Aerosol delivery of synthetic DNA containing CpG motifs in broiler chicks at hatch under field conditions using a commercial-scale prototype nebulizer provided protection against lethal Escherichia coli septicemia.

Synthetic DNA containing CpG motifs (CpG-ODN) are potent innate immune stimulators in neonatal and adult broiler chickens against bacterial septicemia. We have recently demonstrated that intrapulmonary (IPL) delivery of CpG-ODN as microdroplets under laboratory conditions can protect neonatal chickens against lethal Escherichia coli septicemia. The objectives of this study were to develop a commercial-scale poultry nebulizer (CSPN) that can deliver CpG-ODN as microdroplets in neonatal broiler chicks in the hatcheries and study the efficacy of CSPN in inducing immune-protective effects under different environmental conditions in 2 geographical locations in Canada. Three field experiments were conducted in commercial poultry hatcheries during different seasons of the year in Saskatchewan and British Columbia, Canada. Neonatal broiler chicks (n = 8,000/experiment) received CpG-ODN by the IPL route in the CSPN chamber for 30 min, and control chicks received distilled water (DW) for 30 min. Broiler chicks (CpG-ODN-240 chicks/experiment and DW-40 chicks/experiment) were randomly sampled from all locations of the CSPN after nebulization and challenged with a lethal dose of E. coli to examine the CpG-ODN nebulization induced protection. We found a significant level (P < 0.05) of protection in broiler chicks against E. coli challenge, suggesting that the newly built CSPN successfully delivered CpG-ODN via the IPL route. We found that when the CSPN was maintained at humidex 28°C or below and relative humidity (RH) between 40 and 60%, neonatal birds were significantly (P < 0.05) protected against E. coli septicemia after IPL delivery of CpG-ODN. By contrast, protection in chicks was adversely affected when the CSPN was maintained at the humidex of 29°C or higher and RH of 70%. Overall, the present study successfully built a CSPN for CpG-ODN delivery in chicks at the hatchery and revealed that the temperature, humidity, and humidex were critical parameters in CSPN for efficient delivery of CpG-ODN.

Transcription mapping and characterization of proteins produced from early region 4 of porcine adenovirus type 3.

The early region 4 (E4) of porcine adenovirus 3 (PAdV-3) was characterized by Northern blot, rapid amplification of cDNA ends (RACE), RT-PCR and cDNA sequence analysis. Northern blot analysis revealed three different classes of transcripts, which appeared and peaked at different times post-infection. The RT-PCR, RACE and cDNA sequence analysis identified nine major E4 transcripts, all of which shared a 107-bp 5' leader sequence and a 126-bp 3' terminus. These transcripts have one to three introns removed. Interestingly, of the nine major transcripts, there was one fusion transcript of ORFp1 and ORFp7 (ORFp1/7), which codes for a protein of 119 amino acids. All transcripts initiated at nucleotide 33740 of the PAdV-3 genome. To identify proteins, rabbit antiserum was prepared using a bacterial fusion protein encoding p2, p3, p4 or p7 proteins. Serum against p2, p3 and p4 immunoprecipitated proteins of 13.5, 13.6 and 15.3 kDa, respectively, in in-vitro transcribed and translated mRNA and in PAdV-3-infected cells. Serum against p7 immunoprecipitated a protein of 19.8 kDa in in-vitro transcription and translation analysis but recognized two proteins of 19.8 kDa (encoded by ORFp7) and 14 kDa (encoded by the fusion transcript ORF1/7) in PAdV-3-infected cells. The protein encoded by ORFp2 was localized in the nucleus of PAdV-3-infected cells. The proteins encoded by ORFp3 and ORFp7\ORFp1/7 were detected in the cytoplasm of PAdV-3-infected cells. However, the protein encoded by ORFp4 was observed both in the cytoplasm and nucleus of PAdV-3-infected cells.

Characterization and nuclear localization of the fiber protein encoded by the late region 7 of bovine adenovirus type 3.

