Transcription mapping of mouse adenovirus type 1 early region 3

Early region 3 (E3) of mouse adenovirus type 1 was analyzed using S1 nuclease protection and primer extension assays, cDNA sequencing, and genomic sequencing. We present the genomic sequence from 79 to 83 map units of the viral genome, the precise ends and splice sites of the E3 mRNAs, and the predicted protein sequence encoded by the mRNAs. Three major classes of early mRNAs were identified; all were approximately 1 kb long, consisted of three exons, and shared 5' and 3' ends. The three classes had alternative splicing at the junction between the second and third exon. The three proteins predicted by the three mRNAs were slightly similar to the E3 19K glycoprotein of human adenovirus type 3; the longest of the three was the most similar. Open reading frames corresponding to late proteins were also identified in the translated mouse adenovirus type 1 DNA sequence. In mouse adenovirus, as in the human adenoviruses, L4 overlaps E3, and L5 starts just downstream of the E3 region.     

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.     

Monoclonal antibody analysis of bovine herpesvirus-1 glycoprotein antigenic areas relevant to natural infection

Neutralizing antigenic areas on the glycoproteins of bovine herpesvirus-1 (BHV-1) were identified by reciprocal competition radioimmunoassays using monoclonal antibodies. Three interrelated and two independent antigenic areas were identified on the 77-kDa (K) gIV envelope glycoprotein. Antigenic analysis of this protein has not been previously described. Four interrelated and one independent antigenic areas were found on the 97K gIII envelope glycoprotein. A third group of monoclonal antibodies reacting in Western blot with the 74K subunit of gI, a 130K disulfide-linked 74K/55K heterodimer, revealed four interrelated antigenic areas. All of the antigenic areas on all three glycoproteins were reactive with neutralizing monoclonal antibodies and all were targets for antibody-complement lysis. However, antibodies against gIV were the most efficient at neutralizing the virus and rendering infected cells susceptible to antibody-complement lysis. Convalescent sera from experimentally infected calves were used in a competitive radioimmunoassay to confirm that each antigenic area on the gI, gIII, or gIV glycoproteins was a target for bovine antibodies during primary infection with BHV-1.     

Structural, functional, and immunological characterization of bovine herpesvirus-1 glycoprotein gl expressed by recombinant baculovirus.

The major glycoprotein complex gl of bovine herpesvirus-1 was expressed at high levels (36 micrograms per 1 x 10(6) cells) in insect cells using a recombinant baculovirus. The recombinant gl had an apparent molecular weight of 116 kDa and was partially cleaved to yield 63-kDa (glb) and 52-kDa (glc) subunits. This processing step was significantly less efficient in insect cells than the analogous step in mammalian cells, even though the cleavage sites of authentic and recombinant gl were shown to be identical. The oligosaccharide linkages were mostly endoglycosidase-H-sensitive, in contrast to those of authentic gl, which has mostly endoglycosidase-H-resistant linkages and an apparent molecular weight of 130/74/55 kDa. Despite the reduced cleavage and altered glycosylation, the recombinant glycoprotein was transported and expressed on the surface of infected insect cells. These surface molecules were biologically active as demonstrated by their ability to induce cell-cell fusion. Fusion was inhibited by three monoclonal antibodies specific for antigenic domains I and IV on gl. Domain I maps to the extracellular region of the carboxy terminal fragment glc and domain IV to the very amino terminus of the glb fragment, indicating that domains mapping in two distinct regions of gl function in cell fusion. Monoclonal antibodies specific for eight different epitopes recognized recombinant gl, indicating that the antigenic characteristics of the recombinant and authentic glycoproteins are similar. In addition, the recombinant gl was as immunogenic as the authentic gl, resulting in the induction of gl-specific antibodies in cattle.     

