Glycoprotein IV of bovine herpesvirus 1-expressing cell line complements and rescues a conditionally lethal viral mutant.

Glycoprotein IV (gIV) of bovine herpesvirus 1 (BHV-1), a homolog of herpes simplex virus glycoprotein D, represents a major component of the viral envelope and a dominant immunogen. To analyze the functional role of gIV during BHV-1 replication, cell line BUIV3-7, which constitutively expresses gIV, was constructed and used for the isolation of gIV- BHV-1 mutant 80-221, in which the gIV gene was replaced by a lacZ expression cassette. On complementing gIV-expressing cells, the gIV- BHV-1 replicated normally but was unable to form plaques and infectious progeny on noncomplementing cells. Further analysis showed that gIV is essential for BHV-1 entry into target cells, whereas viral gene expression, DNA replication, and envelopment appear unchanged in both noncomplementing and complementing cells infected with phenotypically complemented gIV- BHV-1. The block in entry could be overcome by polyethylene glycol-induced membrane fusion. After passaging of gIV- BHV-1 on complementing cells, a rescued variant, BHV-1res, was isolated and shown to underexpress gIV in comparison with its wild-type parent. Comparison of the penetration kinetics of BHV-1 wild type, phenotypically complemented gIV- BHV-1, and BHV-1res indicated that penetration efficiency correlated with the amount of gIV present in virus particles. In conclusion, we show that gIV of BHV-1 is an essential component of the virion involved in virus entry and that the amount of gIV in the viral envelope modulates the penetration efficiency of the virus.     

Molecular cloning, sequencing, and expression of functional bovine herpesvirus 1 glycoprotein gIV in transfected bovine cells.

The gene encoding bovine herpesvirus 1 (BHV-1) glycoprotein gIV was mapped, cloned, and sequenced. The gene is situated between map units 0.892 and 0.902 and encodes a predicted protein of 417 amino acids with a signal sequence cleavage site between amino acids 18 and 19. Comparison of the BHV-1 amino acid sequence with the homologous glycoproteins of other alphaherpesviruses, including herpes simplex virus type 1 glycoprotein gD, revealed significant homology in the amino-terminal half of the molecules, including six invariant cysteine residues. The identity of the open reading frame was verified by expression of the authentic recombinant BHV-1 gIV in bovine cells by using eucaryotic expression vectors pRSDneo (strong, constitutive promoter) and pMSG (weak, dexamethasone-inducible promoter). Constitutive expression of gIV proved toxic to cells, since stable cell lines could only be established when the gIV gene was placed under the control of an inducible promoter. Expression of gIV was cell associated and localized predominantly in the perinuclear region, although nuclear and plasma membrane staining was also observed. Radioimmunoprecipitation revealed that the recombinant glycoprotein was efficiently processed and had a molecular weight similar to that of the native form of gIV expressed in BHV-1-infected bovine cells. Recombinant gIV produced in the transfected bovine cells induced cell fusion, polykaryon formation, and nuclear fusion. In addition, expression of gIV interfered with BHV-1 replication in the transfected bovine cells.     

Expression of bovine herpesvirus 1 glycoprotein gIV by recombinant baculovirus and analysis of its immunogenic properties.

The gene encoding the gIV glycoprotein of bovine herpesvirus 1 has been inserted into the genome of Autographa californica baculovirus in lieu of the coding region of the A. californica baculovirus polyhedrin gene. Recombinant protein was identified by its reactivity with gIV-specific monoclonal antibodies and expressed at high levels (about 85 micrograms per 2.5 x 10(6) cells) in Spodoptera frugiperda (SF9) cells. The recombinant glycoprotein had an apparent molecular mass of 63 kDa, indicating that it was incompletely glycosylated. However, it was transported to and expressed on the cell surface of infected SF9 cells. Furthermore, reactivity with polyclonal and monoclonal antibodies specific for gIV suggested that most epitopes were functionally unaltered on the recombinant gIV. Immunization of cattle with recombinant gIV in crude, partially purified, or pure form resulted in the induction of neutralizing antibodies to BHV-1, which were reactive with authentic gIV. However, the neutralizing antibody titers were lower than those elicited by an equivalent amount of affinity-purified authentic gIV, which appeared to be mainly due to reduced recognition of one of the neutralizing antigenic domains of gIV, designated domain I. The potential use of this recombinant gIV glycoprotein as a vaccine to bovine herpesvirus 1 infection in cattle is discussed.     

