Evaluation of an African swine fever (ASF) vaccine strategy incorporating priming with an alphavirus-expressed antigen followed by boosting with attenuated ASF virus.

In this study, an alphavirus vector platform was used to deliver replicon particles (RPs) expressing African swine fever virus (ASFV) antigens to swine. Alphavirus RPs expressing ASFV p30 (RP-30), p54 (RP-54) or pHA-72 (RP-sHA-p72) antigens were constructed and tested for expression in Vero cells and for immunogenicity in pigs. RP-30 showed the highest expression in Vero cells and was the most immunogenic in pigs, followed by RP-54 and RP-sHA-p72. Pigs primed with two doses of the RP-30 construct were then boosted with a naturally attenuated ASFV isolate, OURT88/3. Mapping of p30 identified an immunodominant region within the amino acid residues 111-130. However, the principal effect of the prime-boost was enhanced recognition of an epitope covered by the peptide sequence 61-110. The results suggest that a strategy incorporating priming with a vector-expressed antigen followed by boosting with an attenuated live virus may broaden the recognition of ASFV epitopes.

Persistence and Tissue Distribution of Infectious Bursal Disease Virus Serotypes 1 and 2 in Turkeys.

Two experiments were conducted to determine the persistence and tissue distribution of serotypes 1 and 2 of infectious bursal disease virus (IBDV) in specific-pathogen-free and vaccinated turkeys. In Experiment 1, three groups of 2-wk-old turkey poults, including a negative control group, were used. In groups 1 and 2, 13 poults in each group were challenged with either serotype 1 (STC) or serotype 2 (OH) strains using an inoculum of 10(4) 50% embryo infectious dose (EID50)/0.2 ml/bird. Thymus, bursa, spleen, kidney, lungs, liver, pancreas, caecum, and breast and thigh muscles were sampled at predetermined intervals. The bursal tissues from birds inoculated with either serotype were reverse transcriptase-PCR (RT-PCR) positive up to 21 days postinoculation (DPI). In both groups virus isolation from bursas was possible up to 14 DPI. Except for the bursas and spleens in birds inoculated with serotype 1 and bursas in birds inoculated with serotype 2, all other tissues were RT-PCR negative. In Experiment 2, five groups of turkey poults were used. At 4 wk of age, group 1 was challenged with a serotype 1 STC strain and group 2 with serotype 2 OH strain using an inoculum size of 10(2) EID50/0.2 ml for both serotypes. Groups 3 and 4 were vaccinated at 2 wk of age using an inactivated serotype 1 IBDV vaccine. At 2 wk postvaccination, groups 3 and 4 were challenged with STC and OH strains respectively. From group 1, bursal, spleen, and liver tissues were RT-PCR positive up to 14 DPI; breast muscle and kidney tissues were positive up to 7 DPI; and lungs and pancreatic tissues were positive up to 3 DPI. From group 2, bursal tissues were RT-PCR positive up to 14 DPI and lung tissues up to 3 DPI. All of the tissue samples collected from groups 3, 4, and 5 were RT-PCR negative. Virus could not be isolated from RT-PCR positive bursal homogenate. In this work, it was confirmed that the virus persisted in the bursa longer than in any other tissues. The difference in the results between Experiments 1 and 2 could be due to the age of poults at vaccination and the higher inoculum size used in Experiment 1. This study indicates that turkeys are more resistant to IBDV as compared to chickens. Viruses of serotypes 1 and 2 infect turkeys and persist in bursal tissue for 14 days and RNA was detected up to 21 days.

Persistence and tissue distribution of infectious bursal disease virus in experimentally infected SPF and commercial broiler chickens.