Immunization with a dicistronic plasmid expressing a truncated form of bovine herpesvirus-1 glycoprotein D and the amino-terminal subunit of glycoprotein B results in reduced gB-specific immune responses

As an approach to create a divalent DNA vaccine, a truncated secreted version of bovine herpesvirus-1 (BHV-1) glycoprotein D (tgD) and the amino-terminal subunit of glycoprotein B (gBb) were expressed from a dicistronic plasmid, designated pSLIAtgD-IRES-gBb. Intradermal immunization of mice with pSLIAtgD-IRES-gBb or a mixture of plasmids encoding tgD (pSLIAtgD) and gBb (pSLIAgBb) by needle injection or gene gun elicited strong tgD-specific immune responses. However, a significant reduction in gBb-specific immune responses was observed upon immunization of mice with pSLIAtgD-IRES-gBb or a mixture of pSLIAtgD and pSLIAgBb in comparison to immunization with pSLIAgBb alone. This reduction in gBb-specific immune responses induced by pSLIAtgD-IRES-gBb was due to production of low amounts of gBb from pSLIAtgD-IRES-gBb, inefficient processing and transport of gBb, and possibly competition for antigen-presenting cells by tgD and gBb. These results indicate that, although divalent plasmids may be used to express different antigens, the efficacy of vaccination with such plasmids may be influenced by the plasmid design and the characteristics of the expressed antigens.     

Growth behavior of bovine herpesvirus-1 in permissive and semi-permissive cells.

Bovine herpesvirus-1 (BHV-1) can replicate well in bovine-derived cell lines such as Madin Darby bovine kidney (MDBK) but grows poorly in hamster lung (HmLu-1). Virus replication, DNA synthesis, and immediate-early gene expression are severely restricted in HmLu-1. We compared adsorption and penetration of BHV-1 in permissive MDBK and semi-permissive HmLu-1 cells. At a low multiplicity of infection, BHV-1 attached to permissive MDBK cells twice as much as to HmLu-1. The presence of heparin inhibited the attachment of BHV-1 to MDBK cells by about 60%, but over 90% of the attachment was inhibited in HmLu-1. To investigate the penetration of BHV-1, we performed the quantitative measurement of viral DNA by quantitative competitive (QC)PCR in infected cells. In MDBK cells, virions attached to the cell surface, penetrated into the cells and were transported to the nucleus. However in HmLu-1, only a small fraction of the virions attached to the cell surface were allowed to penetrate. Our results indicated that the replication of BHV-1 in semi-permissive HmLu-1 was not dramatically restricted at one certain point but at some various stages including adsorption and penetration.     

Modulation of immune responses to bovine herpesvirus-1 in cattle by immunization with a DNA vaccine encoding glycoprotein D as a fusion protein with bovine CD154

The objective of this study was to determine whether a DNA vaccine encoding bovine CD154 linked to a truncated version of bovine herpesvirus-1 (BHV-1) glycoprotein D (tgD-CD154) induces enhanced tgD-specific immune responses in cattle. In vitro characterization demonstrated that tgD and tgD-CD154 both bind to cultured bovine B cells, whereas only tgD-CD154 induces interleukin-4-dependent proliferation, suggesting that tgD-CD154 specifically binds the CD40 receptor and induces receptor signalling. Calves were immunized with plasmid encoding either tgD or tgD-CD154 by intradermal injection with a needle-free device. After two immunizations, tgD-specific immune responses were observed in both vaccinated groups and after challenge with BHV-1 these responses further increased. Animals immunized with plasmid encoding tgD-CD154 had significantly higher tgD-specific serum titres of immunoglobulins G and A but significantly lower numbers of tgD-specific interferon-gamma-secreting cells than animals immunized with plasmid encoding tgD after BHV-1 challenge. This suggests that the expression of an antigen as a chimeric protein with CD154 can qualitatively alter immune responses in cattle. Since we previously showed that plasmid encoding tgD-CD154 induces significantly enhanced secondary tgD-specific antibody responses in sheep, there appear to be interspecies differences in the immune responses induced by tgD-CD154, which suggests that both proteins in the chimeric molecule may influence protein targeting and the induction of an immune response.     

