VTechWorks Repository :: Browsing by Author "Toth, Thomas E."

Browsing by Author "Toth, Thomas E."

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Applicability of vaccinia virus as cloning and expression vector for bacterial genes: mice immune responses to vaccinia virus expressing Brucella abortus and Listeria monocytogenes antigens
Baloglu, Simge (Virginia Tech, 2001-07-27)
Previous studies by our group showed that vaccinia virus recombinants expressing Brucella abortus (BA) antigens heat shock protein GroEL, 18 kDa protein and Cu/Zn SOD, were unable to induce protective immune responses against Brucella challenge. This dissertation analyzes the possible reasons for this phenomenon, by using other genes/proteins from BA and Listeria monocytogenes (LM), various shuttle plasmids (pSC65, pSC11) and immune response modulators (CpG, IL-12, B7-1). As the first objective, a vaccinia virus recombinant (WRL7/L12), expressing the BA L7/L12 gene was generated. L7/L12 ribosomal protein was used as a T-cell reactive antigen, with protective potential to Brucella challenge. The WRL7/L12 was able to express the gene of interest and induce IgG2A type antibody response, but not a protective immune response against Brucella challenge. As a control, an antigen from LM proven to induce CTL and protective immune responses, was used to test the efficacy of vaccinia virus to induce protection. A portion of hly gene, encoding partial listeriolysin (pLLO), was inserted into the same vaccinia virus stain. This recombinant (WRpLLO) was able to induce protection against a Listeria challenge. Next another vaccinia virus recombinant expressing Brucella abortus Cu/Zn SOD was analyzed. Although a variety of approaches, including the enhancement of the protein expression by the pMCO2 synthetic promoter, booster immunization, addition of the oligomer CpG adjuvant (WRSODCpG) to enhance Th1 type response, were used, the SOD recombinant failed to protect mice against Brucella challenge. Lastly, vaccinia virus produces a family of proteins that bind cytokines, chemokines and interferons to evade the host defensive systems. Therefore, a vaccinia virus strain co-expressing murine IL-12, and cofactor B7-1, were used to generate the recombinant WRIL12L7/L12. In order to further boost the induction of Th 1 type response, the adjuvant CpG was used. A similar recombinant, WRIL12pLLO, was generated with partial hly gene to serve as a positive control for protection. Mice immune responses to these recombinants, with and without adjuvant CpG, were analyzed, and compared with the recombinants generated with vaccinia strain WR. Co-expression of IL12 and B7 abrogated the protective efficacy of the vaccinia/ pLLO recombinant.
Assessment of the Expression of Brucella Abortus Heat Shock Protein, Groel, in Vaccinia Virus to Induce Protection Against a Brucella Challenge in Balb/C Mice
Baloglu, Simge (Virginia Tech, 1997-07-08)
B. abortus is an intracellular facultative bacterial pathogen which causes abortion in cattle and undulant fever in humans. Cattle vaccines such as B. abortus strains 19 and RB51 are live vaccine strains which protect approximately 75% of the vaccinated animals. No effective vaccines are available for the prevention of brucellosis in humans. We are developing vaccinia virus recombinants expressing various B. abortus proteins to prevent brucellosis in susceptible mammalian species. In this work the B. abortus groEL gene encoding the antigenic heat shock protein GroEL was subcloned into vaccinia virus via homologous recombination. Expression of the GroEL protein in vaccinia infected cells in-vivo was confirmed by immunoblotting. Groups of 5 female BALB/C mice were injected with the vaccinia recombinant or appropriate positive and negative control vaccines. Mice were bled and their humoral immune responses assessed. In addition, mice were challenged with virulent B. abortus strain 2308 and protection measured by the rate of splenic clearance of live Brucella. In spite of demonstrating specific GroEL antibodies in recombinant vaccinia injected mice, no significant level of protection was demonstrable. Preliminary lymphocyte transformation assays were carried out to establish if a cell mediated immune response to GroEL was induced in the vaccinated animals.
Cross-Species Infection and Characterization of Avian Hepatitis E Virus
Sun, Zhifeng (Virginia Tech, 2005-01-20)
As novel or variant strains of HEV continue to evolve rapidly both in humans and other animals, it is important to develop a rapid pre-sequencing screening method to select field isolates for further molecular characterization. Two heteroduplex mobility assays (HMA) were developed to genetically differentiate field strains of swine HEV and avian HEV from known reference strains. It was shown that the HMA profiles generally correlate well with nucleotide sequence identities and with phylogenetic clustering between field strains and the reference swine HEV or avian HEV strains. Therefore, by using different HEV isolates as references, the HMA developed in this study can be used as a pre-sequencing screening tool to identify variant HEV isolates for further molecular epidemiological studies. Our previous study showed that avian HEV antibody is prevalent in apparently healthy chickens. A prospective study was conducted on a known seropositive but healthy chicken farm. Avian HEV was identified from the healthy chicken flock. Avian HEV isolates recovered from the healthy chicken share 70-97% nucleotide sequence identities with those isolates which cause hepatitis-splenomegaly (HS) syndrome based on partial helicase and capsid gene regions. Recovery of identical viruses from the experimentally inoculated chickens in the subsequent transmission study further confirmed our field results. The capsid gene of avian HEV isolates from chickens with HS syndrome were also characterized and found to be heterogeneic, with 76-100% nucleotide sequence identities to each other. The study indicates that avian HEV is enzootic in chicken flocks and spread subclinically among chicken populations, and that the virus is heterogeneic. As HEV can not be propagated in vitro, in order to further characterize avian HEV, an infectious viral stock with a known infectious titer must be generated. Bile and feces collected from specific-pathogen-free (SPF) chickens experimentally infected with avian HEV were used to prepare an avian HEV infectious stock. The infectivity titer of this infectious stock was determined, by intravenously inoculating one-week old SPF chickens, to be 5 x 10^4.5 50% chicken infectious doses (CID₅₀) per ml. Seroconversion, viremia as well as fecal virus shedding were observed in the inoculated chickens. Contact control chickens also became infected via direct contact with inoculated ones. Avian HEV infection in chickens was found to be dose-dependent. To determine if avian HEV can infect across species, one-week old SPF turkeys were intravenously inoculated each with 10^4.5(CID₅₀) of avian HEV. The inoculated turkeys seroconverted to avian HEV antibodies at 4-8 weeks postinoculation (WPI). Viremia was detected at 2-6 WPI, and fecal virus shedding at 4-7 WPI in inoculated turkeys. This is the first demonstration of cross-species infection by avian HEV. Little is known regarding the characteristics of the small ORF3 protein largely due to the lack of a cell culture system for HEV. To characterize the small protein, the ORF3 proteins of avian HEV and swine HEV were expressed in Escherchia coli, and purified by BugBuster His-Bind Purification System. Western blot analysis showed that avian HEV ORF3 protein is unique and does not share common antigenic epitopes with those of swine HEV and human HEV. However, swine HEV (genotype 3) and human HEV (genotype 1) ORF3 proteins cross-react with each other antigenically. To determine if the ORF3 protein is a virion protein, infectious stocks of avian HEV and swine HEV were first generated in SPF