Group of fifteen dogs were immunized respectively with 0.25 ml, 0.5 ml and 1.0 ml vaccine respectively containing 0.25108FFU/mL, 0.5108FFU/mL and 1.0108FFU/mL inactivated HEP-dG viruses. virus can improve the expression of G-gene mRNA and the G protein and produce more offspring viruses in cells. The amount of G protein revealed a positive relationship with immunogenicity in mice and dogs. The inactivated HEP-dG recombinant virus induced higher levels of VNA and conferred better protection against virulent RABV in mice and dogs than the inactivated parental virus and a commercial vaccine. The protective antibody persisted for at least 12 months. These data demonstrate that the HEP-dG is stable, induces a strong VNA response and confers protective immunity more effectively than the RABV HEP-Flury strain. HEP-dG could be a potential candidate in the development of novel inactivated rabies vaccines Introduction Rabies remains one of the most important public health problems worldwide, causing more than 60,000 human deaths each year [1]. Rabies is caused by rabies virus (RABV), which is the prototype virus of the genus. Rabies virus (species 1) belongs to the genus Lyssavirus in the family and restriction sites were introduced upstream and downstream of the RV G gene by using primers RVG1 (restriction site is underlined) and RVG2 (restriction site Mouse monoclonal to eNOS is underlined). The fragment was digested with and and cloned into pHEP-3.0 predigested with and em PstI /em . Fig. 1 illustrates the construction of the dG recombinant clone. The resulting plasmid was designated pHEP-dG. Open in a separate window Figure 1 Construction of the full-length cDNA plasmid with double G gene (pHEP-dG) by RT-PCR.(A) Schematic diagram of the two virus genomes (arrows indicate the position of the two pairs of primers. (B) The RT-PCR products of the HEP-dG and the rHEP-Flury were amplified by N1/N2 primers (B1, lane 1 and 2). The RT-PCR products of the HEP-dG by DGG1/DGG2 primers after eight passages in BHK-21 cells (B2, lane 1), but no product was FN-1501 amplified from the cells infected with the rHEP-Flury strain (B2, lane 2). (M, molecular size marker). Rescue of RABV from cDNA clone Recombinant viruses were rescued as described previously [29]. Briefly, BHK-21 cells (1106) grown in a 12-well tissue culture plate were transfected using a superfect transfection kit (Qiagen) with four helper plasmids, -N (0.625 g), -P (0.3125 g), -L (0.125 g), -G (0.1875 g) and the full-length cDNA clone pHEP-dG (2.5 FN-1501 g). At day 6 after transfection, supernatants were transferred to a 96-well plate and incubated for another 6 days. Cells were examined by immunofluorescence staining with FITC-labeled RV N-specific antibody (Fujirabio Inc. Malvern, PA). The culture fluid was collected as virus stock and stored at ?80C until use. Confirmation of the rescued virus by RT-PCR and sequencing To confirm if the rescued RABV was derived from pHEP-dG, RT-PCR was preformed with two pairs of primers. The primer pair N1 (sense) ( em class=”gene” 5-AGTCTCTATAGGTTGAGC-3 /em ) and N2 (antisense) ( em class=”gene” 5-GATGAAATAAGAGTGAGG-3 /em ), corresponding to the positions from nucleotide 506 to 524 and from 930 FN-1501 to 948 of the N gene were used to amplify RABV N gene. Another primer pair DGG1 (sense) ( em class=”gene” 5-AAAGGGTGTTTGAGAGTTGG-3 /em ) corresponding to the positions from nucleotide 1079 to 1099 (based on the first G gene sequence of the HEP-dG genome) and DGG2 (antisense) ( em class=”gene” 5-ACAGGTTGGTACATCCTTCGTCC-3 /em ) corresponding to the positions from nucleotide 149 to 172 (based on the second G gene sequence of the FN-1501 HEP-dG genome) was used to amplify the double G gene of HEP-dG virus. Sequencing of the amplified cDNA fragment was carried out by using TaKaRa reagents. Titration of virus Viral titers were determined by direct fluorescent antibody assay in BHK-21 cells. BHK-21 cells in 96-well plate were inoculated with serial 10-fold dilution of virus and incubated at 37C for 4 days. Cells were fixed with 80% acetone for 30 min and stained with FITC-labeled anti-rabies mAb (Fujirabio). Antigen-positive foci were counted under a fluorescent microscope FN-1501 (OLYMPUS) and calculated as focus forming unit (FFU) per milliliter. Virus growth curve Monolayer cultures of 5106 BHK-21 cells were infected with individual virus at a multiplicity of infection (MOI) of 5. Then the cultures were incubated at 37C. Supernatants were harvested at 1, 2, 3, 4 and 5 days post-inoculation (p.i.), and virus titers determined by.
Group of fifteen dogs were immunized respectively with 0