Role of bovine adenovirus-3 33K protein in viral replication

The L6 region of bovine adenovirus type (BAdV)-3 encodes a nonstructural protein named 33K. To identify and characterize the 33K protein, rabbit polyclonal antiserum was raised against a 33K-GST fusion protein expressed in bacteria. Anti-33K serum immunoprecipitated a protein of 42 kDa in in vitro translated and transcribed mRNA of 33K. However, three proteins of 42, 38, and 33 kDa were detected in BAdV-3 infected cells. To determine the role of this protein in virus replication, a recombinant BAV-33S1 containing insertional inactivation of 33K (a stop codon created at the seventh amino acid of 33K ORF) was constructed. Although BAV-33S1 could be isolated, the mutant showed a severe defect in the production of progeny virus. Inactivation of the 33K gene showed no effect on early and late viral gene expression in cells infected with BAV-33S1. However, formation of mature virions was significantly reduced in cells infected with BAV-33S1. Surprisingly, insertional inactivation of 33K at amino acid 97 (pFBAV-33.KS2) proved lethal for virus production. Although expression of early or late genes was not affected, no capsid formation could be observed in mutant DNA-transfected cells. These results suggest that 33K is required for capsid assembly and efficient DNA capsid interaction.     

Biolistic-mediated gene transfer using the bovine herpesvirus-1 glycoprotein D is an effective delivery system to induce neutralizing antibodies in its natural host

A genetic vaccine consisting of the bovine herpesvirus-1 (BHV-1) glycoprotein D (gD) gene was constructed and administered to cattle using the biolistic (gene-gun) process. Results were compared to standard intramuscular injection of an inactivated whole BHV-1 commercial vaccine. Cattle genetically immunized by the gene-gun-delivered gD subunit vaccine developed high titers of IgG antibodies specific to gD demonstrating that this immunization method is a potent humoral response inducer. Further, gene-gun vaccinated cattle produced high neutralizing antibody titers to BHV-1 similar to levels induced in the commercial vaccine immunized animals. Additionally, cellular immunity was measured by an increased level of IFN-gamma mRNA detected in PBMC of cattle immunized with the gD gene or with the commercial vaccine, whereas augmented levels of IL-4 were not detected following vaccination. Because of its simplicity and effectiveness in inducing an immune response in cattle similar to a commercial vaccine, gene-gun delivery of a subunit BHV-1 gD vaccine would be a viable alternative to current immunization protocols.     

Induction of immune response to bovine herpesvirus-1 with anti-idiotypic antibodies.

Previously, we prepared rabbit anti-idiotypic (anti-Id) antibodies against murine monoclonal antibodies (MAbs) specific for the major bovine herpesvirus-1 (BHV-1) envelope glycoproteins. Glycoprotein III (gIII) contains neutralization epitopes and may be the virus attachment protein. Anti-Id antibodies to a neutralizing MAb that reacts with gIII were purified by sequential immunoaffinity chromatography. Immune responses to the purified anti-Id reagent and BHV-1 were compared in mice. Both groups of mice produced BHV-1-specific neutralizing antibodies. However, lymphocyte proliferative responses and interferon and interleukin-2 production were specific for the respective immunizing antigens. These results suggest that the anti-Id reagent may bear an internal image of a B-cell-stimulating epitope of glycoprotein gIII; however, this epitope does not stimulate a virus-specific cellular immune response in mice.     

Suppository-mediated DNA immunization induces mucosal immunity against bovine herpesvirus-1 in cattle.

Mucosal surfaces are the primary sites for the transmission of infectious agents including viruses, so effective vaccines generally should induce mucosal immunity. Furthermore, noninvasive delivery is desirable because of the ease of application, the high degree of patient compliance, and the improved safety for patients and clinicians due to the elimination of needles. Unfortunately, most of the conventional vaccines are parenterally administered and result in systemic rather than mucosal immunity. Here we present the first report of mucosal immunity by noninvasive DNA immunization in a target species. As an approach to induce mucosal immunity against bovine herpesvirus-1, cows were immunized intravaginally with suppositories containing plasmid coding for glycoprotein D. Serum IgG, as well as IgA both in the serum and in the nasal fluids, were detected, which supports the contention of a common mucosal immune system. This level of immunity was of sufficient magnitude to minimize weight loss and significantly reduce the duration of virus shedding after intranasal viral challenge, which demonstrates the efficacy of suppository-based administration of DNA vaccines to target species. As this is a very practical method of delivery, it has great potential to be applied as vaccine or therapy in a variety of species.     

