Virulence, immunogenicity and reactivation of bovine herpesvirus 1 mutants with a deletion in the gC, gG, gI, gE, or in both the gI and gE gene

Within the framework of developing a marker vaccine against bovine herpesvirus 1 (BHV1), several mutants with deletions in non-essential glycoprotein genes were constructed. Glycoprotein gC, gG, gI and gE single deletion mutants, a gI/gE double deletion mutant and a gE frame-shift mutant were made. The virulence and immunogenicity of these mutants were evaluated in specific-pathogen-free calves. Except for the gC deletion mutant, all mutants were significantly less virulent than the parental wild-type (wt) BHV1 strain Lam. The virulence of the gI and the gI-/gE- mutants was almost completely reduced. Upon challenge infection, the calves of the control group became severely ill, whereas all other calves remained healthy. The reduction of the virus shedding after challenge infection was related to the virulence of the strain of primary inoculation. Virus shedding was almost completely reduced in calves first inoculated with Lam-wt or with gC- and the least reduced in calves inoculated with gI- or gI-/gE-. Six weeks after challenge, all calves were treated with dexamethasone to study whether mutant or challenge virus or both could be reactivated. The gC- and the gG- mutants were reactivated, whereas none of the other mutants were reisolated. Reactivation of challenge virus was reduced in all calves inoculated with mutant viruses. The gC deletion mutant was too virulent and the gI and the gI/gE deletion mutants were the least immunogenic, but based on residual virulence and immunogenicity, both the gG and the gE deletion mutants are candidates for incorporation in live BHV1 vaccines. However, it also depends on the kinetics of the anti-gG and anti-gE antibody response after wild-type virus infection, whether these deletion mutants are really suitable to be incorporated in a marker vaccine.     

Aberrant expression of a cytokeratin in a subset of hepatocytes during chronic WHV infection

The cell culture-adapted KyA strain of equine herpesvirus type 1 (EHV-1) has been found to be attenuated in young horses (Matsumura et al., 1996, Vet. Microbiol. 48, 353-365). The KyA strain lacks at least six genes in its genome, including those encoding glycoproteins gE and gI. To elucidate whether EHV-1 glycoproteins gE and gI play a role in viral virulence, we have constructed an EHV-1 recombinant that has the genes encoding both gE and gI deleted from its genome and its revertant. Growth properties of the deletion mutant virus in vitro were compared with those of the parent and the revertant viruses. Plaque size of the mutant virus in fetal horse kidney (FHK) cells was significantly smaller than those of the parent and the revertant viruses. In one-step growth experiments, however, the yields of infectious virus from FHK cells infected with the deletion mutant, the parent, or the revertant virus were approximately the same. The results suggested that gE and/or gI of EHV-1 promoted cell-to-cell spread of the virus, but that these glycoproteins were not involved in the process of virus maturation and release or in virus attachment and penetration. Subsequently, the virulence of mutant and revertant viruses was examined in young horses. No clinical signs were observed in six horses, including three colostrum-deprived foals inoculated intranasally with the deletion mutant virus, whereas three colostrum-deprived foals inoculated intranasally with the revertant virus manifested clinical signs typical for EHV-1 respiratory infection (i.e., pyrexia, nasal discharge, and swelling of submandibular lymph nodes). The results obtained from in vivo studies revealed that the EHV-1 mutant defective in both gE and gI genes was avirulent in young horses, suggesting that gE and/or gI of the EHV-1 have an important role in EHV-1 virulence. However, the EHV-1 mutant defective in both gE and gI genes induced only a partial protectivity in inoculated foals from manifestation of respiratory symptoms after challenge infection.     

Mutational analysis of the role of glycoprotein I in varicella-zoster virus replication and its effects on glycoprotein E conformation and trafficking

The contributions of the glycoproteins gI (ORF67) and gE (ORF68) to varicella-zoster virus (VZV) replication were investigated in deletion mutants made by using cosmids with VZV DNA derived from the Oka strain. Deletion of both gI and gE prevented virus replication. Complete deletion of gI or deletions of 60% of the N terminus or 40% of the C terminus of gI resulted in a small plaque phenotype as well as reduced yields of infectious virus. Melanoma cells infected with gI deletion mutants formed abnormal polykaryocytes with a disrupted organization of nuclei. In the absence of intact gI, gE became localized in patches on the cell membrane, as demonstrated by confocal microscopy. A truncated N-terminal form of gI was transported to the cell surface, but its expression did not restore plaque morphology or infectivity. The fusogenic function of gH did not compensate for gI deletion or the associated disruption of the gE-gI complex. These experiments demonstrated that gI was dispensable for VZV replication in vitro, whereas gE appeared to be required. Although VZV gI was dispensable, its deletion or mutation resulted in a significant decrease in infectious virus yields, disrupted syncytium formation, and altered the conformation and distribution of gE in infected cells. Normal cell-to-cell spread and replication kinetics were restored when gI was expressed from a nonnative locus in the VZV genome. The expression of intact gI, the ORF67 gene product, is required for efficient membrane fusion during VZV replication.     