chickens and pigs, respectively. Virions were subsequently purified by sucrose density gradient centrifugation and virion proteins were characterized by SDS-PAGE and Western blot analysis. Two major forms of ORF2 proteins of avian HEV were identified: a 56 kDa and an 80 kDa proteins. Multiple immunoreactive forms of ORF2 proteins of swine HEV were also observed: 40 kDa, 53 kDa, 56 kDa and 72 kDa. However, the ORF3 protein was not detected from the native virions of avian HEV or swine HEV. These findings provide direct evidence that ORF2 indeed encodes a structural protein of HEV, whereas ORF3 does not. To search for other potential animal reservoirs for HEV, the prevalence of IgG anti-HEV antibody was determined in field mice caught in chicken farms to assess the possibility of mice as a potential reservoir for HEV infection in chickens. Three different recombinant HEV antigens derived from avian HEV, swine HEV, and human HEV were used in the ELISA assays. The anti-HEV seropositive rates in wild field mice (Mus musculus), depending upon the antigen used, are 15/76 (20%), 39/74 (53%), and 43/74 (58%), respectively. HEV RNA was also detected from 29 fecal and/or serum samples of mice. The HEV sequences recovered from field mice shared 72-100% nucleotide sequence identities with each other, 73-99% sequence identities with avian HEV isolates, and 51-60% sequence identities with representative strains of swine and human HEVs. However, attempts to experimentally infect laboratory mice (Mus musculus) with the PCR-positive fecal materials recovered from the wild field mice were unsuccessful. We also attempted to experimentally infect 10 Wistar rats each with avian HEV, swine HEV, and an US-2 strain of human HEV, respectively. However, the inoculated rats did not become infected as evidenced by the lack of viremia, virus shedding in feces or seroconversion. These data suggest that mice caught in chicken farms are infected by a HEV-like virus, but additional work is needed to determine the origin of the mouse virus as well as the potential role of rodents in HEV transmission. In summary, we developed two HMAs which are useful for differentiation and identification of variant strains of swine and avian HEVs. We genetically identified and characterized an avian HEV strain from apparently healthy chickens in seropositive flocks. We showed that avian HEV can cross species barriers and infect turkeys. Our data indicated that avian and swine HEV ORF2 genes encode structural proteins, whereas ORF3 genes do not. Evidence in this study also showed that HEV or HEV-like agent exists in field mice on a chicken farm.
Design of a Fiber Optic Sensor Array for in Vitro Monitoring of Cellular Processes
West, Douglas (Virginia Tech, 1998-02-26)
Current analysis of the life and death cycles of in vitro cellular systems is based on visual observation methods relying upon morphological changes monitored using a microscope. Data collected from these techniques are not as precise as scientists desire them to be. The methods are discontinuous, indirect, costly, and time and labor intensive. The human element plays a significant part in error propagation as individual style of the researcher lends to skewing the data. Experimental results will differ greatly from laboratory to laboratory just because the methods of monitoring cellular activity are not standardized. The researcher uses experience to determine the best way to collect data quickly and "accurately" according to his or her definition. There is a great need not only to standardize data collection processes, but also to eliminate human error induced by lack of experience or fatigue. This research proposes a fiber optic based monitoring system as a possible solution to eliminate a number of problems with current cellular data collection methods and to increase the data collection rate tremendously since the process could be automated.
Detection of Feline Leukemia Virus in Feline Bone Marrow Using Polymerase Chain Reaction
Stimson, Erin Leigh (Virginia Tech, 2000-03-31)
Latent feline leukemia virus (FeLV) infections, in which proviral DNA is integrated into host DNA, but not actively transcribed, are suspected to be associated with many diseases. Bone marrow is the suspected site of the majority of latent infections. The purpose of this study was to determine if polymerase chain reaction (PCR) could detect FeLV proviral DNA in bone marrow and provide a method of detecting latent infections. Blood and bone marrow samples from fifty cats and bone marrow from one fetus were collected; sixteen had FeLV-associated diseases. Serum ELISA, blood and bone marrow immunofluorescent antibody test (IFA), and blood and bone marrow PCR were performed on each cat, and IFA and PCR on bone marrow of the fetus. Forty-one cats were FeLV negative. Five cats and one fetus were persistently infected with FeLV. Four cats were discordant; two ELISA positive with other tests negative, one bone marrow IFA negative with other tests positive, and one bone marrow IFA positive with other tests negative. No cats were positive on bone marrow PCR only. These results indicate that PCR can detect FeLV in bone marrow, but no cats in this study harbored FeLV only in the bone marrow. Not all cats with FeLV-associated diseases are persistently or latently infected with FeLV.
Development of a New Class of Viral Disinfectants: Enzymatic Inactivation of Sa-11 Rotavirus
Walker, Shawn Christopher (Virginia Tech, 1997-12-09)
The non-enveloped, pH- and heat resistant rotavirus (RV), which is cross species-infective among cattle, swine and humans may cause dehydration and high mortality in the young. Rotaviruses are inactivated only by corrosive and toxic disinfectants. In this study, the effects of bacterial proteases as a new type of disinfectants on simian rotavirus (SA-11) were analyzed. SA-11 rotavirus replicates in cells causing cytopathic effect (CPE) and is similar in protein composition to cattle and swine RV. Preliminary experiments tested the temperature and pH sensitivity of SA-11 rotavirus. At pH 8.5, 45°C was the highest temperature at which no loss in viral titer was seen, and the virus was still infective following treatment at 65°C for 2 hrs. pH sensitivity tests were then conducted for two hours at 45°C, with pH 5 being the lowest and pH 8.5 being the highest at which no loss in titer was observed. Four proteases were then tested for effectiveness at inactivating SA-11 rotavirus at their pH optimal at 45°C. Alcalase was selected as the most efficient protease. Alcalase was found to inactivate SA-11 at 25°C, and pH 8.5 in 3 days, indicating that enzymes were relatively effective at lower temperatures. SA-11 rotavirus virus was then tested for sensitivity to pH at 25°C and 15°C in absence of enzyme. At pH 2, 25°C a ~4 log reduction was seen following 15 min of treatment, with viable virus still remaining after 8 hrs, at 15°C a ~1.75 log reduction was seen following 2 hrs, and a ~4 log reduction following 8 hrs of treatment. At pH 4 and 6, at 25°C and 15°C no effect on SA-11 titer was seen after 120 hrs treatment. The enzyme was then tested at 1.0% and 0.1% enzyme concentration, at 15°C and 25°C, and pH 6 to determine efficacy of enzyme at sub-optimal conditions. Following treatment with 1.0% Alcalase at 25°C a ~3.25 log reduction, and at 15°C, 1.0% Alcalase, a ~1.75 log reduction was seen at 120 hrs. At 15°C, 1.0% Alcalase a ~1.75 log reduction was seen at 120 hrs. Treatment with 0.1% Alcalase at 25°C, pH 6 resulted in ~2.25 log reduction after 120 hrs. At 15°C, 0.1% Alcalase a ~1.25 log reduction was seen following 120 hrs. The results showed that proteases, used as viral disinfectants, were not as effective at inactivating rotaviruses under simulated field conditions as originally hoped, nevertheless the ease of application and moderate but definite efficacy against rotaviruses may help reduce rotaviral infections and severity of clinical signs in young animals.