To identify the protein encoded by the L7 region of bovine adenovirus-3 (BAdV-3), specific antisera were raised by immunizing rabbits with bacterial fusion proteins encoding the N-terminus or C-terminus of the BAdV-3 fiber protein. Immunoprecipitation and Western blot analysis confirmed that the fiber is expressed as a 102 kDa glycoprotein, which is localized to the nucleus of infected cells. To identify the nuclear localization signals (NLS), BAdV-3 fiber deletion mutants and GFP/beta-galactosidase fusion proteins were expressed in transfected cells, and subcellular localization was visualized by immunofluorescence microscopy. Analysis of deletion mutants localized the NLS to the N-terminal 41 amino acids. Analysis of the N-terminal 41 amino acids identified a cluster of basic residues between amino acid 14 and 20. Substitution of the basic residues (16KAKR19) with acidic residues (16EAEE19) resulted in the accumulation of fiber in the cytoplasm. However, 16KAKR19 or 12VYPYKAKRPNI22 were not sufficient for efficient transport of a cytoplasmic protein GFP/beta-galactosidase to the nucleus. The recombinant BAdV-3 expressing mutant fiber containing 16EAEE19 instead of 16KAKR19 was unable to replicate efficiently in Madin-Darby bovine kidney cells, suggesting that the NLS of fiber carries out important in vivo functions.

Bovine adenovirus type 3 E1B(small) protein is essential for growth in bovine fibroblast cells.

In order to study the function of bovine adenovirus type 3 (BAV-3) E1A and E1B(small) proteins, we constructed two mutants: (a) BAV102A carries an in-frame deletion in the coding region for the E1A protein (nt 831-1080); (b) BAV102B carries an insertion of triple stop codons in the E1B region (nt 1654, 178 bp downstream of the E1B(small) start codon), which stops the translation of the E1B(small) gene. BAV102A virus could grow to the wild-type BAV-3 titer in transformed cell line VIDO R2 (HAV-5 E1 transformed) cells, but no progeny virus could be found in fetal bovine retina cells (FBRC). RT-PCR and Western blot analysis showed that neither mRNA transcripts nor protein expression of early genes [E1B(small) and DNA binding protein (DBP)] could be detected in BAV102A infected FBRC. The BAV102B grew 1.5 log less than wild-type BAV-3 in FBRC; however, no BAV102B progeny virus could be observed in bovine fibroblast (BFB) cells. No appreciable difference was observed in DBP transcript synthesis between wild-type BAV-3- or BAV102B-infected FBRC. However, compared to wild-type BAV-3, BAV102B viral DNA synthesis and fiber gene expression were found to be slightly reduced in FBRC. In contrast, compared to wild-type BAV-3, DBP transcripts and viral DNA synthesis were drastically reduced in BAV102B-infected BFB cells. In addition, no fiber gene expression could be detected in BAV102B-infected BFB cells. These results suggest that BAV-3 E1A is essential for virus replication and is required for activating the transcription of other BAV-3 early genes. However, the requirement for E1B(small) protein for BAV-3 replication appears to be cell type-dependent.

Vaccination of pigs with a recombinant porcine adenovirus expressing the gD gene from pseudorabies virus.

Five week old, commercially available large white pigs were vaccinated with either a single dose or two doses of a recombinant porcine adenovirus expressing the glycoprotein D gene from pseudorabies virus (PRV). Pigs were monitored for the development of serum neutralizing antibodies to PRV and challenged 3 weeks after final vaccination. Prior to challenge, pigs given 2 doses of the vaccine demonstrated boosted levels of antibody compared with those given a single dose, and all surviving pigs had increased neutralization titres over pre-challenge levels. Following challenge, pigs were monitored for clinical signs of disease, with blood and nasal swabs collected for virus isolation. All control animals became sick with elevated temperatures for 6 days post challenge, whereas; vaccinated animals displayed an increase in body temperature for only 2-3 days. Control pigs and those given a single dose all lost condition, but the group given 2 doses remained healthy. At postmortem, gross lesions of pneumonia only occurred in control animals and those given a single dose of vaccine. Histology carried out on the brains of all animals demonstrated a difference in severity of infection and frequency of immunohistochemical antigen detection between test animals, with control and single dose groups being most severely affected and pigs given 2 doses the least. Virus isolation studies demonstrated that no viraemia could be detected, but virus was found in nasal swabs from some animals in both groups of vaccinates following challenge.