Bovine cells expressing bovine herpesvirus 1 (BHV-1) glycoprotein IV resist infection by BHV-1, herpes simplex virus, and pseudorabies virus.

We expressed the bovine herpesvirus 1 (BHV-1) glycoprotein IV (gIV) in bovine cells. The protein expressed was identical in molecular mass and antigenic reactivity to the native gIV protein but was localized in the cytoplasm. Expressing cells were partially resistant to BHV-1, herpes simplex virus, and pseudorabies virus, as shown by a 10- to 1,000-fold-lower number of plaques forming on these cells than on control cells. The level of resistance depended on the level of gIV expression and the type and amount of challenge virus. These data are consistent with previous reports by others that cellular expression of the BHV-1 gIV homologs, herpes simplex virus glycoprotein D, and pseudorabies virus glycoprotein gp50 provide partial resistance against infection with these viruses. We have extended these findings by showing that once BHV-1 enters gIV-expressing cells, it replicates and spreads normally, as shown by the normal size of BHV-1 plaques and the delayed but vigorous synthesis of viral proteins. Our data are consistent with the binding of BHV-1 gIV to a cellular receptor required for initial penetration by all three herpesviruses and interference with the function of that receptor molecule.     

Inhibition of antigen-induced and interleukin-2-induced proliferation of bovine peripheral blood leukocytes by inactivated bovine herpes virus 1.

The mechanism by which bovine herpesvirus 1 (BHV-1) predisposes cattle to bacterial pneumonia was investigated by using an in vitro system to demonstrate immunosuppression. At a multiplicity of infection of 0.001, live or inactivated BHV-1 induced a 50% inhibition of the proliferative response of peripheral blood mononuclear leukocytes to antigen (vaccinia virus in vaccinia virus-immunized cattle which were BHV-1 negative) or interleukin-2. At this same multiplicity of infection, the mitogen-induced proliferation of peripheral blood mononuclear leukocytes was unaffected. This inhibition of antigen and interleukin-2-induced proliferative responses could not be reversed by the addition of excess amounts of interleukin-2 and could not be prevented by the addition of indomethacin to block prostaglandin production. Antibodies to BHV-1, especially those specific for glycoproteins gI and gIV, were able to block the inhibitory effect of BHV-1 in these in vitro assays. These results showed that antibody to BHV-1 blocks the immunosuppressive effect of the virus in vitro and suggested that an appropriate antibody response to BHV-1 could protect cattle from virus-induced immunosuppression leading to secondary bacterial pneumonia.     

Bovine natural killer-like cell responses against cell lines expressing recombinant bovine herpesvirus type 1 glycoproteins

The murine retrovirus shuttle vector pZipNeo SV(X)1 was used to construct plasmids encoding the three major surface glycoproteins of bovine herpesvirus-1 (BHV-1). Each plasmid was transfected into D17, a canine osteosarcoma cell line sensitive to lysis by bovine NK-like cells when infected with BHV-1. After selection in G418 sulfate, cell lines expressing the recombinant gene products were sorted by flow microfluorimetry, radioimmunoprecipitated, and analyzed by SDS-PAGE for fidelity as compared to the native viral glycoproteins. Two of the three genetically engineered cell lines (gI and gIV) could successfully serve as targets to detect bovine NK-like cytolysis. These findings support and extend a previous study from our laboratory indicating a role for BHV-1 glycoproteins in the cytolytic response by bovine NK-like cells. Additionally, this study demonstrated that individual proteins are recognized by these effector cells, and that recombinant glycoproteins can direct cytolytic activity in the absence of host cell infection-associated proteins. This is the first known report of Ag directed cytotoxicity by bovine null (non-B, non-T) cells.     