Evidence of Extrahepatic Sites of Replication of the Hepatitis E Virus in a Swine Model
Williams, Trevor Paul Emrys (Virginia Tech, 2001-05-01)
Hepatitis E virus (HEV) is the major cause of enterically transmitted non-A, non-B hepatitis in many developing countries, and is also endemic in many industrialized countries. Due to the lack of an effective cell culture system and a practical animal model, the mechanisms of HEV pathogenesis and replication are poorly understood. It has been speculated that HEV replicates in sites other than the liver. Since HEV is presumably fecal-orally transmitted it is unclear how the virus reaches the liver and extrahepatic replication could be a possible explanation. The recent identification of swine HEV from pigs affords us an opportunity to systematically study HEV replication in a swine model. We experimentally infected specific-pathogen-free (SPF) pigs with two strains of HEV: swine HEV and the US-2 strain of human HEV. Eighteen pigs (group 1) were each inoculated intravenously with swine HEV, nineteen pigs (group 2) with the US-2 strain of human HEV, and seventeen pigs (group 3) as uninoculated controls. To identify the potential extrahepatic sites of HEV replication using the swine model, two pigs from each group were necropsied at 3, 7, 14, 20, 27, and 55 days post inoculation (DPI). Thirteen different types of tissues and organs were collected from each necropsied animal. Reverse transcriptase PCR (RT-PCR) was used to detect the presence of positive strand HEV RNA in each tissue collected during necropsy at different DPIs. A negative strand-specific RT-PCR was standardized and used to detect the replicative, negative-strand of HEV RNA from tissues that tested positive for the positive strand RNA. As expected, positive strand HEV RNA was detected in almost every type of tissue at some time point during viremic period between 3 and 27 DPI. Positive-strand HEV RNA was still detectable in some tissues in the absence of serum HEV RNA from both swine and human HEV inoculated pigs. However, replicative, negative strand of HEV RNA was detected primarily in the small intestine, lymph nodes, colon, and liver. Our results demonstrate for the first time that HEV replicates in tissues other than the liver and that the gastrointestinal tract is also the target of virus infection. The data from this study may have important implications for HEV pathogenesis, xenotransplantation, and the development of an in vitro cell culture system for HEV.
Feline Leukemia Virus Detection in Corneal Tissues of Cats by Polymerase Chain Reaction and Immunohistochemistry
Herring, Ian Phillip (Virginia Tech, 1998-04-27)
Corneal transplantation carries a high rate of success in the domestic cat and is an indicated treatment for specific corneal diseases in this species. The potential for iatrogenic transmission of viral diseases is a well-recognized problem in human corneal transplantation programs and screening donors for certain diseases is routine. Feline leukemia virus (FeLV) is a common agent of disease in domestic cats and available blood tests are highly effective in identification of infected individuals. This study investigates the presence of FeLV within corneal tissues of FeLV infected cats. Seventeen cats were identified to be positive for serum p27 antigen by enzyme-linked immunosorbent assay (ELISA). Twelve of these individuals were found to be positive on peripheral blood by immunofluorescent antibody (IFA) testing. Seventeen ELISA negative cats were identified to serve as negative controls. Full thickness corneal specimens were collected from all subjects and analyzed for the presence of FeLV proviral DNA and gp70 antigen by polymerase chain reaction (PCR) and immunohistochemical (IHC) testing, respectively. Eleven (64.7%) positive corneal PCR results were obtained from 17 ELISA positive cats. Of 12 cats which were both ELISA and IFA positive on peripheral blood, 10 (83.3%) had positive corneal PCR results. All corneal tissues from ELISA negative subjects were PCR negative. IHC staining of corneal sections revealed the presence of FeLV gp70 in corneal tissues of nine (52.9%) ELISA positive cats. Of the 12 cats which were both ELISA and IFA positive on peripheral blood, 8 (66.7%) had positive corneal IHC results. Positive IHC staining was localized to the corneal epithelium. Corneal tissues of all ELISA negative cats and all IFA negative cats were negative on IHC testing. This study reveals FeLV to be present within the corneal epithelium of some FeLV infected cats. Screening potential corneal donors for this virus is warranted. This work was funded by grants from the American College of Veterinary Ophthalmologists, the Virginia Veterinary Medical Association Pet Memorial Fund, and the DSACS Quick Response Fund.