Nuclear localization of the ORF2 protein encoded by porcine circovirus type 2.

Infectious porcine circovirus type 2 (PCV2) was generated following transfection of a porcine retina cell line (VIDO R1) with cloned circovirus DNA. Expression of open reading frame 2 (ORF2) was detected at 24 h postinfection and onwards increasingly throughout the infection by Western blot analysis using ORF2 specific polyclonal antibody. Moreover, the ORF2 protein was also detected in purified PCV2 virus, indicating that ORF2 is a structural component of PCV2 viral capsid. Nuclear localization of PCV2 ORF2 was demonstrated by immunofluorescence assay in PCV2-infected cells. An analysis of the subcellular localization of a series of truncation mutants of ORF2 fused with the green fluorescent protein indicated that the nuclear localization signal of ORF2 was conferred by the N-terminal 41 amino acids. This domain was further analyzed through site-directed mutagenesis, suggesting that the presence of basic amino acid residues at positions 12 to 18 and 34 to 41 are important for the strict nuclear targeting of PCV2 ORF2.

Determination of bovine adenovirus-3 titer based on immunohistochemical detection of DNA binding protein in infected cells.

DNA sequence coding for a portion of DNA binding protein (amino acids 3-58) of bovine adenovirus type-3 (BAV-3) was cloned and expressed in Escherichia coli as a fusion protein with Schistosoma japonicum glutathione S-transferase. The fusion protein was affinity purified and used to immunize rabbits. Immunoprecipitation and Western blot analysis showed that the antiserum could specifically recognize a protein of 48 kDa in BAV-3-infected cells, which was produced both in early and late phases of BAV-3 life cycle. Based on the ability of antiserum to recognize DNA binding protein, a novel assay for BAV-3 quantitation was established. The assay is less time consuming and can be performed on a wide variety of bovine cells. In addition, virus titers determined by this assay are comparable to the standard plaque assay.

121R protein from the E3 region of bovine adenovirus-3 inhibits cytolysis of mouse cells by human tumor necrosis factor.

Based on the DNA sequence and known mRNA structures, the early region 3 (E3) of bovine adenovirus (BAV)-3 has the potential to encode four proteins. One of them (121R) is produced as a 14.5-kD protein throughout infection. Analysis of the 121R protein showed limited homology to a 14.7K protein of human adenovirus (HAV)-5. Interestingly, both anti-14.7K and anti-121R sera immunoprecipitated a 14.5-kD protein from cells infected with BAV-3. To determine if 121R is functionally related to 14.7K, we constructed a recombinant E3-deleted HAV-5 (AdKV121) expressing the BAV-3 E3 121R protein. Mouse C3HA cells infected with HAV-5 mutant dl 758 (deletion of 14.7K) were sensitive to TNF lysis. However, wild-type HAV-5- or recombinant AdKV121-infected cells were resistant to TNF-induced cytolysis. Our result show that the BAV-3 E3 121R protein is serologically and functionally related to the 14.7K protein encoded by the E3 region of HAV-5.

Characterization of DNA binding protein of porcine adenovirus type 3.

To identify and characterize the protein encoded by the E2A region of porcine adenovirus (PAV)-3, DNA sequence coding for a portion (amino acids 102-457) of the DNA binding protein (DBP) open reaching frame was cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase protein of Schistosoma japonica. The affinity-purified fusion protein was used to immunize rabbits. Immunoprecipitation/Western blot analysis demonstrated that the antisera specifically recognized a protein of 50 kD in PAV-3-infected cells. Immunoperoxidase staining detected the DBP protein predominantly in the nucleus of the cells. Western blot analysis demonstrated that DBP was detected as early as 6 h after infection and remained detectable throughout the infection. Based on these results, a novel assay for quantitation of PAV-3 was established. The assay is less time consuming and can be performed in different porcine cells. In addition, virus titers determined by this assay are comparable to the standard plaque assay.

