringa Oleifera leaf extract on vaccinated and non-vaccinated Hubbard chickens experimentally infected with Newcastle virus

In veterinary medicine plant based medicine is achieving a huge importance worldwide. This research was subjected to rectify the hydrophilic Moringa Oleifera alcoholic leaves extract could improve the immune system in vaccinated and non-vaccinated broiler Hubbard chickens experimentally exposed to Newcastle disease (ND) virus. Seventy five chicks with age one day old were splitted randomly into five groups equally in distribution with fifteen chick in each group. Group I was untreated unvaccinated (control negative group) while group IV was infected group with NDV (control positive group). The experimental Groups II and V were given daily oral treatment of hydrophilic alcoholic leaves extract of M. oleifera at 200 mg/kg body weight until day 21 of age while groups III and V were ND vaccinated with La Sota strain of ND vaccines. The four groups (II, III, IV, V) were infected with ND virus velogenic strain (VNDV) on day 21. Following to infection, Monitoring of birds were done daily for clinical signs, postmortem examination, morbidity and mortality. Cellular, humeral immune response and phagocytic activity were evaluated and the data were statistically analyzed using (SPSS). Total and differential cell numbers as well as Haemagglutination inhibition (HI) titre increased in the extract treated and vaccinated group which give total protection against NDV much more than treated and unvaccinated group. As a result it could be recommended to use M. Olifera extract from the first day of rearing in Hubbard chicken with ND vaccination program as a prophylactic treatment in protection of birds against ND infection.     

Phenotypic and Genotypic Characterization of Avian Escherichia coli O86:K61 Isolates Possessing a Gamma-Like Intimin

Escherichia coli O86:K61 has long been associated with outbreaks of infantile diarrhea in humans and with diarrheal disease in many animal species. Studies in the late 1990s identified E. coli O86:K61 as the cause of mortality in a variety of wild birds, and in this study, 34 E. coli O86:K61 isolates were examined. All of the isolates were nonmotile, but most elaborated at least two morphologically distinct surface appendages that were confirmed to be type 1 and curli fimbriae. Thirty-three isolates were positive for the eaeA gene encoding a gamma type of intimin. No phenotypic or genotypic evidence was obtained for elaboration of Shiga-like toxins, but most isolates possessed the gene coding for the cytolethal distending toxin. Five isolates were selected for adherence assays performed with tissue explants and HEp-2 cells, and four of these strains produced attaching and effacing lesions on HEp-2 cells and invaded the cells, as determined by transmission electron microscopy. Two of the five isolates were inoculated orally into 1-day-old specific-pathogen-free chicks, and both of these isolates colonized, invaded, and persisted well in this model. Neither isolate produced attaching and effacing lesions in chicks, although some pathology was evident in the alimentary tract. No deaths were recorded in inoculated chicks. These findings are discussed in light of the possibility that wild birds are potential zoonotic reservoirs of attaching and effacing E. coli.     

Comparison of techniques for the detection of avian infectious bronchitis virus as a contaminant of vaccines

Techniques for detecting various levels of both field and vaccine strains of infectious bronchitis virus in a deliberately contaminated Newcastle disease vaccine were compared using chick embryos, chick kidney cells, chick tracheal organ cultures and chickens with a view to determining the most appropriate method for screening vaccines for freedom from IBV contamination. Techniques examined included detection of abnormalities and deaths in embryos, cytopathic effect in chick kidney cells, ciliostasis in chick tracheal organ cultures and clinical signs and virus isolation in chickens as well as the fluorescent antibody test, negative contrast electron microscopy and serology where appropriate. Results showed that the techniques capable of detecting both strains of infectious bronchitis virus were, in order of sensitivity, the fluorescent antibody test on allantoic cells from infected embryos, ciliostasis and direct electron microscopy of allantoic fluid. One surprising feature was the poor results obtained using chickens. Some detection was achieved with tracheal virus isolation and tracheal organ cultures prepared from inoculated birds and to a lesser extent with histology and clinical signs, but no technique detected the field strain.     

Viral Susceptibility Range of the Fathead Minnow (Pimephales promelas) Poikilothermic Cell Line

The viral susceptibility range of a poikilothermic cell line derived from the fathead minnow (Pimephales promelas) (FHM) to infection by a number of homoiothermic viruses representing most of the presently recognized viral groups and a member of the psittacosis-lymphogranuloma-trachoma group of agents was studied. All infectious agents, except poliovirus types 1 and 3, infectious bursal agent, and an avian infectious bronchitis virus (IBV) strain, readily multiplied in the FHM cell culture system, producing a detectable cytopathic effect. Although inconclusive evidence was obtained with two other avian IBV strains, these results indicated the ability of the FHM cell culture system to readily support the propagation of a variety of cytopathogenic homoiothermic viral agents.     

