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PubMed2005

Vaccination against very virulent infectious bursal disease virus using recombinant T4 bacteriophage displaying viral protein VP2.

In order to develop a desirable inexpensive, effective and safe vaccine against the very virulent infectious bursal disease virus (vvIBDV), we tried to take advantage of the emerging T4 bacteriophage surface protein display system. The major immunogen protein VP2 from the vvIBDV strain HK46 was fused to the nonessential T4 phage surface capsid protein, a small outer capsid (SOC) protein, resulting in the 49 kDa SOC-VP2 fusion protein, which was verified by sodium dodecylsulfate polyacrylamide gel electrophoresis and Western blot. Immunoelectromicroscopy showed that the recombinant VP2 protein was successfully displayed on the surface of the T4 phage. The recombinant VP2 protein is antigenic and showed reactivities to various monoclonal antibodies (mAbs) against IBDV, whereas the wild-type phage T4 could not react to any mAb. In addition, the recombinant VP2 protein is immunogenic and elicited specific antibodies in immunized specific pathogen free (SPF) chickens. More significantly, immunization of SPF chickens with the recombinant T4-VP2 phage protected them from infection by the vvIBDV strain HK46. When challenged with the vvIBDV strain HK46 at a dose of 100 of 50% lethal dose (LD50) per chicken 4 weeks after the booster was given, the group vaccinated with the T4-VP2 recombinant phage showed no clinical signs of disease or death, whereas the unvaccinated group and the group vaccinated with the wild-type T4 phage exhibited 100% clinical signs of disease and bursal damages, and 30%-40% mortality. Collectively, the data herein showed that the T4-displayed VP2 protein might be an inexpensive, effective and safe vaccine candidate against vvIBDV.

Animals↗
PubMed2002

Confirmation of the existence of two distinct genetic groups of infectious bursal disease virus in Australia.

OBJECTIVE: To characterise infectious bursal disease viruses (IBDVs) prevalent at major commercial sites throughout Australia and to compare the nucleic acid sequences of local strains of IBDV with those of characterised overseas strains. DESIGN: Samples of bursae were collected from 20 broiler farms that belonged to different poultry companies in New South Wales (NSW), Queensland (Qld), Victoria (Vic), Westem (WA) and South Australia (SA). METHOD: Bursae were collected from broilers between 24 and 35 days of age. Bursal tissue was homogenised and tested for the presence of IBDV antigen using four monoclonal antibodies (Mabs) which detect antigenic variation in IBDV strains. The nucleotide sequences of the hypervariable region (HVR) within the VP2 gene of IBDVs was determined and the deduced amino acid sequences compared with three vaccine strains and six previously characterised Australian IBDV strains. The deduced amino acid sequences were also compared with the published amino acid sequences of overseas strains. The phylogenetic relationships between Australian strains and overseas strains were then determined. RESULTS: IBDV was detected in birds from 14 out of 20 farms sampled. Typing with four Mabs showed that all viruses from Vic (6) and SA (10) were antigenic variants, whereas all viruses from NSW (29), Qld (4) and WA (5) were classical-like strains. Nucleotide sequencing of one sample from each of the 14 farms on which IBDV was detected confirmed results obtained with Mabs. The amino acid sequences of all Australian viruses differed from the amino acid sequences of foreign IBDV strains. Phylogenetic analysis showed that Australian IBDV viruses belonged to two distinct genetic groups. Very virulent (vv) IBDV strains belonged to a third genetic group, and overseas classical and variant strains belonged to a fourth genetic group. CONCLUSIONS: The results confirmed previous findings that there are two groups of IBDV strains circulating in commercial broilers in Australia. The majority are classical-like strains that are antigenically and genetically similar to vaccine strains 002/73 and V877. These classical strains were prevalent in broilers in three states, NSW, Qld and WA. The second group of strains are antigenic variants that were only found in broilers in two states, Vic and SA. All Australian IBDVs characterised to date are genetically distinct and can be differentiated from all other overseas strains. This enables identification of incursion of any exotic strain into Australian poultry, be it classical, US variant or wIBDV strains.

Amino Acid Sequence↗
PubMed2003

Ability of Lactococcus lactis to export viral capsid antigens: a crucial step for development of live vaccines.

