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

attempt to control the major manifestations [abortion and nervous Symptoms] attributed to listeria monocytogenes infection in sheep

A trial to control abortion and nervous symptoms in sheep was carried out through an experiment applied on 2 groups of pregnant ewes. First group included 6 ewes immunized S/C with 2ml suspension of local isolated field living L. monocytogenes [diluted 8X10 [9]/ml] and challenged with 2 ml of the same strain of L. monocytogenes [2xl0 [2]/ml]. This group revealed gradual increase in the antibody titre till delivery using serum agglutination test [SAT] and FLISA. In addition, failure to reisolate L. monocytogenes in the rectal and vaginal swabs and no significant lesions characteristic for L. monocytogenes infection were detected pathologically. The second group [control group] consisted of 3 pregnant ewes, two of which challenged as the first group and the third ewe still alive and delivered normally and its lambs challenged as control group. The antibody titre of the 2 challenged pregnant ewes of this group [one died with nervous symptoms and the other aborted] was not present till challenge and increase somewhat after challenge. Moreover, L. monocytogenes was re isolated from rectal and vaginal swabs and from collected organs of dead ewe and from placenta of aborted one and from foeti. Histopathologically, the dead ewe revealed focal gliaosis, necrosed neurons, neurophagia, endothelial cell hyperplasia and perivascular infiltration by few number of lymphocytes and glia cells in the brain. Necrotic hepatocytes in the liver, follicular depletion in the spleen, exocytosis and focal areas of necrobiotic changes in the uterine epithelium and necrosis in the tips of the villi of the placenta. The foeti of dead and aborted ewes showed foci of coagulative necrosis, hemolysed R.B.C.s and hemosidrosis in the liver. In this experimental work, a recommendation of using live preparation of L. monocytogenes vaccine has afforded a hope in control of listeriosis in pregnant ewes. There is a significant increase in antibody response in the serum of immunized dams and their lambs suggested the role of dam vaccination in protection of neonate against listeriosis