Molecular detection, isolation and characterization of avian rotavirus from broiler chicken

Chicken rotaviruses [CRVs] were detected, identified and characterized in broiler chicken with diarrhea for the first time in Egypt Fecal samples were collected from 85 naturally occurring diarrheal outbreaks in commercial chicken broilers fmis that are located in a wide range of geographical areas including many Egyptian govemorates mainly 6th Octobar, Elfayom, Giza, Qaluobea, Menofla and Elmansoura during year 2008. CRV was detected in the fecal samples by ELISA using monoclonal antibodies [Mabs] against VP6, Electron Microscopy [EM], Reverse Transcription-Polymerase Chain Reaction [RTPCR] and the virus was isolated using SPF chickens. The obtained results confirmed the isolation and identification of group A chicken rotavirus while the molecular characterization analysis using different primers sets suggested that the isolated chicken rotavirus does not belong to the same cluster of Chi rotavirus strain but most likely more related to Po-13 strain [mammalian like chicken rotavirus strains]. This study reports the importance of rotaviruses in broiler chickens with delayed growth and diarrhea

Improved quality control protocol for fowl pox virus vaccines based on the use of PCR and sequence analysis to detect REV and ALV as contaminants

In the present study, twenty one Fowl pox virus [FPV] vaccines were collected in the period between 2003-2005 and tested using the currently used and an improved quality control protocols. Results of currently used quality control protocol revealed negativity of all tested vaccines for the presence of contaminants and the results were satisfactory. Using PCR to detect Reticuloendotheliosis virus [REV] as contaminant in such vaccines revealed negative result except one suspected contaminated vaccine. Inoculation of this vaccine in egg, chicken embryo fibroblasts [CEFs], Specific pathogen free [SPF] chicks for three passages and testing of samples collected from inoculated host revealed positive amplification using REV specific primers. Sequence analysis of the obtained amplification fragment for REV revealed its negativity and confirmed the non specific amplification of such primers which were previously published in several PCR studies for REV. Using avian leucosis virus [ALV] sets of primers to detect groups A, B, C, D and J in a PCR reaction revealed positive amplification of ALV fragment and confirming the contamination of tested vaccine with ALV. The study proposes the importance of using PCR followed by sequence analysis of the amplified product to confirm the contaminations fonnd in the FPV vaccines

Construction of a recombinant baculovirus expressing the major capsid protein [VP6] of bovine rotavirus

The present study reports the expression of VP6, the major inner capsid protein of bovine rotavirus Nebraska calf diarrhea virus [NCDV] strain in a baculovirus expression system. The full-length DNA copies of RNA segment 6 [coding for VP6 protein] of NCDV were inserted into a baculovirus expression vector. A recombinant baculovirus carrying the VP6 gene was constructed through homologous recombination between the baculovirus recombinant plasmid carrying the VP6 gene and Autographa californica nuclear polyhedrosis virus [AcNPV] under the control of the polyhedrin promotor. Infection of Spodoptera frugiperda [Sf9] cells with the recombinant baculovirus expressing VP6 protein revealed a high-level of expression when tested by immunoflurescence and solid phase ELISA tests using BRV-specific polyclonal antibodies. The VP6 expressed protein was detected in Coomassie blue stained SDS-PAGE and produced a detectable band in Western blot assay. The high degree of reactivity with BRV-specific polyclonal antibodies confirmed that the antigenic determinants of the expressed protein were unaltered. The use of the in vitro expressed VP6 protein in the field diagnosis and vaccine development to control rotavirus infection is of considerable intere

Expression of the bovine coronavirus spike glycoprotein subunits in insect cells using recombinant baculoviruses

In the present study, the bovine coronavirus [BCV] spike glycoprotein cleavage products [S1 and S2] were individually expressed in Spodoptera frugiperda [Sf9] insect cells, using a baculovirus expression system. The coding sequence of S1 and S2 gene fragments were amplified by RT-PCR and cloned into the baculovirus shuttle vector pBlueBac4.5/V5-His TOPO [R] TA. The cloned fragments were inserted into the genome of Autographa californica nuclear polyhydrosis virus [AcMNPV] under control of the polyhedrin promoter, through a process of homologous recombination between the shuttle vector and a linearized replication-defective baculovirus DNA [Bac-N-Blue[TM]]. Recombinant baculoviruses were selected by plaque purification; verified for the presence of target sequences using PCR and propagated for generation of high-titer viral stocks. Infection of insect cells with the recombinant baculoviruses revealed high-level expression of the target proteins as indicated by immunofluoresent test and solid phase ELISA using BCV-specific polyclonal antiserum