This study was initiated to determine the persistence, distribution, and quantification of infectious bursal disease virus (IBDV) in lymphoid and nonlymphoid tissues of specific-pathogen-free (SPF) and commercial broiler chickens. Two serotype 1 strains, STC classic and IN variant, were independently used in the experiments. Five separate experiments were conducted using 2- and 4-wk-old SPF chickens, 2- and 4-wk-old in ovo-vaccinated commercial broilers, and 2-wk-old commercial broilers having maternally derived anti-IBDV antibodies. Pooled data from five experiments revealed that SPF chickens had a significantly higher incidence of IBDV-positive reverse transcriptase PCR (RT-PCR) results than commercial chickens (multivariable logistic regression, adjusted odds ratio = 15.28; 95% confidence limits [CL] = 9.53, 24.51, P < 0.0001). In many cases, the viral RNA (vRNA) persisted longer in in ovo-vaccinated commercial broilers bearing maternally derived antibodies compared with similar broilers not vaccinated in ovo. The STC strain was more frequently detected in tissues than the IN strain (chi-square P < 0.0001). In lymphoid tissues, STC and IN strains were detected for the longest duration in bursal tissues followed by spleen, thymus, and bone marrow. In nonlymphoid tissues, STC and IN strains were detected the longest in cecum followed by liver, kidney, pancreas, lungs, thigh, and breast muscles. Compared with bursal tissues, muscle and bone marrow tissues were significantly less likely to yield an IBDV-positive RT-PCR result (P < 0.0001). Although STC vRNA was detected up to 42 days postinoculation (DPI) in bursal homogenates of SPF chickens, virus isolation from bursal homogenates using embryonated chicken eggs was only possible up to 28 DPI. Similarly, STC vRNA was detected up to 42 DPI in bursal tissues of commercial broilers, but infectious virus could be isolated only up to 21 DPI. The IN strain was isolated up to 10 DPI from bursal homogenates of SPF chickens and broilers, but vRNA was detected up to 35 DPI in SPF chickens and 21 DPI in broilers. This study emphasizes that the detection ofvRNA is not indicative of the presence of infectious virus, and virus isolation has to be performed to prove the presence of infectious virus.

Efficacy of the transfluthrin-based personal insect repellent kit (PIRK) against the ixodid ticks Ixode s scapularis, Amblyomma americanum and Dermacentor variabilis.

An assay series was performed to assess the contact and spatial efficacy of the Personal Insect Repellent Kit (PIRK) against three species of ixodid ticks. The PIRK, a portable, passive device comprised of an inert physical substrate incorporated with the active ingredient (AI) transfluthrin (TF), has demonstrated spatial efficacy against flying insects, including three species of mosquitoes, sand flies and stable flies. The device is the only TF end-use product registered with the EPA. Here we report the first studies to explore potential of the PIRK to control Ixodes scapularis, Amblyomma americanum and Dermacentor variabilis. Dose-response assays confirmed toxicity of TF to larvae of all species in the µg/ml range following a 30-min exposure period. Nymphs and adults exhibited irritancy and avoidance behaviors on contact with the PIRK. Greater than 90% knockdown (KD) of I. scapularis nymphs and adults was observed after a 10-s exposure, and of A. americanum nymphs and adults after 10-s and 120-s exposure, respectively. Additionally, greater than 90% mortality was observed in I. scapularis nymphs and adults after 10-s and 40-s exposure, respectively. In spatial assays, the PIRK caused KD and post-exposure mortality of adult female I. scapularis exposed at a range of 5-28 cm. These results suggest both contact and spatial capacity of the PIRK, with greatest potency to nymphs versus adults and the prostriate tick I. scapularis versus the metastriate species A. americanum and D. variabilis. Future studies will explore spatial activity at a range of distances and exposure times, in the presence and absence of host cues and under semi-field conditions.

Differential modulation of cytokine, chemokine and Toll like receptor expression in chickens infected with classical and variant infectious bursal disease virus.

Infectious bursal disease (IBD) is an important immunosuppressive disease of chickens. The causative agent, infectious bursal disease virus (IBDV), consists of two serotypes, 1 and 2. Serotype 1 consists of classic IBDV (cIBDV) and variant IBDV (vIBDV). Both of these strains vary in antigenicity and pathogenesis. The goal of this study was to compare the immunopathogenesis of cIBDV and vIBDV. Three-week-old specific pathogen free chickens were inoculated intraocularly with standard challenge strain (STC) (cIBDV) and a variant strain Indiana (IN) (vIBDV). The cIBDV produced more pronounced bursal damage, inflammatory response and infiltration of T cells as compared to vIBDV. There were significant differences in the expression of innate (IFN-α and IFN-ß), proinflammatory cytokine and mediator (IL-6 and iNOS) in cIBDV- and vIBDV-infected bursas. The expression of chemokines genes, IL-8 and MIP-α was also higher in cIBDV-infected chickens during the early phase of infection. The expression of Toll like receptor 3 (TLR3) was downregulated at post inoculation days (PIDs) 3, 5, and 7 in the bursas of vIBDV-infected chickens whereas TLR3 was upregulated at PIDs 3 and 5 in cIBDV-infected bursas. In vIBDV-infected bursa, TLR7 expression was downregulated at PIDs 3 and 5 and upregulated at PID 7. However, TLR7 was upregulated at PIDs 3 and 7 in cIBDV-infected bursas. The expression of MyD88 was downregulated whereas TRIF gene expression was upregulated in cIBDV- and vIBDV-infected bursa. These findings demonstrate the critical differences in bursal lesions, infiltration of T cells, expression of cytokines, chemokines and TLRs in the bursa of cIBDV-and vIBDV-infected chickens.