Bovine herpesvirus 1 attachment to permissive cells is mediated by its major glycoproteins gI, gIII, and gIV.

A bovine herpesvirus 1 (BHV-1) gIII deletion mutant (gIII-) was produced by means of recombinant DNA that retained the ability to replicate in cell culture. However, the gIII- mutant was functionally defective, showing impaired attachment to permissive cells, a delay in virus replication, and reduced extracellular virus production. The attachment defect exhibited by the gIII- mutant is an indication of the role played by gIII in the normal infection process. This was shown by dramatically decreased binding of radiolabelled gIII- virus to permissive cells and a slower adsorption rate, as measured by plaque formation, than the wild-type (wt) virus. Furthermore, treatment of the gIII- virus with neomycin increased virus adsorption and plaque formation by severalfold, whereas neomycin treatment had no effect on the wt virus. This observation showed that the gIII- mutant was strictly defective in adsorption but fully competent to produce productive infections once induced to attach. The gIII- mutant showed greater sensitivities than did the wt virus to anti-gI and anti-gIV antibody-mediated neutralization. Analyses with panels of monoclonal antibodies to gI and gIV revealed that the epitopes gI-IV and gIV-III were the main targets for enhanced neutralization. This provided evidence that gI and gIV may also participate in virus attachment. Finally, when affinity-purified gI, gIII, and gIV were tested for their ability to inhibit virus adsorption, gIII had the most pronounced inhibitory effect, followed by gI and then gIV. gIII was able to completely inhibit wt virus adsorption, and at a high concentration, it also partially inhibited the gIII- mutant. gI and gIV inhibited wt and gIII- mutant adsorption to a comparable extent. Our results collectively indicate that gIII plays a predominant role in virus attachment, but gI and gIV also contribute to this process. In addition, a potential cooperative mechanism for virus attachment with these three proteins is presented.     

Lymphocyte proliferative responses to separated bovine herpesvirus 1 proteins in immune cattle.

The immune response to bovine herpesvirus 1 (BHV-1) infection can protect cattle from subsequent challenge with the virus. This protection involves a variety of defensive strategies, and the activation of most of these defenses requires the recognition of viral proteins by the cellular immune system. To identify some of the BHV-1 proteins recognized by T lymphocytes, we measured in vitro proliferative responses to individual proteins. Viral proteins were separated by gel electrophoresis followed by Western immunoblotting, and immunoblots were evaluated for serological reactions. Unstained blotted fractions were processed into antigen-bearing particles for analysis in blastogenesis assays. Purified BHV-1 proteins obtained by immunoadsorbent chromatography were processed and included for comparison in both enzyme-linked immunosorbent and proliferation assays. The tegument protein VP8 and the glycoprotein gIV appeared to be the antigens which most consistently stimulated the proliferation of lymphocytes from BHV-1-immunized animals. Positive blastogenic responses were also detected to gI, gIII, and to one or more uncharacterized, low-molecular-weight proteins in some of the cattle tested. These results indicate that T-lymphocyte proliferative responses to BHV-1 proteins are detectable in immune cattle and may be important in protection from BHV-1 infection.     

Bovine herpesvirus 1 VP22 enhances the efficacy of a DNA vaccine in cattle

For this study, the intercellular trafficking ability of bovine herpesvirus 1 (BHV-1) VP22 was applied to improve the efficacy of a DNA vaccine in calves. A plasmid encoding a truncated version of glycoprotein D (tgD) fused to VP22 was constructed. The plasmid encoding tgD-VP22 elicited significantly enhanced and more balanced immune responses than those induced by a plasmid encoding tgD. Furthermore, protection against a BHV-1 challenge was obtained in calves immunized with the plasmid encoding tgD-VP22, as shown by significant reductions in viral excretion. However, less significant protection was observed for animals vaccinated with the tgD-expressing plasmid, correlating with the lower level of immunity observed prechallenge. This is the first report of the use of VP22 as a transport molecule in the context of a DNA vaccine for a large animal species.     