Generation of Baculovirus-Brucella Abortus Heat Shock Protein Recombinants; Mice Immune Responses Against the Recombinants, and B. Abortus Superoxide Dismutase and L7/L12 Recombinant Proteins
Bea, Joo-eun (Virginia Tech, 1999-02-05)
Brucella abortus is capable of resisting the microbicidal mechanisms of phagocytic cells and growing within phagocytic cells, usually macrophages. B. abortus, like several other intracellular bacteria responds to the hostile environment in macrophages by producing heat shock proteins (HSPs) which are induced by environmental stresses. Bacterial HSPs are very immunogenic, eliciting both cellular and humoral immune responses in the infected host. The significance of host cellular and protective immune responses directed against these proteins is currently unresolved. Baculovirus recombinants were generated in Sf9 insect cells for B. abortus HSPs and the protein expression was optimized. Humoral (Western blot), cell mediated (CMI, IFN-g- release by splenocytes, and CD3+CD4+, CD3+CD8+ T cell/ total splenocytes ratios) and protective immune responses of BALB/c mice (challenge with virulent B. abortus 2308) against these recombinants, against B. abortus superoxide dismutase (SOD) and ribosomal L7/L12 proteins, inoculated alone or in various combinations with complete Freund's, Ribi and recombinant IL-12 as adjuvants, were analyzed. Vaccinia virus-GroEL recombinant as priming immunogen, followed by baculovirus-GroEL-Ribi booster, was explored. Androstenediol, an immune up-regulator, was tested for its ability to induce resistance against challenge. None of the mice inoculated with individual, divalent or trivalent HSP-expressing Sf9 cells combined with Freund's were protected against challenge and the Sf9 cell-induced response masked the recombinant protein-specific CMI responses. Recombinant HSPs were purified and combined with Ribi. Although significant IFN-g release was induced by immunization with the HtrA-Ribi combination, no mice were protected against challenge. Priming with vaccinia virus-GroEl recombinant and boosting with purified baculovirus-GroEL protein-Ribi combination did not induce protection. Androstenediol did not enhance in vivo resistance to challenge. IL-12 alone did not activate splenocytes but induced significant IFN-g release in mice when combined with killed B. abortus RB51 vaccine, purified recombinant HtrA or purified SOD proteins, or L7/L12 expressing Escherichia coli cells. Significant protection was induced by SOD combined with IL-12. No correlation was seen between IFN-g release by splenocytes and protection against challenge in the SOD/IL-12-immunized mice. The results suggest that B. abortus HSPs are not highly immunogenic in mice and though various immune responses may be induced by one or another HSPs, protective immune response, unfortunately, is not among them. The results of this study reflect the difficulties in experimenting with immune responses against single or a limited number of recombinant B. abortus proteins. This is particularly true when the task includes induction of a protective immune response and finding significant correlation between different types of immune response assays.
Genetic and Immunological Analyses of a Brucella abortus Protein Exhibiting Lectin-like Properties
Vemulapalli, Tracy H. (Virginia Tech, 2000-01-28)
Brucella abortus is a facultative, intracellular zoonotic pathogen, which can cause undulant fever in humans and abortion in cattle. Despite all of the progress in brucellosis research, there are still many unanswered questions regarding the molecular mechanisms involved in the pathogenesis of Brucella infections. To better understand the Brucella antigens involved in virulence and/or immunity, genetic and immunologic characterization of a 16 kDa protein of B. abortus was performed. Using PCR methods, the gene encoding the 16 kDa protein was cloned and sequenced. PCR and Southern blot analysis revealed that the gene is conserved among the 6 nomen species of Brucella. Overexpression of this protein in B. abortus vaccine strain RB51 was achieved using Brucella groE and sodC promoters as well as its own promoter. Protection and clearance studies were performed in mice to determine the role of this protein in Brucella immunity and pathogenesis. Inoculation with either strain RB51 overexpressing the 16 kDa protein or a DNA vaccine encoding the 16 kDa protein gene failed to provide significant protection. No difference was noted between the splenic clearance of B. abortus strain 2308 and its recombinant overexpressing the 16 kDa protein. A mutant of strain 2308 (2308D16) was created by disrupting the 16 kDa protein's gene with a chloramphenicol resistance cassette. Western blot analysis indicated that the O antigen profile of strain 2308D16 differed from that of strain 2308. Mice cleared strain 2308D16 faster than strain 2308 indicating the potential attenuation of the disruption mutant. Purified 16 kDa protein was obtained by overexpressing it in E. coli via the pRSET expression system. Western blotting results initially identified this protein as an immunoglobulin-binding protein. Hemagglutination assay revealed that the 16 kDa protein exhibits lectin-like properties. Preliminary studies using hemagglutination inhibition identified mannose as a possible sugar to which the 16 kDa protein can interact. The lectin-like properties exhibited by the 16 kDa protein appears to influence smooth lipopolysaccharide production, and thereby may be involved in virulence.
Identification of genotype 3 hepatitis E virus (HEV) in serum and fecal samples from pigs in Thailand and Mexico, where genotype 1 and 2 HEV strains are prevalent in the respective human populations
Cooper, K.; Huang, Fang-Fang; Batista, L.; Rayo, C. D.; Bezanilla, J. C.; Toth, Thomas E.; Meng, Xiang-Jin (American Society for Microbiology, 2005-04)
Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important public health concern in many developing countries. Increasing evidence indicates that hepatitis E is a zoonotic disease. There exist four major genotypes of REV, and HEV isolates identified in samples from pigs belong to either genotype 3 or 4. Genotype 1 and 2 HEVs are found exclusively in humans. To determine whether genotype 1 and 2 HEVs also exist in pigs, a universal reverse transcription-PCR assay that is capable of detecting all four REV genotypes was used to test for the presence of REV RNA in serum and/or fecal samples from pigs in Thailand, where genotype 1 human REV is prevalent, and from pigs in Mexico, where genotype 2 human REV was epidemic. In Thailand, swine REV RNA was detected in sera from 10/26 pigs of 2 to 4 months of age but not in sera from 50 pigs of other ages. In Mexico, swine HEV RNA was detected in 8/125 sera and 28/92 fecal samples from 2-to 4-month-old pigs. Antibodies to swine REV were also detected in about 81% of the Mexican pigs. A total of 44 swine REV isolates were sequenced for the open reading frame 2 gene region. Sequence analyses revealed that all swine REV isolates identified in samples from pigs in Thailand and Mexico belong to genotype 3. Phylogenetic analyses revealed that minor branches associated with geographic origin exist among the swine REV isolates. The results indicated that genotype 1 or 2 swine HEV does not exist in pigs from countries where the respective human REV genotype I or 2 is prevalent. It is likely that only genotype 3 and 4 REV strains have zoonotic potential.
In vitro cytotoxic activity of equine lymphocytes on equine herpesvirus-1 infected allogenic fibroblasts
Edens, Lucy Marie (Virginia Tech, 1994-05-15)
The objectives of this study were to: 1) develop a technique to analyze the in vitro cytotoxic activity of lymphocytes from adult horses against equine herpes virus-1 (EHV-1) infected allogenic equine dermal fibroblasts (EDF); 2) evaluate the ability of a 72 hour in vitro incubation with interleukin-2 (I L-2) to enhance the lymphocytic cytolytic activity against EHV-1 infected EDF; 3) compare the cytotoxic activity among lymphocytes isolated from pregnant mares and non-pregnant mares against EHV-1 infected EDF; 4) ascertain if any correlations existed between the percent cytotoxicity and percentage of lymphocytes phenotypically identified by five different mouse-anti-equine monoclonal antibodies; and 5) determine if any correlation existed between virus-neutralizing antibody titers and the percent cytotoxicity. Results of the study indicate that in vitro cytotoxic activity of equine lymphocytes against EHV-1 infected allogenic fibroblasts can be measured with a standard 4 hour 51Cr release assay. This activity was enhanced by an in vitro incubation with IL-2. The cytolytic activity of freshly isolated lymphocytes was greater for non-pregnant than pregnant mares. However, after IL-2 stimulation the cytolytic activity was greater for lymphocytes from pregnant mares. A positive correlation was not detected between the percentage of phenotypically identified cells and the percent cytoxicity, although several negative correlations were present. This suggests that the cytotoxic activity was either not mediated by any of the phenotypically identified cell populations or that the activity was mediated by several different cell populations. No correlation was detected between virus neutralizing antibody titers and the percent cytotoxicity.