Antigenic regions of African swine fever virus phosphoprotein P30.

African swine fever (ASF) is one of the most complex and lethally haemorrhagic viral diseases of swine, affecting all breeds and ages of pigs. In the absence of ASF vaccines, reliable laboratory diagnosis and restricted biosecurity are critical for disease prevention and control. A detection of ASF-specific antibodies in an unvaccinated pig is a good marker for the diagnosis of ASF. The immunoperoxidase test (IPT) is a sensitive test for detecting ASF virus (ASFV) antibodies. However, due to the complexity of the procedure, the IPT is only suitable to be used as a confirmatory test. The ASFV p30 protein-based enzyme-linked immunosorbent assay (ELISA) is widely used for ASFV antibody screening, but the sensitivity is not comparable to the IPT. It is essential to have a better understanding of the antigenic properties of ASFV p30 to improve p30-based serologic tests. In this study, we developed a panel of 21 monoclonal antibodies (mAbs) against ASFV p30. With 14 out of the 21 mAbs, we defined 4 antigenic regions that contain at least 4 linear epitopes. Nine of the 14 mAbs mapped to antigenic regions 3 and 4 reacted with p30 in all serologic methods tested in this study, such as indirect immunofluorescence assay (IFA), ELISA and Western blot. The antigenic regions 3 and 4 are highly conserved and immunodominant in host antibody response. These mAbs and the defined p30 antigenic regions 3 and 4 provide valuable tools for the development and improvement of ASF serologic assays.

Epitope mapping of African swine fever virus (ASFV) structural protein, p54.

In the absence of a vaccine for African swine fever virus (ASFV), diagnostic tools are critical for early detection and implementation of control measures. Along with other immunogenic proteins, p54 is a good serological target for conducting ASF detection and surveillance. In this study, a panel of 12 mouse monoclonal antibodies (mAbs) was prepared against a baculovirus-expressed p54(60-178) polypeptide. Further screening showed that five mAbs were positive for reactivity against ASFV-infected cells and recombinant p54 proteins. Mapping studies using five polypeptides and 12 oligopeptides, showed that mAb #154-1 recognized a conserved polypeptide sequence, p54(65-75), and was placed into Group 1. Mabs #143-1 and #7 recognized a region covered by p54(93-113) and were placed into Group 2. Group 3 consisted of mAbs #101 and #117, which recognized p54(118-127). Sera from pigs infected with the low virulent OURT 88/3 strain recognized the same p54 region covered by the Group 3 mAbs. When tested in a neutralization format, only mAb #143-1 showed neutralization activity above background. Together, the results identify important antigenic and immunogenic regions located on p54, which provide new tools for improving ASFV diagnostics.

Development and characterization of monoclonal antibodies against p30 protein of African swine fever virus.

Among the structural proteins that compose the virion of African swine fever virus (ASFV), p30 is one of the most immunogenic proteins and is produced during early stage of ASFV infection. These two characteristics make p30 a good target for diagnostic assays to detect ASFV infection. In this study, we describe a panel of newly generated p30-specific monoclonal antibodies (mAbs). The reactivity of these mAbs was confirmed by immunoprecipitation and Western blot analysis in Vero cells infected with alphavirus replicon particles that express p30 (RP-p30). Furthermore, this panel of mAbs recognized ASFV strains BA71 V (Genotype I) and Georgia/2007 (Genotype II) in immunofluorescence assays on virus-infected Vero cells and swine macrophages, respectively. These mAbs also detected p30 expression by immunohistochemistry in tissue samples from ASFV-infected pigs. Epitope mapping revealed that a selected mAb from the panel recognized a linear epitope within the 32-amino acid region, 61-93. In contrast, two of the mAbs recognize the C-terminal region of the protein, which is highly hydrophilic, enriched in glutamic acid residues, and predicted to contain an intrinsically disordered protein region (IDPR). This panel of mAbs and mAb-based diagnostic assays potentially represent valuable tools for ASFV detection, surveillance and disease control.

Linear epitopes in African swine fever virus p72 recognized by monoclonal antibodies prepared against baculovirus-expressed antigen.