Expression of bovine herpesvirus 1 glycoproteins gI and gIII in transfected murine cells.

Genes encoding two of the major glycoproteins of bovine herpesvirus 1 (BHV-1), gI and gIII, were cloned into the eucaryotic expression vectors pRSVcat and pSV2neo and transfected into murine LMTK- cells, and cloned cell lines were established. The relative amounts of gI or gIII expressed from the two vectors were similar. Expression of gI was cell associated and localized predominantly in the perinuclear region, but nuclear and plasma membrane staining was also observed. Expression of gI was additionally associated with cell fusion and the formation of polykaryons and giant cells. Expression of gIII was localized predominantly in the nuclear and plasma membranes. Radioimmunoprecipitation in the presence or absence of tunicamycin revealed that the recombinant glycoproteins were proteolytically processed and glycosylated and had molecular weights similar to those of the forms of gI and gIII expressed in BHV-1-infected bovine cells. However, both recombinant glycoproteins were glycosylated to a lesser extent than were the forms found in BHV-1-infected bovine cells. For gI, a deficiency in N-linked glycosylation of the amino-terminal half of the protein was identified; for gIII, a deficiency in O-linked glycosylation was implicated. The reactivity pattern of a panel of gI- and gIII-specific monoclonal antibodies, including six which recognize conformation-dependent epitopes, was found to be unaffected by the glycosylation differences and was identical for transfected or BHV-1-infected murine cells. Use of the transfected cells as targets in immune-mediated cytotoxicity assays demonstrated the functional recognition of recombinant gI and gIII by murine antibody and cytotoxic T lymphocytes. Immunization of mice with the transfected cells elicited BHV-1-specific virus-neutralizing antibody, thus verifying the antigenic authenticity of the recombinant glycoproteins and the important role of gI and gIII as targets of the immune response to BHV-1 in this murine model system.     

Role of N-linked glycans in antigenicity, processing, and cell surface expression of bovine herpesvirus 1 glycoprotein gIV.

Glycoprotein gIV, a structural component of bovine herpesvirus type 1, stimulates high titers of virus-neutralizing antibody. The protein contains three potential sites for the addition of N-linked carbohydrates. Three mutants were constructed by oligonucleotide-directed mutagenesis, in each case changing one N-linked glycosylation site from Asn-X-Thr/Ser to Ser-X-Thr/Ser. A fourth mutant was altered at two sites. The altered forms of the gIV gene were cloned into a vaccinia virus transfer vector to generate recombinant vaccinia viruses expressing mutant proteins. Analysis of these mutants revealed that only two (residues 41 and 102) of the three (residues 41, 102, and 411) potential sites for the addition of N-linked glycans are actually utilized. Absence of glycans at residue 41 (gN1) showed no significant effect on the conformation of the protein or induction of a serum neutralizing antibody response. However, mutant proteins lacking glycans at residue 102 (gN2) or residues 41 and 102 (gN1N2) showed altered reactivity with conformation-dependent gIV-specific monoclonal antibodies. These mutants also induced significantly lower serum neutralizing antibody responses than wild-type gIV. Nonetheless, each of the mutant proteins were modified by the addition of O-glycans and transported to the cell surface. Our results demonstrate that absence of N-linked glycans at one (residue 102) or both (residues 41 and 102) utilized N-linked glycosylation sites alters the conformation but does not prevent processing and transport of gIV to the cell surface.     

Characterization of the bovine herpesvirus 4 major immediate-early transcript.