Investigation of Haemophilus somnus Virulence Factors: Lipooligosaccharide Sialylation and Inhibition of Superoxide Anion Production
Howard, Michael D. (Virginia Tech, 2005-04-05)
Virulent strains of the bovine opportunistic pathogen Haemophilus somnus (Histophilus somni) cause multi-systemic diseases in cattle. One of the reported virulence factors that H. somnus may use to persist in the host is resistance to intracellular killing. It is reported in this dissertation that H. somnus significantly (P <0.001) inhibited production of superoxide anion (O2-) by bovine mammary and alveolar macrophages as well as by polymorphonuclear leukocytes. Inhibition of O2- production was time- and dose-dependent and did not occur after incubation with Escherichia coli, H. influenzae, or Brucella abortus. Non-viable H. somnus, purified lipooligosaccharide (LOS), or cell-free supernatant from mid-log phase cultures did not inhibit O2- production, indicating that O2- inhibition required contact with live H. somnus. Commensal isolates of H. somnus were less capable or incapable of inhibiting macrophage O2- production compared to isolates tested from disease sites. H. somnus shares conserved epitopes in its LOS with Neisseria gonorrhoeae, N. meningitidis, and H. influenzae, and can also undergo structural phase variation of these LOS epitopes. Sialylation of the terminal galactose of H. somnus LOS is another reported virulence mechanism. Current sequencing of the genomes of H. somnus strains 2336 (pathogenic) and 129Pt (commensal) has enabled in silico identification of three open reading frames (ORFs) involved in sialylation. The ORFs-1 (hsst-I) and -2 (hsst-II) had BLASTx homology to sialyltransferases, while ORF-3 (neuAhs) had BLASTx homology to CMP-sialic acid synthetases. These ORFs were amplified by PCR and cloned into the expression vector pCWOri+. Thin layer chromatography of the hsst-I gene product showed this sialyltransferase exhibited preference for sialylation of terminal N-acetyllactosamine (LacNAc, beta-Gal-[1,4]-beta-GlcNAc-R). However, Hsst-II preferentially sialylated lacto-N-biose (LNB, beta-Gal-[1,3]-beta-GlcNAc-R). In this study, phase variation of the terminal linkage in isolate 738 from a 3 linked galactose (LNB) to a 4 linked galactose (LacNac) was demonstrated. Such variation of a glycose linkage appears to be a novel mechanism of LOS phase variation. Furthermore, the ability of sialylated strain 738 LOS vs de-sialylated strain 738 LOS to induce Toll-like receptor 4 signaling was decreased by 28%, as determined by ELISA for Macrophage Inflammatory Protein-2. Therefore, sialylated LOS may aid H. somnus to avoid host innate immunity.
Mechanism of Pathogenesis and Replication of an Avian Strain of the Hepatitis E Virus in a Chicken Model
Billam, Padma (Virginia Tech, 2007-04-16)
Hepatitis E is an acute, enterically transmitted disease of public health importance. The mechanism of pathogenesis of HEV is poorly understood due to the lack of an in vitro cell culture system and an ideal animal model system. With the discovery of avian HEV and its association with a hepatic disease (Hepatitis-Splenomegaly syndrome), chickens provide an excellent small homologous animal model system to study this important virus. The objectives of this dissertation were to utilize chickens as a model system to study the pathogenesis and replication of avian HEV under the natural route of infection, to identify potential extrahepatic replication sites, to determine and analyze the complete genomic sequence of the avirulent strain of avian HEV, and to study the compartive pathogenesis of the two isolates of avian HEV, the prototype pathogenic and avirulent strains of avian HEV. We attempted to experimentally infect specific-pathogen-free (SPF) adult chickens by the natural fecal-oral route in order to systematically study HEV pathogenesis and replication and to characterize the clinical course and pathological lesions associated with avian HEV infection. Sixty-week-old, specific-pathogen-free (SPF) chickens were inoculated with 5 x104.5 50% chicken infectious dose of avian HEV by oronasal route and IV route. All oronasally- and IV- inoculated chickens had seroconverted to avian HEV antibodies and fecal virus shedding was detected variably from 1 to 20 DPI in the IV group, and from 10 to 56 DPI in the oronasal group. Avian HEV RNA was detected in serum, bile, and liver samples earlier during the course of infection in IV-inoculated chickens than in oronasally-inoculated ones. Gross liver lesions including subcapsular hemorrhages and enlargement of right intermediate lobe and microscopic hepatic lesions in the liver characterized by lymphocytic periphlebitis and phlebitis were observed in inoculated chickens. This is the first report of experimental HEV infection via its natural route in a homologous animal model system. Very little is known about HEV pathogenesis and it has been hypothesized that HEV replicates in tissues other than liver. The replicating negative-strand viral RNA was detected by negative-strand-specific RT-PCR in liver, serum, colon, cecum, jejunum, ileum, duodenum and cecal tonsils,but not in other non-GIT tissues. Immunohistochemistry using an avian HEV capsid protein-specific anti-peptide antibody revealed positive signal in liver and GIT tissues including colon, jejunum, ileum, cecum, cecal tonsils and pancreas. The detection of avian HEV capsid antigen and replicative negative-strand viral RNA in the GIT tissues indicates that HEV replicates in the GI tract following infection by fecal-oral route. The complete genomic sequence of an avirulent strain of avian HEV was determined using primer walking strategy. The full-length genome of the avirulent strain is 6649 nts in length and has a nucleotide sequence identity of 90.1% with the prototype pathogenic strain. Numerous non-silent mutations were observed in ORF1, the region coding for the nonstructural proteins. Six unique non-silent mutations were identified in the capsid-encoding ORF2 region and the ORF3 had four non-silent mutations. Phylogenetic analysis based on full-length genomic sequence revealed that the avirulent strain is clustered together with the pathogenic avian HEV and represents a branch distinct from mammalian HEVs. In order to study the comparative pathogenesis between the pathogenic and avirulent strains of avian HEV, an infectious stock of the avirulent avian HEV was generated and infectivity titer was determined to be 5 x 102.5 CID50 per ml by experimentally infecting young SPF chickens. Six-week-old SPF chickens were inoculated with one of two strains of avian hepatitis E viruses, pathogenic avian HEV recovered from a chicken with HS syndrome and avirulent avian HEV isolated from a healthy chicken to study comparative pathogenesis. Most of the chickens seroconverted by 3 wpi in both pathogenic avian HEV and avirulent avian HEV groups. Avian HEV RNA was detected in feces and serum of the chickens from both the inoculated group from 1 wpi. Microscopic liver lesions included lymphocytic periphlebitis and phlebitis the overall hepatic lesion mean score was higher for the pathogenic avian HEV group compared to the avirulent avian HEV and control groups, suggestive of attenuation In summary, SPF chickens were experimentally infected with avian HEV by natural route to study the systematic pathogenesis and replication. Non-liver replication sites of avian HEV were also identified in a chicken model. The complete genomic sequence of an apparently avirulent strain of avian hepatitis E virus was determined and the comparative pathogenesis of avian hepatitis E virus isolates from a chicken with HS syndrome and from a healthy chicken was also studied by experimental infections in young SPF chickens. The results from this dissertation research have important implications for the understanding of HEV pathogenesis.