Mutational analysis of early region 4 of bovine adenovirus type 3.

The primary objective of characterizing bovine adenovirus type 3 (BAV3) in greater detail is to develop it as a vector for gene therapy and vaccination of humans and animals. A series of BAV3 early region 4 (E4) deletion-mutant viruses, containing deletions in individual E4 open reading frames (Orf) or combinations of Orfs, were generated by transfecting primary fetal bovine retinal cells with E4-modified genomic DNA. Each of these mutants was further analyzed for growth kinetics, viral DNA accumulation, and early-late protein synthesis. Mutant viruses carrying deletions in Orf1, Orf2, Orf3, or Orf4 showed growth characteristics similar to those of the E3-deleted BAV3 (BAV302). DNA accumulation and early/late protein synthesis were also indistinguishable from those of BAV302. However, mutant viruses carrying a deletion in Orf5, Orfs 1-3 (BAV429), or Orfs 3-5 (BAV430) were modestly compromised in their ability to grow in bovine cells and express early/late proteins. E4 mutants containing larger deletions, Orfs 1-3 (BAV429) and Orfs 3-5 (BAV430), were further tested in a cotton rat model. Both mutants replicated as efficiently as BAV3 or BAV302 in the lungs of cotton rats. BAV3-specific IgA and IgG responses were detected in serum and at the mucosal surfaces in cotton rats inoculated with mutant viruses. In vitro and in vivo characterization of these E4 mutants suggests that none of the individual E4 Orfs are essential for viral replication. Moreover, successful deletion of a 1.5-kb fragment in the BAV3 E4 region increased the available insertion capacity of replication-competent BAV3 vector (E3-E4 deleted) to approximately 4.5 kb and that of replication-defective BAV3 vector (E1a-E3-E4 deleted) to approximately 5.0 kb. This is extremely useful for the construction of BAV3 vectors that express multiple genes and/or regulatory elements for gene therapy and vaccination.

Analysis of early region 1 of porcine adenovirus type 3.

To identify the proteins encoded by the porcine adenovirus 3 (PAV-3) E1 region, rabbit antisera were prepared using a bacterial fusion protein encoding E1A, E1B(small), or E1B(large) protein. Sera against E1A, E1B(small), and E1B(large) immunoprecipitated a protein of 35, 23, and 53 kDa, respectively, in in vitro translated and transcribed mRNA and PAV-3 infected cells. To determine the role of E1 proteins in PAV-3 replication, we constructed vectors with a deletion(s) in the E1 region. Mutant PAV211, containing deletions in E1A and E3, grew to titers similar to wild-type in VIDO R1 cells (E1A complementing) but not in swine testicular (ST) cells. No early protein (E1B(small), DNA binding protein) expression could be detected in PAV211 infected ST cells by Western blots. Mutant PAV212, containing deletions in E1B(small) and E3, grew to wild-type titers in VIDO R1 or ST cells. These deletions were successfully rescued, resulting in recombinant PAV214, containing deletions in E1A, E1B(small), and E3. However, mutant PAV-3, containing a triple stop codon inserted in the E1B(large) coding sequence, could not be isolated. Next, we constructed a recombinant PAV216 by inserting the green fluorescent protein gene flanked by a promoter and a poly(A) in the E1A region of the PAV214 genome. Both PAV214 and PAV216 replicate as efficiently as wild-type in VIDO R1 cells. These results suggested that (a) E1A is essential for virus replication and is required for the activation of other PAV-3 early genes, (b) E1B(small) is not essential for replication of PAV-3, and (c) E1B(large) is essential for virus replication. Moreover, the PAV216 vector can be used for the expression of a transgene.

Recombinant bovine adenovirus type 3 expressing bovine viral diarrhea virus glycoprotein E2 induces an immune response in cotton rats.