The major immediate-early (IE) RNA of bovine herpesvirus 4 (BHV-4) has been identified and characterized by analyzing cytoplasmic polyadenylated RNA isolated from Madin-Darby bovine kidney cells infected with BHV-4(DN-599) in the presence of cycloheximide. Hybridization of cDNA to Southern blots of viral DNA, Northern (RNA) blot analysis, and S1 nuclease analyses showed that the major BHV-4 IE RNA is a spliced, 1.7-kb RNA, which is transcribed from right to left on the restriction map of the BHV-4 genome from DNA contained in the 8.3-kb HindIII fragment E. The major IE RNA contains three small exons at its 5' end, spliced to a 1.3-kb 3' exon. This RNA is present in much-reduced amounts when cells are infected in the absence of cycloheximide. However, late in infection, the major IE RNA gene region encodes abundant RNAs which differ in structure from the major IE RNA. Nucleotide sequence analysis of the gene encoding the major IE RNA revealed an open reading frame encoding 284 amino acids. A homology search of amino acid sequence data bases showed that a 141-amino-acid region near the amino terminus of the predicted amino acid sequence is similar to sequences near the amino terminus of herpes simplex virus type 1 IE110. This region of homology includes CXXC pairs, which could be involved in zinc finger structures. The region encoding this putative zinc finger domain is also found in RNAs transcribed from this IE region late in infection, but it is spliced to different sequences than those used in IE RNA. Thus, the major IE region of the BHV-4 genome could encode a family of proteins sharing a zinc finger domain.     

Bovine monoclonal anti-idiotypes induce antibodies specific for a synthetic peptide bearing a neutralizing epitope of bovine herpesvirus 1 glycoprotein gI (gB).

Bovine monoclonal anti-Id mimicking a neutralizing epitope of bovine herpesvirus-1 (BHV-1) glycoprotein gI were developed. An epitope present on the 74K subunit of gI identified by a murine mAb 1E11 was selected for this study. Bovine lymphocytes from the prefemoral lymph node of a heifer immunized with mAb 1E11 were fused with SP-2/0, a nonsecreting murine cell-line. Two bovine x murine hybridomas secreting bovine monoclonal anti-Id specific for the Id of 1E11 were stabilized. These anti-Id inhibited the binding of 1E11 to purified glycoprotein gI in a dose-dependent fashion. Naive mice immunized with the anti-Id produced anti-anti-Id (Ab3) that reacted with BHV-1 glycoprotein gI in a RIA, and neutralized BHV-1 infection in vitro. The Ab3 also showed reactivity to the 74K subunit of authentic gI glycoprotein in a Western blot analysis, and to the synthetic peptide bearing the 1E11 epitope in a RIA. These results substantiate the presence of the population of anti-Ab2 that functionally resemble antibodies specific for the immunizing Ag BHV-1 in Ab3, and demonstrate the ability of these anti-Id to elicit BHV-1-specific antibody response.     

Bovine herpesvirus 1 UL49.5 homolog gene encodes a novel viral envelope protein that forms a disulfide-linked complex with a second virion structural protein.

We previously reported that the genome of bovine herpesvirus 1 (BHV-1) contains an open reading frame (ORF) homologous to the herpes simplex virus UL49.5 ORF, and as with the herpes simplex virus UL49.5 ORF, the deduced amino acid sequence of the BHV-1 UL49.5 homolog (UL49.5h) contains features characteristic of an integral membrane protein, implying that it may constitute a functional gene encoding a novel viral envelope protein. This communication reports on the identification of the BHV-1 UL49.5h gene product. By employing an antibody against a synthetic BHV-1 UL49.5h peptide and an UL49.5h gene deletion mutant, the primary product of BHV-UL49.5h gene was identified as a polypeptide with a size of approximately 9 kDa; in both infected cells and isolated virions, the UL49.5h products were found to exist in three forms; monomer, disulfide-linked homodimer, and disulfide-linked heterodimer containing a second viral protein with a size of about 39 kDa. O-Glycosidase digestion and [3H]glucosamine labelling experiments showed that the UL49.5h protein is not glycosylated. Although the deduced amino acid sequence contains putative sites for myristylation and phosphorylation, we were unable to detect either modification. Surface labelling and trypsin digestion protection experiments showed that the BHV-1 UL49.5h protein was present on the surface of infected cells and on the surface of mature virions. Nonionic detergent partition of isolated virions revealed that the UL49.5h protein is more tightly associated with the virion tegument-nucleocapsid structure than envelope protein gD. The results from this study demonstrate that the BHV-1 UL49.5h gene encodes a nonglycosylated virion envelope protein which may associate with virion internal structures by forming a complex with the 39-kDa virion structural protein.     