Protein p72 is the major capsid protein of African swine fever virus (ASFV) and is an important target for test and vaccine development. Monoclonal antibodies (mAbs) were prepared against a recombinant antigenic fragment, from amino acid (aa) 20-303, expressed in baculovirus. A total of 29 mAbs were recovered and tested by immunofluorescent antibody (IFA) staining on ASFV Lisbon-infected Vero cells. Six antibodies were IFA-positive and selected for further characterization. Epitope mapping was performed against overlapping polypeptides expressed in E. coli and oligopeptides. Based on oligopeptide recognition, the mAbs were divided into 4 groups: mAb 85 (aa 165-171); mAbs 65-3 and 6H9-1 (aa 265-280); mAbs 8F7-3 and 23 (aa 280-294); and mAb 4A4 (aa 290-303). All mAbs were located within a highly conserved region in p72. This panel of antibodies provides the opportunity to develop new assays for the detection of ASFV antibody and antigen.

Genetically edited pigs lacking CD163 show no resistance following infection with the African swine fever virus isolate, Georgia 2007/1.

African swine fever is a highly contagious, often fatal disease of swine for which there is no vaccine or other curative treatment. The macrophage marker, CD163, is a putative receptor for African swine fever virus (ASFV). Pigs possessing a complete knockout of CD163 on macrophages were inoculated with Georgia 2007/1, a genotype 2 isolate. Knockout and wild type pen mates became infected and showed no differences in clinical signs, mortality, pathology or viremia. There was also no difference following in vitro infection of macrophages. The results do not rule out the possibility that other ASFV strains utilize CD163, but demonstrate that CD163 is not necessary for infection with the Georgia 2007/1 isolate. This work rules out a significant role for CD163 in ASFV infection and creates opportunities to focus on alternative receptors and entry mechanisms.

Fas/FasL and perforin-granzyme pathways mediated T cell cytotoxic responses in infectious bursal disease virus infected chickens.

Infectious bursal disease (IBD) is a highly contagious disease of chickens which leads to immunosuppression. In our previous study it was demonstrated that, possibly, CD4(+) and CD8(+) T cells may employ perforin and granzyme-A pathway for the clearance of IBDV-infected bursal cells. In this study, we evaluated the cytotoxic T cell responses involving two independently functioning but complementary mechanisms: Fas-Fas ligand and perforin-granzyme pathways in IBDV-infected chickens. As demonstrated previously, infection of chickens with IBDV was accompanied by influx of CD8(+) T cells in the bursa and spleen. There was an upregulation in the gene expression of cytolytic molecules: Fas and Fas ligand (FasL), perforin (PFN) and granzyme-A (Gzm-A) in bursal and in the splenic tissues of IBDV inoculated chickens. Additionally, for the first time, we detected Fas, Fas ligand, Caspase-3 and PFN producing CD8(+) T cells in the bursa and spleen of IBDV-infected chickens. The infiltration and activation of CD8(+) T cells was substantiated by the detection of Th1 cytokine, IFN-γ. These data suggest that T cells may be involved in the clearance of virus from the target organ bursa and peripheral tissues such as spleen. The findings of these studies provide new insights into the pathogenesis of IBD and provide mechanistic evidence that the cytotoxic T cells may act through both Fas-FasL and perforin-granzyme pathways in mediating the clearance of virus-infected cells.

Expression of perforin-granzyme pathway genes in the bursa of infectious bursal disease virus-infected chickens.

Infectious bursal disease (IBD) is an economically important immunosuppressive disease of chickens. The IBD virus (IBDV) actively replicates in B cells and causes severe bursal damage. Generally, T cells are refractory to infection with IBDV but are known to promote virus clearance. However, the mechanisms of T cell mediated viral clearance are not well understood. In this study, we evaluated the molecular mechanisms of cytotoxic T cell responses in the pathogenesis of IBD in chickens. Infection of chickens with IBDV was accompanied by the infiltration of CD4(+) and CD8(+) T cells in the bursa. There was an upregulation in the gene expression of important cytolytic molecules; perforin (PFN), granzyme-A (Gzm-A), DNA repair and apoptotic proteins; high mobility proteins group (HMG) and poly (ADP-ribose) polymerase (PARP) in the bursa of Fabricius (BF) whereas expression of NK (natural killer) lysin was downregulated. Importantly, PFN producing CD4(+) and CD8(+) T cells were also detected in the bursa of IBDV-infected chickens by immunohistochemistry. The Th1 cytokines, IL-2 and IFN-γ expression was also strongly upregulated, suggesting the activation of T cells. The findings of this study highlight the mechanisms of IBD pathogenesis and the role of cytotoxic T cells in the clearance of virus-infected cells.