Recombinant bovine adenovirus is being developed as a live vector for animal vaccination and for human gene therapy. In this study, two replication-competent bovine adenovirus 3 (BAV-3) recombinants (BAV331 and BAV338) expressing bovine viral diarrhea virus (BVDV) glycoprotein E2 in the early region 3 (E3) of BAV-3 were constructed. Recombinant BAV331 contains chemically synthesized E2 gene (nucleotides modified to remove internal cryptic splice sites) under the control of BAV-3 E3/major late promoter (MLP), while recombinant BAV338 contains original E2 gene under the control of human cytomegalovirus immediate early promoter. Since E2, a class I membrane glycoprotein, does not contain its own signal peptide sequence at the 5' end, the bovine herpesvirus 1 (BHV-1) glycoprotein D signal sequence was fused in frame to the E2 open reading frame (ORF) for proper processing of the E2 glycoprotein in both the recombinant viruses. Recombinant E2 protein expressed by BAV331 and BAV338 recombinant viruses was recognized by E2-specific monoclonal antibodies as a 53-kDa protein, which also formed dimer with an apparent molecular weight of 94 kDa. Insertion of an E2-expression cassette in the E3 region did not effect the replication of recombinant BAV-3s. Intranasal immunization of cotton rats with these recombinant viruses generated E2-specific IgA and IgG responses at the mucosal surfaces and in the serum. In summary, these results show that the pestivirus glycoprotein can be expressed efficiently by BAV-3. In addition, mucosal immunization with replication-competent recombinant bovine adenovirus 3 can induce a specific immune response against the expressed antigen.

Optimization of bovine coronavirus hemagglutinin-estrase glycoprotein expression in E3 deleted bovine adenovirus-3.

Adenoviral vectors expressing foreign genes have many desirable properties in applications such as vaccination. Recently, we have generated replication-competent (E3 deleted) bovine adenovirus-3 (BAV-3) recombinants expressing significant amounts of glycoprotein D (gD) of bovine herpesvirus-1 (a DNA virus). However, attempts to express the RNA virus genes using the same strategy were not successful. In an effort to optimize the expression, we have constructed several BAV-3 recombinants carrying the hemagglutinin esterase (HE) gene of bovine coronavirus (BCV) in the E3 region with or without exogenous transcription control elements. The expression studies suggest that the introduction of a 137 bp chimeric intron upstream of the HE cDNA is able to increase the level of HE gene expression. The introduction of a SV40 early promoter or human cytomegalovirus (HCMV) immediate early (IE) promoter into the expression cassette changed the kinetics of the HE expression. However, the recombinant BAV-3 containing HE under the HCMV IE promoter replicated less efficiently than the wild-type BAV-3. These studies should prove useful in expression of other RNA viral genes in the E3 region of BAV-3 expression system.

The immunogenicity and efficacy of replication-defective and replication-competent bovine adenovirus-3 expressing bovine herpesvirus-1 glycoprotein gD in cattle.

Replication-competent and replication-defective bovine adenovirus type 3 recombinants expressing the bovine herpesvirus type 1 (BHV-1) glycoprotein D (gD) were tested for induction of gD specific immune responses in calves using intratracheal (1st and 2nd immunization) and sub-cutaneous (3rd immunization) route of immunization. The replication-defective recombinant BAV501 induced systemic immune responses against gD as low titers of anti gD-IgG were detected in the serum. However, the efficacy of the replication-competent BAV3.E3gD to induce gD-specific antibodies in the serum and the nasal secretions was superior to that of replication-defective BAV501 when both viruses were given at the same dosage. Partial protection from challenge was induced in calves immunized with replication-competent BAV3.E3gD. A dramatic increase in the titers of anti-gD IgG and IgA levels, both in serum and nasal secretions, following BHV-1 challenge (anamnestic response) suggested that the animals immunized with replication-defective BAV501 had been primed for gD-specific antibody responses.

Adenoviruses as vectors for delivering vaccines to mucosal surfaces.