Interference of bovine herpesvirus 1 (BHV-1) in sorbitol-Induced apoptosis

In order to determine the ability of bovine herpesvirus type 1 (BHV-1) to suppress apoptosis, we examined the effects of BHV-1 infection on sorbitol-induced apoptosis on Madin-Darby bovine kidney (MDBK) cells. BHV-1 suppresses sorbitol-induced apoptosis in a manner similar to that of herpes simplex virus type 1 (HSV-1), indicating that BHV-1 has one or more anti-apoptotic genes. To elucidate the molecular mechanisms of apoptosis, expression of some genes encoding apoptosis-inhibiting and -promoting factors were analyzed on BHV-1 infected cells during the process of sorbitol-induced apoptosis. Our results revealed that the expression of bcl-2 and bcl-x(L) decreased after 5 and 3 h p.i., respectively; while bax and procaspase-3 expression increased with respect to control as a function of p.i. times and at 7 h p.i. they were not observed. We further show that the expression of p53 gene was also enhanced, suggesting that this apoptotic mechanism is p53 dependent. From these results, we propose that BHV-1 has one or more genes encoding apoptosis-inhibiting factors which interfere with the involvement of bcl-2 gene family members and apoptotic pathway, depending upon caspase-3, triggered by sorbitol.     

IL—18DNA免疫对HIV—1核酸疫苗诱导的免疫应答的影响

为了研究白细胞介素-18(IL-18)基因对人免疫缺陷病毒(HIV-1)核酸疫苗诱导免疫应答的影响,将人IL-18基因插入到真核表达载体pVAX1中,构建了真核表达载体pVAX1-IL-18;将pCI-neoGAG联合pVAX1-IL-18或者pCI-neoGAG单独免疫Balb/c小鼠,检测免疫小鼠的特异性抗体和IFN-γ,同时观察免疫小鼠脾淋巴细胞增殖和小鼠特异性细胞毒性T淋巴细胞(CTL)反应。酶切及测序结果表明成功地构建了人IL-18基因真核表达载体;与pCI-neoGAG免疫组比较,pCI-neoGAG联合pVAX1-IL-18免疫组小鼠血清的抗HIV-1p24抗体滴度降低(P<0.01);而与pCI-neoGAG免疫组比较,pCI-neoGAG联合pVAX1-IL-18免疫组小鼠血清的IFN-γ升高(P<0.01);pCI-neoGAG联合pVAX1-IL-18免疫组小鼠的脾淋巴细胞增殖实验刺激指数(SI)以及特异性CTL活性均高于pCI-neoGAG免疫组(P<0.01)。IL-18基因联合HIV-1核酸疫苗免疫小鼠,可能增强特异性Th1细胞和CTL反应,白细胞介素-18基因对体液免疫有抑制作用。     

The Us9 gene of bovine herpesvirus 1 (BHV-1) effectively complements a Us9-null strain of BHV-5 for anterograde transport, neurovirulence, and neuroinvasiveness in a rabbit model

The alphaherpesvirus envelope protein Us9 is a type II viral membrane protein that is required for anterograde spread of bovine herpesvirus 5 (BHV-5) infection from the olfactory receptor neurons to the brain. In a rabbit seizure model, Us9-deleted BHV-5 failed to invade the central nervous system (CNS) following intranasal infection. However, when injected directly into the olfactory bulb, retrograde-spread infection from the olfactory bulb (OB) to the piriform cortex and other areas connected to the OB was not affected. In contrast to BHV-5, wild-type BHV-1 failed to invade the CNS following intranasal infection. In this study, we show that mature BHV-1 Us9 is a 30- to 32-kDa protein, whereas mature BHV-5 Us9 is an 18- to 20-kDa protein. In vitro, BHV-1 Us9 is expressed at 3 h postinfection (hpi), whereas BHV-5 Us9 is expressed at 6 hpi. Despite these differences, BHV-1 Us9 not only complemented for BHV-5 Us9 and rescued the anterograde-spread defect of the BHV-5 Us9-deleted virus but conferred increased neurovirulence and neuroinvasiveness in our rabbit seizure model. Rabbits infected with BHV-5 expressing BHV-1 Us9 showed severe neurological signs at 5 days postinfection, which was 1 to 2 days earlier than BHV-5 wild-type or Us9-reverted BHV-5 virus. The data underscore the importance of both Us9 genes for virion anterograde transport and neuroinvasiveness. However, Us9 is not the determinant of the differential neuropathogenesis of BHV-1 and BHV-5.     