The food grade bacterium Lactococcus lactis is a potential vehicle for protein delivery in the gastrointestinal tract. As a model, we constructed lactococcal strains producing antigens of infectious bursal disease virus (IBDV). IBDV infects chickens and causes depletion of B-lymphoid cells in the bursa of Fabricius and subsequent immunosuppression, morbidity, or acute mortality. The two major IBDV antigens, i.e., VP2 and VP3, that form the viral capsid were expressed and targeted to the cytoplasm, the cell wall, or the extracellular compartment of L. lactis. Whereas VP3 was successfully targeted to the three compartments by the use of relevant expression and export vectors, VP2 was recalcitrant to export, thus confirming the difficulty of translocating naturally nonsecreted proteins across the bacterial membrane. This defect could be partly overcome by fusing VP2 to a naturally secreted protein (the staphylococcal nuclease Nuc) that carried VP2 through the membrane. Lactococcal strains producing Nuc-VP2 and VP3 in various bacterial compartments were administered orally to chickens. The chickens did not develop any detectable immune response against VP2 and VP3 but did exhibit an immune response against Nuc when Nuc-VP2 was anchored to the cell wall of lactococci.

Animals↗
PubMed1994

Isolation of suppressors of temperature-sensitive folding mutations.

Mutations in the tailspike gene (gene 9) of Salmonella typhimurium phage P22 have been used to identify amino acid interactions during the folding of a polypeptide chain. Since temperature-sensitive folding (tsf) mutations cause folding defects in the P22 tailspike polypeptide chain, it is likely that mutants derived from these and correcting the original tsf defects (second-site intragenic suppressors) identify interactions during the folding pathway. We report the isolation and identification of second-site revertants to tsf mutants.

Bacteriophage P22↗
PubMed1972

Sendai virus-induced transcriptase from infected cells: polypeptides in the transcriptive complex.

A Sendai virus-induced transcriptase-template complex was isolated from the cytoplasm of infected cells by combined sedimentation and isopycnic centrifugation. This transcriptive complex banded at 1.27 g/cm(3) in D(2)O-sucrose gradients. It contained two polypeptides, the viral nucleocapsid structure unit (molecular weight, 60,000) and the largest virion polypeptide (molecular weight, 75,000). The buoyant density, chemical composition, and electron microscopic appearance of the transcriptive complex indicate a structure like that of viral nucleocapsids.

Animals↗
PubMed1990

Coexpression by vaccinia virus recombinants of equine herpesvirus 1 glycoproteins gp13 and gp14 results in potentiated immunity.

The equine herpesvirus 1 glycoprotein 14 (EHV-1 gp14) gene was cloned, sequenced, and expressed by vaccinia virus recombinants. Recombinant virus vP613 elicited the production of EHV-1-neutralizing antibodies in guinea pigs and was effective in protecting hamsters from subsequent lethal EHV-1 challenge. Coexpression of EHV-1 gp14 in vaccinia virus recombinant vP634 along with EHV-1 gp13 (P. Guo, S. Goebel, S. Davis, M. E. Perkus, B. Languet, P. Desmettre, G. Allen, and E. Paoletti, J. Virol. 63:4189-4198, 1989) greatly enhanced the protective efficacy in the hamster challenge model over that obtained with single recombinants. The inoculum doses (log10) required for protection of 50% of hamsters were 6.1 (EHV-1 gp13), 5.2 (EHV-1 gp14), and less than 3.6 (vaccinia virus recombinant expressing both EHV-1 glycoproteins [gp13 and gp14]).

Amino Acid Sequence↗
PubMed1990

Identification of human rotavirus serotype by hybridization to polymerase chain reaction-generated probes derived from a hyperdivergent region of the gene encoding outer capsid protein VP7.

We have synthesized 32P-labeled hybridization probes from a hyperdivergent region (nucleotides 51 to 392) of the rotavirus gene encoding the VP7 glycoprotein by using the polymerase chain reaction method. Both RNA (after an initial reverse transcription step) and cloned cDNA from human rotavirus serotypes 1 through 4 could be used as templates to amplify this region. High-stringency hybridization of each of the four probes to rotavirus RNAs dotted on nylon membranes allowed the specific detection of corresponding sequences and thus permitted identification of the serotype of the strains dotted. The procedure was useful when applied to rotaviruses isolated from field studies.

Antigens, Viral↗
PubMed1994

Protein coding assignment of avian reovirus strain S1133.

Avian reovirus S1133 encodes 10 primary translation products, 8 of which are structural components of the viral particle and 2 of which are nonstructural proteins. The identity of the gene that codes for each of these polypeptides was determined by in vitro translation of denatured individual genome segments.

Animals↗
PubMed1996

Immunization with viruslike particles induces long-term protection of rabbits against challenge with cottontail rabbit papillomavirus.