Immunization of mucosal surfaces has become an attractive route of vaccine delivery because of its ability to induce mucosal immunity. Although various methods of inducing mucosal immunity are being developed, our laboratory has focused on developing adenoviruses as replication-competent and replication-incompetent vectors. The present report will summarize our progress in sequencing the entire bovine adenovirus-3 genome and identifying regions which can be deleted and subsequently used as insertion sites for foreign genes in developing recombinant viral vaccines. Using these recombinant viruses, we demonstrated the 'proof-of-principle' in developing mucosal immunity and, more importantly, inducing protection against bovine herpes virus in a natural host-cattle. Finally, we demonstrated that immunity and protection occurred even in animals that had pre-existing antibodies to the vector.

Replication-defective bovine adenovirus type 3 as an expression vector.

Although recombinant human adenovirus (HAV)-based vectors offer several advantages for somatic gene therapy and vaccination over other viral vectors, it would be desirable to develop alternative vectors with prolonged expression and decreased toxicity. Toward this objective, a replication-defective bovine adenovirus type 3 (BAV-3) was developed as an expression vector. Bovine cell lines designated VIDO R2 (HAV-5 E1A/B-transformed fetal bovine retina cell [FBRC] line) and 6.93.9 (Madin-Darby bovine kidney [MDBK] cell line expressing E1 proteins) were developed and found to complement the E1A deletion in BAV-3. Replication-defective BAV-3 with a 1.7-kb deletion removing most of the E1A and E3 regions was constructed. This virus could be grown in VIDO R2 or 6.93.9 cells but not in FBRC or MDBK cells. The results demonstrated that the E1 region of HAV-5 has the capacity to transform bovine retina cells and that the E1A region of HAV-5 can complement that of BAV-3. A replication-defective BAV-3 vector expressing bovine herpesvirus type 1 glycoprotein D from the E1A region was made. A similar replication-defective vector expressing the hemagglutinin-esterase gene of bovine coronavirus from the E3 region was isolated. Although these viruses grew less efficiently than the replication-competent recombinant BAV-3 (E3 deleted), they are suitable for detailed studies with animals to evaluate the safety, duration of foreign gene expression, and ability to induce immune responses. In addition, a replication-competent recombinant BAV-3 expressing green fluorescent protein was constructed and used to evaluate the host range of BAV-3 under cell culture conditions. The development of bovine E1A-complementing cell lines and the generation of replication-defective BAV-3 vectors is a major technical advancement for defining the use of BAV-3 as vector for vaccination against diseases of cattle and somatic gene therapy in humans.

Transcription map and expression of bovine herpesvirus-1 glycoprotein D in early region 4 of bovine adenovirus-3.

Early region 4 (E4) of bovine adenovirus type 3 (BAV-3) was analyzed by Northern blotting, RT-PCR analysis, cDNA sequencing, and S1 nuclease protection assays. The transcriptional map of the E4 region of BAV-3 has marked dissimilarities from those of mouse adenovirus-1, ovine adenovirus-287, and human adenovirus-2, for which the transcriptional maps have been constructed. The E4 region of BAV-3, located between 98.6 and 89.8 MU transcribes seven distinct classes of bovine adenovirus type 3 mRNA. The seven mRNA species formed by the removal of one to three introns share both the 3' end and a short 5' leader (25 nucleotides). The E4 mRNAs can encode at least five unique polypeptides, namely, 143R1, 69R, 143R2, 268R, and 219R. Isolation of a replication-competent recombinant "BAV404" containing 1.9-kb insertion [glycoprotein (gD) of bovine herpesvirus 1, under the control of a SV40 early promoter and poly(A)] in the region between E4 and the right ITR suggested that this region is nonessential for BAV-3 replication. Expression of gD by BAV404 recombinant virus was confirmed by immunoprecipitation with gD-specific monoclonal antibodies. Analysis of the kinetics of protein expression indicated that gD is expressed at both early and late times postinfection. These results suggest that: (a) E4 produces seven 5'-3' coterminal mRNAs and (b) the right terminal region of BAV-3 can be used for the expression of vaccine antigens.

Experimental inoculation of heifers with bovine adenovirus type 3.

