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Comparison between dot-immunoblotting assay and clinical sign determination method for quantifying avian infectious bronchitis virus vaccine by titration in embryonated eggs.

ABSTRACT: A sensitive and specific method for measuring the vaccine titer of infectious bronchitis virus (IBV) is important to commercial manufacturers for improving vaccine quality. Typically, IBV is titrated in embryonated chicken eggs, and the infectivity of the virus dilutions is determined by assessing clinical signs in the embryos as evidence of viral propagation. In this study, we used a dot-immunoblotting assay (DIA) to measure the titers of IBV vaccines that originated from different pathogenic strains or attenuation methods in embryonated eggs, and we compared this assay to the currently used method, clinical sign evaluation. To compare the two methods, we used real-time reverse transcription-PCR, which had the lowest limit of detection for propagated IBV. As a clinical sign of infection, dwarfism of the embryo was quantified using the embryo: egg (EE) index. The DIA showed 9.41% higher sensitivity and 15.5% higher specificity than the clinical sign determination method. The DIA was particularly useful for measuring the titer of IBV vaccine that did not cause apparent stunting but propagated in embryonated chicken eggs such as a heat-adapted vaccine strain. The results of this study indicate that the DIA is a rapid, sensitive, reliable method for determining IBV vaccine titer in embryonated eggs at a relatively low cost.

SUBMITTER: Yuk SS

PROVIDER: S-EPMC7119534 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Comparison between dot-immunoblotting assay and clinical sign determination method for quantifying avian infectious bronchitis virus vaccine by titration in embryonated eggs.

Yuk Seong-Su SS Kwon Jung-Hoon JH Noh Jin-Yong JY Hong Woo-Tack WT Gwon Gyeong-Bin GB Jeong Jei-Hyun JH Jeong Sol S Youn Ha-Na HN Heo Yong-Hwan YH Lee Joong-Bok JB Park Seung-Yong SY Choi In-Soo IS Song Chang-Seon CS

Journal of virological methods 20160121

A sensitive and specific method for measuring the vaccine titer of infectious bronchitis virus (IBV) is important to commercial manufacturers for improving vaccine quality. Typically, IBV is titrated in embryonated chicken eggs, and the infectivity of the virus dilutions is determined by assessing clinical signs in the embryos as evidence of viral propagation. In this study, we used a dot-immunoblotting assay (DIA) to measure the titers of IBV vaccines that originated from different pathogenic s ...[more]

PMID: 26802454

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Project description:Avian infectious bronchitis (IB) is an acute, highly infectious and contagious viral disease of chickens caused by avian infectious bronchitis virus (IBV) belonging to the genus Coronavirus and family Coronaviridae. It can affect all age groups of birds. The toll-like receptors (TLRs) are a major class of innate immune pattern recognition receptors that have a key role in immune response and defense against various infections.The TLRs are essential for initiation of innate immune responses and in the development of adaptive immune responses. An in ovo model was employed to study the antiviral activity of TLR ligands (Pam3CSK4, LPS and CpG ODN) on replication of IBV. It was hypothesized that optimum dose and specific timing of TLR ligands may reduce viral load of IBV in specific pathogen free (SPF) embryonated chicken eggs (ECEs). Further, the mechanism involved in the TLR-mediated antiviral response in chorioallantoic membrane (CAM) of ECEs was investigated. The ECEs of 9-11 days old were treated with different doses (high, intermediate and low) of TLR-2 (Pam3CSK4), TLR-4 (LPS) and TLR-21 (CpG ODN) ligands. In addition, to know the timing of TLR ligand treatment, six time intervals were analyzed viz. 36, 24 and 12?h prior to infection, time of infection (co-administration of TLR ligands and avian IBV) and 12 and 24?h post-IBV infection. For studying the relative expression of immuno-stimulatory genes (IFN-?, IFN-?, IFN-?, IL-1?, iNOS and OAS) in CAM, TLR ligands were administered through intra-allantoicroute and CAM were collected at 4, 8 and 16?h post treatment. The results demonstrated that intermediate dose of all the three TLR ligands significantly reduced virus titers and used in the present study. However, the LPS reduced virus titer pre- and post-IBV infection but Pam3CSK4 and CpG ODN reduced only pre-IBV infection. Further analysis showed that TLR ligands induced IFN-?, IL-1? and IFN stimulated genes viz. iNOS and OAS genes in CAM. The present study pointed towards the novel opportunities for rational design of LPS as immuno-stimulatory agent in chickens with reference to IBV. It may be speculated that in ovo administration of these TLR ligands may enhance resistance against viral infection in neonatal chicken and may contribute towards the development of more effective and safer vaccines including in ovo vaccines.

Project description:Avian infectious bronchitis virus (IBV) infection is one of the major viral respiratory diseases of chickens. Better understanding of the molecular basis of viral pathogenesis should contribute significantly towards the development of improved prophylactic, therapeutic and diagnostic reagents to control infections. In the present investigation, transcriptional profiles were analyzed by using RNA recovered from the lung tissue of IBV infected 18-day-old chicken embryos at 6, 24, 48 and 72 h post IBV infection. This microarray analysis was completed using avian cDNA arrays comprised of fragments of 1191 unique chicken and turkey gene transcripts. These arrays were generated from normalized cDNA subtraction libraries that were derived from avian pneumovirus (APV) infected chicken embryo fibroblast (CEF) cultures and tissues obtained from APV infected turkeys subtracted with their respective uninfected cultures and tissues. Of the 1191 unique genes represented on the array, the expression of a total of 327 genes (27% of total) were altered by two-fold or more from 6 through 72 h post-infection. A comparative analysis of IBV regulated genes with genes previously reported to change in expression following infection with other avian respiratory viruses revealed both conserved and unique changes. Real-time qRT-PCR was used to confirm the regulated expression of genes related to several functional classes including kinases, interferon induced genes, chemokines and adhesion molecules, vesicular trafficking and fusion protein genes, extracellular matrix protein genes, cell cycle, metabolism, cell physiology and development, translation, RNA binding, lysosomal, protein degradation and ubiquitination related genes. Microarray analysis served as an efficient tool in facilitating a comparative analysis of avian respiratory viral infections and provided insight into host transcriptional changes that were conserved as well as those which were unique to individual pathogens.

Dataset Information

Comparison between dot-immunoblotting assay and clinical sign determination method for quantifying avian infectious bronchitis virus vaccine by titration in embryonated eggs.

Publications

Comparison between dot-immunoblotting assay and clinical sign determination method for quantifying avian infectious bronchitis virus vaccine by titration in embryonated eggs.

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