Genetic analysis of the 3' early region transformation and replication functions of bovine papillomavirus type 1

Cell transformation by BPV-1 is dependent on sequences confined to a region comprising only 69% of the viral DNA. Analysis of this subgenomic fragment of the viral genome has revealed a composite of functions. To further study this region of the BPV-1 genome, we constructed mutants affecting the 3' half of the transforming region. These mutations have revealed a plasmid maintenance function for the E2 ORF and a requirement that the 3' half of E5 be intact for in vitro mouse cell transformation. Cotransfection experiments revealed that phenotypic complementation (wild type transformation efficiency and autonomous replication of viral DNA) resulted in recombination between input plasmids in the majority of cases. However, rare cases were encountered where the wild type phenotype was observed with no evidence of recombination between input plasmids. These data suggest trans-acting functions of the E2 and E5 ORFs in viral DNA replication and the E5 ORF in cell transformation.     

Occurrence of bovine herpesvirus-1 DNA in nucleosomes and chromatin of bovine herpesvirus-1-infected cells: identification of a virion-associated protein in chromatin of infected cells

During virus replication a fraction of the intranuclear DNA of bovine herpesvirus-1 (BHV-1) was present in the nucleosomal structure of infected eukaryotic cells, and virion proteins were associated with the chromatin of virus infected cells. Synthesis of BHV-1 DNA in bovine embryonic lung (BEL) cells was found to begin four to six hours post-infection (p.i.) and to continue until at least 24 hours p.i. Chromatin isolated from infected cell nuclei at ten hours p.i. contained both BHV-1 viral and cell DNA. No BHV-1 DNA was found in mock-infected cell chromatin. Micrococcal nuclease cleavage products of both mock-infected and BHV-1-infected BEL cell nuclei produced monomers and multimers of unit fragment size which were indistinguishable from each other and displayed a typical nucleosome pattern on agarose gels. Southern analyses of micrococcal nuclease digests of infected cell nuclei indicated that some of the intranuclear BHV-1 DNA was present in a nucleosomal form. Three new proteins (with approximate molecular weights: 125,000, 42,000, and 17,000) were identified in chromatin isolated from BHV-1-infected BEL cells at ten hours p.i. These proteins were not present in mock-infected BEL cell chromatin. The 17,000 molecular weight protein was recognized by BHV-1 virion specific antisera. Neither of the two larger proteins appear to bind DNA from BHV-1. The smallest protein co-migrates with cellular histones, but no DNA binding proteins with the same molecular weight were found in the virion.     

Intracellular trafficking of VP22 in bovine herpesvirus-1 infected cells

The intracellular trafficking of different VP22-enhanced yellow fluorescent protein (EYFP) fusion proteins expressed by bovine herpesvirus-1 (BHV-1) recombinants was examined by live-cell imaging. Our results demonstrate that (i) the fusion of EYFP to the C terminus of VP22 does not alter the trafficking of the protein in infected cells, (ii) VP22 expressed during BHV-1 infection translocates to the nucleus through three different pathways, namely early mitosis-dependent nuclear translocation, late massive nuclear translocation that follows a prolonged cytoplasmic stage of the protein in non-mitotic cells, and accumulation of a small subset of VP22 in discrete dot-like nuclear domains during its early cytoplasmic stage, (iii) the addition of the SV40 large-T-antigen nuclear localization signal (NLS) to VP22-EYFP abrogates its early cytoplasmic stage, and (iv) the VP22 ¹³¹PRPR¹³⁴ NLS is not required for the late massive nuclear translocation of the protein, but this motif is essential for the targeting of VP22 to discrete dot-like nuclear domains during the early cytoplasmic stage. These results show that the amount of VP22 in the nucleus is precisely regulated at different stages of BHV-1 infection and suggest that the early pathways of VP22 nuclear accumulation may be more relevant to the infection process as the late massive nuclear influx starts when most of the viral progeny has already emerged from the cell.