The Modulating Effects of Dietary Fiber and Short-Chain Fatty Acids on Enterocyte Differentiation, Maturation and Turkey Coronavirus Infection
Tirawattanawanich, Chanin (Virginia Tech, 2001-05-17)
In a number of mammalian species, susceptibility to enteric coronavirus infection has been shown to be age-related. This is thought to be associated with enterocyte maturation and receptor protein expression. One of the factors that can influence differentiation and maturation of enterocytes is the availability of short-chain fatty acids (SCFA) in the intestinal lumen. These compounds are by-products of the bacterial fermentation of dietary fiber and serve as the primary energy source for enterocyte metabolism. The overall objective of this dissertation was to evaluate the effects of dietary fiber and short-chain fatty acids on enterocyte differentiation, maturation, and susceptibility to coronavirus infection in turkeys. Initial work involved the development of an indirect immunoperoxidase assay (IPA) for the identification and localization of turkey coronavirus (TCV) in paraffin-embedded, acid-ethanol fixed tissue. IPA was found to be superior to indirect immunofluorescent antibody test (IFA) for this and other diagnostic purposes. To evaluate cellular differentiation and maturation, an SDS-PAGE/immunoblot technique was developed to determine relative levels of villin expression in turkey embryos. Villin is an actin-bound cytoskeletal protein known to be expressed in increasing quantities at the apical surfaces of maturing enterocytes. Villin expression level was found to increase linearly as a function of embryo age. Villin localization was performed by IPA on paraffin-embedded, acid-alcohol fixed tissue. As enterocytes (embryos) matured, villin was found to concentrate at the apical surfaces and eventually at the basolateral membranes. Experiments were also conducted to see what effect in ovo butyrate administration would have on developing embryonic enterocytes. Butyrate has been shown to enhance differentiation of non-neoplastic and neoplastic cells in culture as well as promote healing of damaged intestinal epithelium in human. Villin expression was significantly enhanced in embryos receiving 0.2 and 0.3 M butyrate 36 hours post-administration. Butyrate appeared to enhance villin expression and therefore enterocyte maturation in a dose-dependent manner. Susceptibility of turkey embryos to TCV infection as a function of age and butyrate treatment was investigated as well as epithelial localization of TCV infection in poults. The level of TCV infection of epithelium was found to increase with embryo age between 17 and 23 days. Poults showed higher levels of infection on the distal 2/3 of villi and no evidence of infection in the intestinal crypts. Butyrate administration in 21-day-old embryos followed by TCV inoculation caused a significant increase of the number of infected cells per villus. This data suggested that butyrate might be used as a means to manipulate enterocyte susceptibility to TCV infection. In the final set of experiments, the effects of fiber-fortified poult diets containing 5% cellulose or 5% guar gum on luminal SCFA levels, enterocyte maturation, and TCV infection were investigated. SCFA levels in cecal contents were determined by gas chromatography. Enterocyte maturation was assessed by the determination of villin expression on immunoblot and the severity of TCV infection was determined by IPA and lesion score. Fiber-fortified diets enhanced SCFA production and villin expression, but contrary to embryo studies, TCV infection appeared to be reduced. In general, poults performed better on the diet containing cellulose. Mechanisms regarding the roles of dietary fiber and SCFA in enterocyte differentiation, maturation, and TCV susceptibility are proposed as well as future directions for research. The in ovo and poults system used in this research may serve as models for further investigation of the influences of host and dietary factors on enteric viral infection and recovery.
Molecular and Serological Epidemiology of Swine Hepatitis E Virus from Pigs in Two Countries
Cooper, Kerri Lee (Virginia Tech, 2004-07-14)
Hepatitis E virus (HEV), the causative agent of hepatitis E, is endemic in many developing countries. However, sporadic cases of acute hepatitis E have also been reported in industrialized countries including the United States. Increasing evidence suggested that hepatitis E is zoonotic. Swine HEV was discovered in 1997 from a pig in the United States and has the ability to cross species barrier and infect humans. There are four major genotypes of HEV worldwide and swine HEV identified to date in different countries belongs to either genotypes 3 or 4. Thus far, genotypes 1 (Asian strains) and 2 (a single Mexican strain) of HEV are exclusively found in humans. To determine if genotypes 1 and 2 of HEV also exist in pigs we tested serum and/or fecal samples for from pigs of different age groups in Thailand, and from pigs 2-4 months-of-age in two states (Sonora, Sinaloa) in Mexico. A universal RT-PCR was first standardized to detect all 4 different genotypes of HEV. Swine HEV RNA was detected from in 10/26 pigs at 2-4 months-of-age but not in pigs of 1-, 6-month old, adult/sow pigs from Thailand. In Mexico, swine HEV RNA was detected in 8 of 125 serum samples, 28 of 92 fecal samples of 2-4 month-old pigs. Antibodies to swine HEV were detected in 101 of 125 (80.8%) Mexican pigs. A total of 44 swine HEV isolates were amplified and sequenced for the ORF2 capsid gene region. Sequence analyses revealed that all the swine HEV isolates identified from pigs in Thailand and Mexico belong to genotype 3. Overall, the Mexican swine HEV isolates shared 89-100% sequence identity to each other, and about 89-92% identity with the prototype genotype 3 US swine HEV. The Thailand swine HEV isolates displayed 97-100% nucleotide sequence identity with each other, and 90-91% identity with the prototype genotype 3 swine HEV. Phylogenic analysis revealed that minor branches do exist among Mexican swine HEV isolates. The results from this study indicated that genotype 1 or 2 swine HEV does not exist in pig from countries where human genotypes 1 and 2 HEVs are prevalent.