Combined use of ELISA and Western blot with recombinant N protein is a powerful tool for the immunodiagnosis of avian infectious bronchitis

Background

The avian infectious bronchitis virus (IBV) remains a significant source of loss in the poultry industry and early diagnosis is required to prevent the disease from spreading. This study examined the combined use of an ELISA and Western blot (WB) to detect antibodies against the nucleocapsid protein (N) of IBV. The coding sequence for N was amplified by RT-PCR and expressed in Escherichia coli. A soluble recombinant N protein (rN) of approximately 50?kDa was obtained. A total of 389 sera were tested against the rN in ELISA and the results were compared with those of the commercial IDEXX IBV Ab test. ELISA-rN achieved a 90.34% sensitivity and 90.16% specificity. WB confirmed all false negative sera in ELISA-rN or IDEXX test as truly positive. The current study indicate that the combined use of rN in ELISA and WB is a powerful tool for the immunodiagnosis of avian infectious bronchitis.

Methods

Constructed recombinant pAE/n expression vectors were used to transform E. coli BL21(DE3) Star competent cells (Invitrogen). The rN of infectious bronchitis virus was purified by affinity chromatography using HisTrap HP 1?mL columns pre-packed with pre-charged Ni Sepharose in the ÄKTAprime Automated Liquid Chromatography system (GE Healthcare). A total of 389 serum samples from chickens were used to develop and evaluate the ELISA-rN test. To standardize the indirect ELISA development, serum dilutions (1:100, 1:200 and 1:400) and different concentrations of purified rN antigen (50, 100 and 200?ng/well) were tested. Positive and negative sera for IBV were used as controls. The results were compared with those obtained from a commercial kit. Serum samples scored as negative with the commercial kit but as positive with the ELISA-rN were further analysed by Western blot analyses using the rN protein as an antigen. The results of the ELISA-rN were compared to the commercial kit results using receiver-operating characteristics curves, area under the curve, and confidence intervals with the software GraphPad Prism version 6.0 for Windows (GraphPad Software, USA).

Results

The expected cDNA fragment of approximately 1240?bp was successfully amplified by PCR using primers designed to select for the coding region of the N protein. The rN was expressed as a soluble protein to avoid the refolding steps and, after purification a yield of 10?mg/L of rN was obtained. The SDS-PAGE results demonstrated the presence of two distinct bands that had a molecular mass of approximately 45 and 50 KDa. Out of 244 sera that scored positive in the commercial ELISA IDEXX IBV Ab Test, 220 were also positive in the ELISA-rN, yielding an ELISA-rN test sensitivity of 90.16%. Out of 145 sera that scored negative in the IDEXX IBV Ab Test, 131 also scored negative in the ELISA-rN, indicating a specificity of 90.34%. Sera that tested negative in the ELISA-rN and positive in the commercial test also reacted with the rN protein in Western blot.

Conclusions

The association between the ELISA and Western blot techniques developed in this study with a subunit of IBV (rN) were able to detect antibodies that the commercial ELISA did not detect suggesting that the ELISA-rN has greater sensitivity.

     

Complete Genome Sequence of an Infectious Bronchitis Virus Chimera between Cocirculating Heterotypic Strains

To date, multiple serotypes and genotypes of infectious bronchitis virus (IBV) have been isolated and identified. In order to provide more information on the viral evolution of IBVs, a new virulent strain named GX-NN09032, isolated from Guangxi, China, in 2009, was sequenced, and phylogenetic and recombination analyses were conducted. Furthermore, potential recombination events associated with GX-NN09032 were found in four IBV strains, including GX-YL5, DY07, CK/CH/SD09/005, TC07-2. The present study suggested that GX-NN09032 might contribute to the emergence of modern IBV variants through recombination.     

The hydrophobic domain of infectious bronchitis virus E protein alters the host secretory pathway and is important for release of infectious virus

The coronavirus (CoV) E protein plays an important role in virus assembly. The E protein is made in excess during infection and has been shown to have ion channel activity in planar lipid bilayers. However, a role in infection for the unincorporated E or its ion channel activity has not been described. To further investigate the function of the infectious bronchitis virus (IBV) E protein, we developed a recombinant version of IBV in which the E protein was replaced by a mutant containing a heterologous hydrophobic domain. The mutant virus, IBV-EG3, was defective in release of infectious virus particles. Further characterization of IBV-EG3 revealed that damaged particles appeared to accumulate intracellularly. The phenotype of IBV-EG3 suggested that the hydrophobic domain of IBV E may be important for the forward trafficking of cargo, so we determined whether IBV E facilitated the delivery of cargo to the plasma membrane. Surprisingly, we found that IBV E, but not EG3, dramatically reduced the delivery of cargo to the plasma membrane by impeding movement through the Golgi complex. Furthermore, we observed that overexpression of IBV E, but not EG3, induced the disassembly of the Golgi complex. Finally, we determined that the delivery of IBV S to the plasma membrane was reduced in cells infected with wild-type-IBV compared to those infected with IBV-EG3. Our results indicated that the hydrophobic domain of IBV E alters the host secretory pathway to the apparent advantage of the virus.