Rabbits were immunized with recombinant baculovirus-produced virus-like particles (VLPs) of cottontail rabbit papillomavirus (CRPV) to determine whether these antigens could induce long-term protection against experimental challenge with CRPV. Infectious CRPV and human papillomavirus type 11 L1 VLPs were used as positive and negative control immunogens, respectively. Three groups of immunized animals were challenged with 10-fold serial dilutions of infectious CRPV at 2 weeks, 6 months, and 12 months after immunizations. Antibody titers in serum reached 1:10,000 immediately after the final booster immunization and then decayed to 1:150 at 6 months and 1:100 at 12 months in unchallenged rabbits. Serum neutralization titers followed similar kinetics. Papillomas grew on control-immunized rabbits at sites challenged with 10(-1) (100% of sites), 10(-2) (96% of sites), 10(-3) (63% of sites), and 10(-4) (13% of sites) dilutions of virus. At 2 weeks after CRPV L1 VLP immunizations, the rabbits were completely protected against virus challenge. At both 6 and 12 months after CRPV L1 VLP immunizations, strong protection was also observed. In the last two groups, three of seven rabbits were completely protected and only 4 of 14 or 29% of sites challenged with 10(-1 dilution of virus grew papillomas. Papillomas growing at these four sites were also reduced in size (3.5 +/- 0.7 mm) at 50 days postchallenge compared with sites challenged with 10(-1) dilution on control-immunized rabbits (13.2 +/- 4.2 mm). The results demonstrate that strong and long-lasting protection against experimental challenge with papillomaviruses can be achieved with VLP immunogens.

Animals↗
PubMed1998

Subunit rotavirus vaccine administered parenterally to rabbits induces active protective immunity.

Virus-like particles (VLPs) are being evaluated as a candidate rotavirus vaccine. The immunogenicity and protective efficacy of different formulations of VLPs administered parenterally to rabbits were tested. Two doses of VLPs (2/6-, G3 2/6/7-, or P[2], G3 2/4/6/7-VLPs) or SA11 simian rotavirus in Freund's adjuvants, QS-21 (saponin adjuvant), or aluminum phosphate (AlP) were administered. Serological and mucosal immune responses were evaluated in all vaccinated and control rabbits before and after oral challenge with 10(3) 50% infective doses of live P[14], G3 ALA lapine rotavirus. All VLP- and SA11-vaccinated rabbits developed high levels of rotavirus-specific serum and intestinal immunoglobulin G (IgG) antibodies but not intestinal IgA antibodies. SA11 and 2/4/6/7-VLPs afforded similar but much higher mean levels of protection than 2/6/7- or 2/6-VLPs in QS-21. The presence of neutralizing antibodies to VP4 correlated (P < 0.001, r = 0.55; Pearson's correlation coefficient) with enhanced protection rates, suggesting that these antibodies are important for protection. Although the inclusion of VP4 resulted in higher mean protection levels, high levels of protection (87 to 100%) from infection were observed in individual rabbits immunized with 2/6/7- or 2/6-VLPs in Freund's adjuvants. Therefore, neither VP7 nor VP4 was absolutely required to achieve protection from infection in the rabbit model when Freund's adjuvant was used. Our results show that VLPs are immunogenic when administered parenterally to rabbits and that Freund's adjuvant is a better adjuvant than QS-21. The use of the rabbit model may help further our understanding of the critical rotavirus proteins needed to induce active protection. VLPs are a promising candidate for a parenterally administered subunit rotavirus vaccine.

Adjuvants, Immunologic↗
PubMed2000

Horizontal transmissible protection against myxomatosis and rabbit hemorrhagic disease by using a recombinant myxoma virus.

We have developed a new strategy for immunization of wild rabbit populations against myxomatosis and rabbit hemorrhagic disease (RHD) that uses recombinant viruses based on a naturally attenuated field strain of myxoma virus (MV). The recombinant viruses expressed the RHDV major capsid protein (VP60) including a linear epitope tag from the transmissible gastroenteritis virus (TGEV) nucleoprotein. Following inoculation, the recombinant viruses induced specific antibody responses against MV, RHDV, and the TGEV tag. Immunization of wild rabbits by the subcutaneous and oral routes conferred protection against virulent RHDV and MV challenges. The recombinant viruses showed a limited horizontal transmission capacity, either by direct contact or in a flea-mediated process, promoting immunization of contact uninoculated animals.

Animals↗
PubMed2001

A recombinant newcastle disease virus with low-level V protein expression is immunogenic and lacks pathogenicity for chicken embryos.