Molecular Characterization of Animal Strains of Hepatitis E Virus (HEV): Avian HEV and Swine HEV
Huang, Fang-Fang (Virginia Tech, 2004-12-01)
Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important public health concern in many developing countries. It mainly infects young adults and has a mortality of up to 25% in pregnant women. Although hepatitis E is only sporadic in industrialized countries including the United States, a relative high seroprevalence rate has been reported in healthy individuals. Evidence suggests that there exist animal reservoirs for HEV and HEV transmission is zoonotic. Animal strains of HEV, swine HEV and avian HEV have been identified from a pig and a chicken, respectively, in the United States. Studies showed that swine HEV and avian HEV are genetically and antigenically related to human HEV, and that pigs and chickens are useful animal models to study HEV replication, pathogenesis and cross-species infection. The objectives of this dissertation were to genetically characterize both avian HEV and swine HEV, to determine their serological and molecular epidemiology in the United States, to assess the ability of avian HEV cross-species infection in non-human primates, to determine the full-length genomic sequence and genome organization, and to construct an infectious cDNA clone of avian HEV. The prevalence of swine HEV infections in US swine herds and the heterogeneity of swine HEV isolates from different geographic regions of the United States were determined. We found that 35% pigs and 54% swine herds were positive for swine HEV RNA. Partial capsid gene region of twenty-seven US swine HEV isolates was sequenced and was showed to share 88%-100% nucleotide sequence identity to each other and 89-98% identity with the prototype US swine HEV, but only <79% identity with Taiwanese swine HEV isolates and most known human strains of HEV worldwide. All US swine HEV isolates belong to the same genotype 3 with the prototype US swine HEV and the two US strains of human HEV. Similarly, the prevalence of avian HEV infections in US chicken flocks and the heterogeneity of avian HEV isolates were also determined. Helicase gene region of eleven field isolates of avian HEV from chickens with hepatitis-splenomegaly (HS) syndrome was sequenced and was found to share 78-100% nucleotide sequence identities with each other, 79-88% identities with the prototype avian HEV, 76-80% identities with Australian chicken big liver and spleen disease virus (BLSV), and 56-61% identities with other known strains of mammalian HEV. A relative high prevalence of anti-avian HEV antibodies was found in apparently healthy chicken flocks in 5 states. Like swine HEV, the seropositivity of avian HEV in adult chickens was higher than that in young chickens. To genetically characterize the avian HEV genome, we determined the full-length genomic sequence of avian HEV, which is 6,654 bp in length excluding the poly (A) tail, and 600 bp shorter than that of mammalian HEVs. Avian HEV has similar genomic organization with human and swine HEVs, but shared only about 50% nucleotide sequence identity with mammalian HEVs in the complete genome. Significant genetic variations such as deletions and insertions, particularly in the ORF1 of avian HEV, were observed, but motifs in the putative functional domains of the ORF1 were relatively conserved between avian HEV and mammalian HEVs. Phylogenetic analyses based on the full-length genomic sequence revealed that avian HEV represents a branch distinct from human and swine HEVs. Since swine HEV infects non-human primates and possibly humans, the ability of avian HEV cross-species infection in non-human primates was also assessed. However, unlike swine HEV, avian HEV failed to infect two rhesus monkeys under experimental conditions. With the availability of the complete genome sequence of avian HEV, we constructed three full-length cDNA clones of avian HEV and tested their infectivity by in vitro transfection of the LMH chicken liver cells and by in vivo intrahepatic inoculation of specific-pathogen-free (SPF) chickens. The results showed that all 3 cDNA clones of avian HEV were infectious both in vitro and in vivo, as the capped RNA transcripts from each of the clones were replication-competent in transfected LMH cells and developed active infection in inoculated SPF chickens. In summary, avian HEV and swine HEV infections are enzootic in chicken flocks and in swine herds in the United States, respectively. Like human HEV, swine HEV and avian HEV isolates from different geographic regions are also genetically heterogenic. Complete genomic sequence analyses showed that avian HEV is related to, but distinct from, human and swine HEVs. Unlike swine HEV, avian HEV is probably not transmissible to non-human primates. Infectious cDNA clones of avian HEV have been successfully constructed. The availability of the infectious clones for a chicken strain of HEV now affords us an opportunity to study the mechanisms of HEV replication, pathogenesis and cross-species infection.
Molecular characterization of the major envelope protein of porcine reproductive and respiratory syndrome virus (PRRSV) and evaluation of its use for a diagnostic assay, vaccine development, and the examination of quasispecies evolution
Key, Kijona Farthing (Virginia Tech, 2007-04-19)
Porcine reproductive and respiratory syndrome (PRRS) is a viral disease that has devastated the global swine industry since the mid 1980s. Although modified live vaccines (MLVs) are typically used for the prevention of clinical disease, they are not always fully effective. Additionally, acute PRRS outbreaks, characterized by more severe clinical signs, have appeared in herds that were previously vaccinated. In this dissertation, we further analyzed the pathogenesis of PRRSV through genetic characterization, assay development, and quasispecies evaluation using the PRRSV ORF5 gene while also attempting to develop an improved PRRS vaccine. To explore the possible mechanism for the emergence of acute PRRS, the open reading frame 5 (ORF5) gene encoding the major envelope protein (GP5) of acute PRRSV isolates was characterized. Sequence and phylogenetic analyses revealed that seven of the acute PRRS virus (PRRSV) isolates were related to other N. American PRRSV isolates while one isolate, 98-37120-2, was very closely related to and may have been derived from the MLV, RespPRRS. We also developed a heteroduplex mobility assay (HMA) for quickly identifying PRRSV field isolates with significant nucleotide sequence identities (â d98%) with the MLVs based on the amplification, denaturation, and reannealing of the ORF5 gene of the field isolates with those of MLV reference strains. All of the field isolates that were highly related to RespPRRS (â T2% nucleotide sequence divergence) were identified by the HMA to form homoduplexes with the reference RespPRRS MLV. We also developed a unique strategy for infecting pigs with PRRSV, known as in vivo transfection, by bypassing the traditional in vitro cell culture step required for in vivo studies. We demonstrated that inoculation of RNA transcripts of a PRRSV infectious cDNA clone directly into the lymph nodes and tonsils of pigs produces active PRRSV infection. Using this method, we also examined the quasispecies populations of PRRSV. Finally, we evaluated the ability of Salmonella choleraesuis to express the PRRSV GP5, and tested its immunogenicity in mice. Based on our data, there was no indication of Salmonella replication in the mice or any evidence of antibody production against S. choleraesuis or PRRSV GP5.