Nine 2-year-old heifers having BAd3-neutralizing antibody titers between 1:120 and 1:1080 were individually exposed intranasally to an aerosol of 10(8) pfu of wild type (wt) bovine adenovirus type 3 (BAd3). Four animals were kept as non-inoculated controls. The heifers were examined daily for rectal temperature, weight gain/loss, nasal and ocular discharges, and other clinical signs for 10 d post-inoculation. None of the animals showed any sign of clinical disease. Virus excretion was observed in one animal only on Day 3 post-inoculation. All BAd3-inoculated heifers demonstrated a significant (P < 0.005, paired t-test) rise in BAd3-specific serum IgG, IgG1, or IgG2 ELISA titers and virus-neutralizing antibody titers compared to the titers before inoculation. All virus-inoculated animals demonstrated increased levels of BAd3-specific IgA ELISA titers in nasal secretions. These results suggest that in the presence of circulating BAd3-neutralizing antibodies, intranasal inoculation of cattle with wt BAd3 would result in inapparent infection.

Mucosal immunization of calves with recombinant bovine adenovirus-3: induction of protective immunity to bovine herpesvirus-1.

To determine the potential of replication-competent (E3-deleted) bovine adenovirus-3 (BAV-3) as a delivery system for vaccine antigens in calves, we evaluated the ability of recombinant BAV-3 expressing different forms of of bovine herpesvirus-1 (BHV-1) glycoprotein gD to protect against BHV-1 infection in calves that had pre-existing BAV-3 specific antibodies. Three- to four-month-old calves, vaccinated intranasally with recombinant BAV-3 expressing full-length gD (BAV3.E3gD) or a truncated version of gD (gDt) (BAV3.E3gDt), or with E3-deleted BAV-3 (BAV3.E3d; control), were challenged with BHV-1 strain 108. Vaccination with BAV3.E3gD or BAV3.E3gDt induced gD-specific antibody responses in serum and nasal secretions, and primed calves for gD-specific lymphoproliferative responses. In addition, all calves developed complement-independent neutralizing antibodies against BHV-1. Protection against viral challenge was observed in calves vaccinated with recombinant BAV3.E3gD or BAV3.E3gDt as shown by a significant reduction in body temperature and clinical disease, and a partial reduction in the amount and duration of virus excretion in nasal secretions. These results indicate that replication-competent BAV-3-based vectors can induce protective immune responses in calves (the natural host) that have pre-existing BAV-3-specific antibodies.

Transcription mapping and characterization of 284R and 121R proteins produced from early region 3 of bovine adenovirus type 3.

We established the transcription map of early region (E) 3 of bovine adenovirus 3 (BAV-3) by Northern blot, S1 nuclease protection assays, cDNA sequencing, and RT-PCR analysis. Five major classes of mRNAs were identified, which shared the 3' ends. Four classes of mRNAs transcribed from the E3 promoter also shared the 5' end, while one major class of mRNA transcribed from the major late promoter contained a tripartite leader sequence at the 5' end. These five transcripts have the potential to encode four proteins, namely 284R, 121R, 86R, and 82R. To identify the proteins, rabbit antiserum was prepared using a bacterial fusion protein encoding 284R or 121R protein. Serum against 284R immunoprecipitated protein of 26-32 kDa in in vitro translated and transcribed mRNA and three proteins of 48, 67, and 125 kDa from BAV-3-infected cells. Western blots and enzymatic digestions confirmed that the 284R protein is a glycoprotein, which contains only N-linked oligosaccharides, both high mannose (48 kDa) and complex types (67 kDa). Serum against 121R immunoprecipitated a protein of 14.5 kDa from in vitro translated and transcribed mRNA and BAV-3-infected cells. Although 121R protein shows limited sequence similarity to a 14.7-kDa protein of human adenovirus 5, the 284R protein appears to be unique to BAV-3. Since proteins encoded by the E3 region appear to influence adenovirus pathogenesis, the 284R protein may contribute to the unique pathogenic properties of BAV-3.