Newcastle disease virus (NDV) edits its P-gene mRNA by inserting a nontemplated G residue(s) at a conserved editing site (3'-UUUUUCCC-template strand). In the wild-type virus, three amino-coterminal P-gene-derived proteins, P, V, and W, are produced at frequencies of approximately 68, 29, and 2%, respectively. By applying the reverse genetics technique, editing-defective mutants were generated in cell culture. Compared to the wild-type virus, mutants lacking either six nucleotides of the conserved editing site or the unique C-terminal part of the V protein produced as much as 5, 000-fold fewer infectious progeny in vitro or 200,000-fold fewer in 6-day-old embryonated chicken eggs. In addition, both mutants were unable to propagate in 9- to 11-day-old embryonated specific-pathogen-free (SPF) chicken eggs. In contrast, a mutant (NDV-P1) with one nucleotide substitution (UUCUUCCC) grew in eggs, albeit with a 100-fold-lower infectious titer than the parent virus. The modification in the first two mutants described above led to complete abolition of V expression, whereas in NDV-P1 the editing frequency was reduced to less than 2%, and as a result, V was expressed at a 20-fold-lower level. NDV-P1 showed markedly attenuated pathogenicity for SPF chicken embryos, unlike currently available ND vaccine strains. These findings indicate that the V protein of NDV has a dual function, playing a direct role in virus replication as well as serving as a virulence factor. Administration of NDV-P1 to 18-day-old embryonated chicken eggs hardly affected hatchability. Hatched chickens developed high levels of NDV-specific antibodies and were fully protected against lethal challenge, demonstrating the potential use of editing-defective recombinant NDV as a safe embryo vaccine.

Animals↗
PubMed2001

A novel nonnucleoside inhibitor specifically targets cytomegalovirus DNA maturation via the UL89 and UL56 gene products.

3-Hydroxy-2,2-dimethyl-N-[4([[5-(dimethylamino)-1-naphthyl]sulfonyl]amino)-phenyl]propanamide (BAY 38-4766) is a novel selective nonnucleoside inhibitor of cytomegalovirus (CMV) replication with an excellent safety profile. This compound and structural analogues inhibit neither viral DNA synthesis nor viral transcription and translation. Accumulation of dense bodies and noninfectious enveloped particles coincides with inhibition of both concatemer processing and functional cleavage at intergenomic transitions, pointing to interference with viral DNA maturation and packaging of monomeric genome lengths. Resistant virus populations, including a murine CMV (MCMV) isolate with 566-fold-decreased drug sensitivity, were selected in vitro. Sequencing of the six open reading frames (ORFs) known to be essentially involved in viral DNA cleavage and packaging identified mutations in ORFs UL56, UL89, and UL104. Construction of MCMV recombinants expressing different combinations of murine homologues of mutant UL56, UL89, and UL104 and analysis of drug susceptibilities clearly demonstrated that mutant ORFs UL89 exon II (M360I) and M56 (P202A I208N) individually confer resistance to BAY 38-4766. A combination of both mutant proteins exhibited a strong synergistic effect on resistance, reconstituting the high-resistance phenotype of the in vitro mutant. These findings are consistent with genetic mapping of resistance to TCRB (2,5,6-trichloro-1-beta-D-ribofuranosyl benzimidazole) (P. M. Krosky et al., J. Virol. 72:4721-4728, 1998) and provide further indirect evidence that proteins encoded by UL89 and UL56 function as two subunits of the CMV terminase. While these studies also suggest that the molecular mechanism of BAY 38-4766 is distinct from that of benzimidazole ribonucleosides, they also offer an explanation for the excellent specificity and tolerability of BAY 38-4766, since mammalian DNA does not undergo comparable maturation steps.

Animals↗
PubMed2002

Complete, long-lasting protection against lethal infectious bursal disease virus challenge by a single vaccination with an avian herpesvirus vector expressing VP2 antigens.

Marek's disease herpesvirus is a vaccine vector of great promise for chickens; however, complete protection against foreign infectious diseases has not been achieved. In this study, two herpesvirus of turkey recombinants (rHVTs) expressing large amounts of infectious bursal disease virus (IBDV) VP2 antigen under the control of a human cytomegalovirus (CMV) promoter or CMV/beta-actin chimera promoter (Pec promoter) (rHVT-cmvVP2 and rHVT-pecVP2) were constructed. rHVT-pecVP2, which expressed the VP2 antigen approximately four times more than did rHVT-cmvVP2 in vitro, induced complete protection against a lethal IBDV challenge in chickens, whereas rHVT-cmvVP2 induced 58% protection. All of the chickens vaccinated with rHVT-pecVP2 had a protective level of antibodies to the VP2 antigen at the time of challenge, whereas only 42 and 67% of chickens vaccinated with rHVT-cmvVP2 or the conventional live IBDV vaccine, respectively, had the antibodies. The antibody level of chickens vaccinated with rHVT-pecVP2 increased for 16 weeks, and the peak antibody level persisted throughout the experiment. The serum antibody titer at 30 weeks of age was about 20 or 65 times higher than that of chickens vaccinated with rHVT-cmvVP2 or the conventional live vaccine, respectively. rHVT-pecVP2, isolated consistently for 30 weeks from the vaccinated chickens, expressed the VP2 antigen after cultivation, and neither nucleotide mutations nor deletion in the VP2 gene was found. These results demonstrate that the amount of VP2 antigen expressed in the HVT vector was correlated with the vaccine efficacy against lethal IBDV challenge, and complete protective immunity that is likely to persist for the life of the chickens was induced.

Animals↗