Low-volume jet injection for intradermal immunization in rabbits

Background

This study tested a low-volume (20–30 μl/20–30 μg DNA) jet injection method for intradermal delivery of a DNA vaccine. Jet injection offers the advantages of a needle-less system, low-cost, rapid preparation of the injected DNA solution, and a simple delivery system. More than one construct can be injected simultaneously and the method may be combined with adjuvants.

Results

Low-volume jet injection targeted delivery of a DNA solution exclusively to the dermis and epidermis of rabbits. A three injection series of plasmid DNA, encoding the Hepatitis B Surface Antigen stimulated a humoral immune response in 2/5 rabbits. One rabbit developed a significant rise in antibody titer after 1 injection and one following 2 injections. There were no significant differences between jet injection and particle bombardment in the maximal antibody titers or number of injections before response. A three injection series of the same plasmid DNA by particle bombardment elicited a significant rise in antibody titer in 3/5 rabbits. One rabbit developed antibody after 1 injection and two after 3 injections. In contrast, 0/5 rabbits receiving DNA by needle and syringe injection responded. In the jet injection and particle bombardment groups, gene expression levels in the skin did not predict response. While immune responses were similar, luciferase gene expression levels in the skin following particle bombardment were 10–100 times higher than jet injection.

Conclusion

Low-volume jet injection is a simple, effective methodology for intradermal DNA immunization.     

Bovine herpesvirus 5 (BHV-5) Us9 is essential for BHV-5 neuropathogenesis

Bovine herpesvirus 5 (BHV-5) is a neurovirulent alphaherpesvirus that causes fatal encephalitis in calves. In a rabbit model, the virus invades the central nervous system (CNS) anterogradely from the olfactory mucosa following intranasal infection. In addition to glycoproteins E and I (gE and gI, respectively), Us9 and its homologue in alphaherpesviruses are necessary for the viral anterograde spread from the presynaptic to postsynaptic neurons. The BHV-5 Us9 gene sequence was determined, and the predicted amino acid sequence of BHV-5 Us9 was compared with the corresponding Us9 sequences of BHV-1.1. Alignment results showed that they share 77% identity and 83% similarity. BHV-5 Us9 peptide-specific antibody recognized a doublet of 17- and 19-kDa protein bands in BHV-5-infected cell lysates and in purified virions. To determine the role of the BHV-5 Us9 gene in BHV-5 neuropathogenesis, a BHV-5 Us9 deletion recombinant was generated and its neurovirulence and neuroinvasive properties were compared with those of a Us9 rescue mutant of BHV-5 in a rabbit model. Following intranasal infection, the Us9 rescue mutant of BHV-5 displayed a wild-type level of neurovirulence and neural spread in the olfactory pathway, but the Us9 deletion mutant of BHV-5 was virtually avirulent and failed to invade the CNS. In the olfactory mucosa containing the olfactory receptor neurons, the Us9 deletion mutant virus replicated with an efficiency similar to that of the Us9 rescue mutant of BHV-5. However, the Us9 deletion mutant virus was not transported to the bulb. Confocal microscopy of the olfactory epithelium detected similar amounts of virus-specific antigens in the cell bodies of olfactory receptor neuron for both the viruses, but only the Us9 rescue mutant viral proteins were detected in the processes of the olfactory receptor neurons. When injected directly into the bulb, both viruses were equally neurovirulent, and they were transported retrogradely to areas connected to the bulb. Taken together, these results indicate that Us9 is essential for the anterograde spread of the virus from the olfactory mucosa to the bulb.