Document Type

Dissertation - University Access Only

Award Date


Degree Name

Doctor of Philosophy (PhD)

Department / School



Pestiviruses are enveloped positive stranded RNA viruses from the genus pestivirus of Flaviviridae. The genomes are 12.3-12.5 kb, and encode 11-12 proteins produced by co- or post-translational processing of the viral polyproteins with viral and host cell proteases (Thiel et al., 1996). Three main pestivirus types [bovine viral diarrhea virus (BVDV) type 1 (BVDV-1), BVDV-2 and BDV] are causes of economically important enteric, respiratory, reproductive, and immune system diseases in wild and domestic ruminants worldwide (Bolin, 1996). Fifty years after the identification of BVDV, it remains a worldwide problem for the bovine industry. Vaccination and non-vaccination control measures for BVDV are far from satisfactory in spite of the availability of both modified live virus (ML V) and inactivated virus vaccines. Control efforts have been hindered by the genetic and antigenic variability between pestiviruses and by interspecies transmission. Test and eradication approaches for the control of pestiviruses are not feasible in many parts of the world because of the lack of strong economic support for the eradication program and because of problems controlling ammal movements. For these reasons, we suggested that a reasonable control program for pestivirus infections is a multifaceted task that should include proper herd management, proper diagnosis of pestivirus infections, proper typing of the pestivirus isolates and implementation of effective vaccination strategies. In this investigation, we started our work with the aim of developing an effective vaccine to be a nucleus for a vaccination strategy that can be used in both bovine and ovine species. We adopted a DNA vaccine approach because DNA vaccines can be produced, quality-controlled, maintained, and administered with relative-ease and low cost. DNA vaccines can also overcome many of the problems associated with neonatal vaccination. Phylogenetic analysis was done on several bovine, ovine isolates and two cytopathic Egyptian camel isolates to determine the isolates more suitable for inclusion in the vaccine design. Genotyping of the two camel pestiviruses revealed that Giza4 and Giza7 belonged to BVDV-1 and BVDV-2, respectively. Giza7 Ems nucleotide sequences were 82-88 %, 68.7-72 %, 66.5-67.2%, 68 % and 64 % homologous with BVDV-2, BVDV-1, BDV, CSFV and Reindeer-I viruses, respectively. Giraffe-I pestivirus sequence homology to Giza7 was 70 %, higher than homology values of BVDV-1 Oregon and BVDV-1 NADL. The predicted amino acid sequence homologies of Giza7 were 84.9-90.2 %, 77.4-81.9 %, 73.6 % and 73.6 % for BVDV-2, BVDV-1, BDV and CSFV, respectively. Giraffe-I and Reindeer-I isolates showed 75.l % and 72.1 %, amino acid homology to Giza 7, respectively. Sequence comparison of short stretches of Giza7 NS3 using BLASTn showed a high degree of homology with BVDV-2 pestiviruses. This was the first report of a BVDV-2 infection in African dromedary camels. We proposed that Giza? could have originated from one of two sources, import and interspecies transmission or evolution from an unknown African pestivirus. Because Giza? is closely related to a subgroup of BVDV-2 viruses that have been reported in the US, the adaptation to camels could have been due to interspecies transmission. The observation that Giza7 had a higher nucleotide, but not amino acid, sequence homology with Giraffe-I compared to BVDV-1 strains Oregon and NADL may indicate evolution from an unlmown African pestivirus. The questions surrounding the origin of Giza7 and other incidences of interspecies transmission underlined the need for developing multitype pestivirus vaccination. Selection of the pestivirus genotype-representatives to include in the vaccine proved to be challenging. During the typing of the camel pestivirus, it became evident that some typing techniques are not capable of typing all pestiviruses. In addition, some of the viruses tested were reported to undergo heterologous recombinations with different pestivirus genotypes and, most seriously, some virus preparations were not pure and contained more than one pestivirus type. We used a short region of the pestivirus NS3 that contained the information required to differentiate the major pestivirus genotypes to develop pestivirus NS3-type specific molecular beacons. This approach allowed simultaneous detection and typing of single-type as well as mixed BVDV-l/BVDV-2 RNA in a single tube RT-PCR using a single set of universal pestivirus NS3 primers. The technique allowed the detection of BVDY RNA extracted from 1-3 TCIDso virus suspension Analysis of the available NS3 ovine and swine pestivirus sequences showed that this approach could also be used for simultaneous detection and typing of these pestiviruses. Nucleotide and amino acid sequence analysis of the NS3-typing region in comparison to other typing regions of the pestivirus genome indicated the presence of a continuum of genetic and antigenic mutations within pestiviruses across genotypes. We recommend using NS3 typing as an anchor point for pestivirus genotyping in diagnostic settings and for developing of virus-characteristic genetic profiles to allow tracking vaccinal and field isolates in the environment. Based on nucleotide and amino acid sequence analysis, BVDV-1 Singer, BVDV2, BDV ID211 and BDV CB5 were selected as donors for the immunogens used in the DNA vaccine experiment to broaden the capacity for immune response across several different types of pestiviruses. The purity of these pestivirus isolates were determined using the molecular beacons technique. A series of mammalian expression plasmids containing genes from these viruses were constructed. The BVDV-1 and BVDV-2 constructs contained C and Ems genes of BVDV-1 Singer and BVDV-2 125, respectively, and pestivirus type 3 constructs contained the C protein of BDV ID211 and BDV CB5 stains. Both the C and Ems genes were used because they have been shown to induce strong strain cross-reactive and MHC-restricted CD4+ and CD8+ immune responses. Pestivirus proteins were expressed in NIH/3T3 cells to verify pestivirus proteins-expression from the mammalian expression plasmid. Three groups of Balb/c mice were immunized with 1) a pool of the three pestivirus expression plasmids, 2) a commercial inactivated BVDV type 1 and 2 vaccine, or 3) the parent plasmid. DNA vaccinates exhibited a primary lymphoproliferative response to BVDV-1 Singer the 1st and 2nd week after the primary immunization. Another peak in the lymphoproliferative response was observed the 3rd week after the booster immunization. BVDV-2 lymphorpoliferative response peaked 3 weeks after the initial immunization and 3 and 4 weeks after the booster immuniztion. The lymphoproliferative responses of animals receiving the commercial vaccine were lower than those observed in the animals receiving the DNA vaccine. The lymphoproliferative responses to the type 3 viruses were high 1 week after primary immunization, and gradually declined to the control level over 2 weeks. Lymphoproliferative responses to type 3 viruses peaked again the yc1 and 4th week after the booster immunization. The plasmid vaccination resulted in the production of non-neutralizing antibodies to the homologous pestivirus strains. Neutralizing antibodies were produced against BVDV-1 Singer and BVDV-2 125, but not against BDV ID21 l or BDV CB5. Overall, our results warrant future studies to examine the effect of plasmid vaccination on protecting farm ruminant from pestivirus infections. We recommended conducting the initial experiments in sheep. This multi-plasmid-DNA vaccine approach could provide the basis for a multi-species pestivirus control program.

Library of Congress Subject Headings

Ruminants -- Virus diseases DNA vaccines



Number of Pages



South Dakota State University