Molecular mechanisms of porcine circovirus 2 replication and pathogenesis
Juhan, Nicole McKeown (Virginia Tech, 2007-04-20)
The non-pathogenic porcine circovirus type 1 (PCV1) was originally isolated as a persistent contaminant of the porcine kidney cell line PK-15. Whereas, porcine circovirus type 2 (PCV2) causes postweaning multisystemic wasting syndrome (PMWS) in pigs, which is devastating to the swine industry. My objectives were to determine the effect of maternally derived antibodies on PCV2 infection, assess the role of 2 amino acid substitutions in the PCV2 capsid protein in PCV2 attenuation, evaluate the effect of Rep gene exchange between PCV1 and PCV2 on growth characteristics of a chimeric PCV2, and evaluate the role of open reading frame (ORF) 3 of PCV2 in virus replication and pathogenesis in pigs. Under field conditions, PCV2 infection is widespread and most breeding pigs are seropositive. Assessment of the role of PCV2 maternal antibodies in preventing PCV2 infection in piglets provided evidence that higher levels of maternal antibody provide more protection to piglets. Two amino acid substitutions in the PCV2 capsid protein that enhanced virus replication in vitro and attenuated the virus in vivo were evaluated for their pathogenicity in pigs. The results indicated that P110A and R191S are collectively responsible for virus attenuation. PCV1 replicates better in PK-15 cells and grows at least 1-log titer higher than PCV2. A chimeric PCV with the rep gene of PCV1 replacing that of PCV2 in the genomic backbone of PCV2 replicated more rapidly than PCV1 and PCV2, and more efficiently than PCV2, although to a titer similar to PCV1. The ORF3 of PCV2 is believed to encode a protein involved in apoptosis. The ORF3 start codon was mutated from ATG to GTG and the resulting mutant muPCV2 was infectious in vitro and in pigs; therefore ORF3 is dispensable for virus replication. The pathogenicity of muPCV2 was compared with PCV2 in vivo. Delayed viremia and seroconversion, decreased viral loads, lower level of IgG antibodies, and lower amounts of PCV2 antigen in mesenteric lymph nodes suggested attenuation of muPCV2. However, there was no significant difference in histological or gross lesions in tissues between PCV2- and muPCV2-inoculated groups. The role of ORF3 in attenuation needs to be further elucidated.
Molecular Pathogenesis and Development of a Genetically Engineered Vaccine for Type-2 Porcine Circovirus
Fenaux, Martijn (Virginia Tech, 2004-03-26)
Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), whereas the ubiquitous porcine circovirus type 1 (PCV1) is nonpathogenic for pigs. Since its initial detection in a Canadian commercial swine herd in 1991, PMWS has been detected in all swine producing regions of the world and is now a serious economic problem to the swine industry. The objectives of this dissertation were to biologically, genetically and experimentally characterize both PCV1 and PCV2, to identify the genetic determinant(s) for virulence and replication, and to develop an effective genetically-engineered vaccine against PCV2 infection and PMWS. The genetic heterogeneity of PCV2 and PCV1 isolates from different geographic origins were determined. We found that, although PCV1 and PCV2 genomes were very conserved, some minor genomic variation exists among PCV1 isolates and PCV2 isolates. The nonpathogenic PCV1 and pathogenic PCV2 share only about 76% nucleotide sequence identity but have similar genomic organization. The highest sequence variability among PCV isolates is found in the immunogenic ORF2 capsid gene. Based on the sequence data in this dissertation, a universal polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay was developed that is capable of detecting all known PCV isolates and differentiating between infections by nonpathogenic PCV1 and pathogenic PCV2. In order to study the structural and functional relationship of PCV genes and to develop a genetically-engineered vaccine, we constructed infectious DNA clones of both PCV1 and PCV2. By using the PCV2 infectious clone, we showed that pigs can be infected by direct intrahepatic injection of PCV2 infectious DNA clone. The pathological lesions and clinical disease associated with PCV2 infection were more definitively characterized by using the infectious DNA clone. We found that PCV2 is the primary but not the sole causative agent of PMWS, as the full spectrum of clinical PMWS was not reproduced by the infectious PCV2 DNA clone although pathological lesions characteristic of PMWS were reproduced. A chimeric vaccine was constructed by cloning the immunogenic capsid gene of the pathogenic PCV2 into the genomic backbone of the non-pathogenic PCV1 virus. We showed that the resulting chimeric PCV1-2 vaccine virus, retained the non-pathogenic nature of PCV1 but induced a protective immune response against a wild-type PCV2 challenge. In vaccinated pigs, the chimeric PCV1-2 vaccine reduced PCV2 viremia length and serum virus loads and reduced pathological lesions such as lymphoid depletion (LD) and histiocytic replacement (HR) in lymphoid tissues, inflammation and discoloration of the lymph nodes. The amounts of PCV2 antigen and PCV2 genomic copy loads in lymph node tissues were also significantly reduced. Our results indicated that the attenuated chimeric PCV1-2 virus induces protective immunity against PCV2 infection and thus could serve as an effective vaccine against PCV2 and PMWS. To improve the safety of the vaccine, we attempted to identify the genetic determinant(s) for PCV2 virulence. An isolate of PCV2 was serially passaged for 120 times in PK-15 cells. After 120 passages, a total of two amino acid mutations were identified in the capsid protein of the passage 120 virus (VP120), P110A and R191S. Compared to other known PCV1 and PCV2 sequences, the two amino acid mutations in PCV2 VP120 are unique. The VP120 virus was biologically characterized in vitro and experimentally characterized in specific-pathogen-free (SPF) pigs. The two amino acid mutations resulted in an enhanced replication ability of PCV2 VP120 in PK-15 cells and an attenuated phenotype in infected pigs. The P110A and R191S mutations in the capsid protein either alone or collectively are likely important for PCV2 virulence and replication. In summary, we genetically characterized PCV2 isolates from different geographic regions and developed a PCR-RFLP assay. We constructed and characterized infectious DNA clones of PCV1 and PCV2, and developed a genetically engineered vaccine against PCV2 infection. We also identified the genetic determinants for PCV2 virulence and replication. The vaccine developed in this study, when it becomes available, will help the swine industry control this important pathogen.

Browsing by Author "Toth, Thomas E."

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