In vivo immune evasion mediated by the herpes simplex virus type 1 immunoglobulin G Fc receptor

Herpes simplex virus (HSV) glycoproteins gE and gI form an immunoglobulin G (IgG) Fc receptor (FcgammaR) that binds the Fc domain of human anti-HSV IgG and inhibits Fc-mediated immune functions in vitro. gE or gI deletion mutant viruses are avirulent, probably because gE and gI are also involved in cell-to-cell spread. In an effort to modify FcgammaR activity without affecting other gE functions, we constructed a mutant virus, NS-gE339, that has four amino acids inserted into gE within the domain homologous to mammalian IgG FcgammaRs. NS-gE339 expresses gE and gI, is FcgammaR-, and does not participate in antibody bipolar bridging since it does not block activities mediated by the Fc domain of anti-HSV IgG. In vivo studies were performed with mice because the HSV-1 FcgammaR does not bind murine IgG; therefore, the absence of an FcgammaR should not affect virulence in mice. NS-gE339 causes disease at the skin inoculation site comparably to wild-type and rescued viruses, indicating that the FcgammaR- mutant virus is pathogenic in animals. Mice were passively immunized with human anti-HSV IgG and then infected with mutant or wild-type virus. We postulated that the HSV-1 FcgammaR should protect wild-type virus from antibody attack. Human anti-HSV IgG greatly reduced viral titers and disease severity in NS-gE339-infected animals while having little effect on wild-type or rescued virus. We conclude that the HSV-1 FcgammaR enables the virus to evade antibody attack in vivo, which likely explains why antibodies are relatively ineffective against HSV infection.     

Surface-based labeling of cortical anatomy using a deformable atlas

With regard to BHV1 eradication programs in cattle it is important to know whether sheep can be a reservoir of BHV1. We therefore performed an experiment that consisted of three phases. In phase 1, 10 sheep were inoculated with high doses of BHV1 and kept in close contact with 5 sheep and 5 calves. All inoculated sheep excreted BHV1 between 8 and 15 days post inoculation and seroconverted. Although BHV1 was isolated from the nasal mucosa of 3 out of 5 sentinel sheep, none of the sentinel sheep produced antibodies against BHV1. One sentinel calf excreted BHV1 through days 12-17; the remaining 4 calves excreted BHV1 between days 18 and 24 suggesting that the first calf was infected by sheep and the remaining 4 sentinel calves were infected by that calf and not by sheep. The bacic reproduction ratio (R0) of BHV1 between sheep and calves was estimated at 0.1, and among calves it was estimated at > or = 9. In phase 2, all inoculated sheep were treated with dexamethasone and kept in close contact with 5 sheep and 5 calves. All dexamethasone treated sheep re-excreted BHV1 over a 6- to 9-day period. None of the sentinel animals seroconverted. In phase 3, the sentinel sheep and calves of phase 1 were kept in two groups and were treated with dexamethasone. None of the sentinel sheep re-excreted BHV1, whereas 3 out of 5 sentinel calves did. It is concluded that while BHV1 infection in sheep is possible, BHV1 does not spread from sheep easily to cattle.     

Stimulus specific adaptation in inferior temporal and medial temporal cortex of the monkey

We studied the early immunity induced by a live glycoprotein E (gE) negative bovine herpesvirus 1 (BHV1) marker vaccine. Three groups of specific-pathogen-free calves were either not vaccinated, or vaccinated two days or two hours before the introduction of a calf that was intranasally infected with wild-type BHV1 the day before. We quantified the shedding of gE-negative vaccine virus and of wild-type virus, using a double-staining immunoassay. In calves vaccinated two hours before the introduction of the infected calf, the shedding of wild-type virus was reduced, compared with that of the unvaccinated control calves. The shedding of wild-type virus was most significantly reduced in the calves that were vaccinated two days before: only very small amounts of wild-type virus were isolated. Wild-type virus was not detected at all in the samples from one of the five calves of that group. Furthermore, this calf was the only one in which we did not detect antibodies against gE. Hence, intranasal vaccination with a live gE-negative vaccine induced early immunity against a BHV1 contact infection. This suggests that this vaccine can be used efficaciously in the early stages of a BHV1 outbreak.     

Protective immunity of bovine herpesvirus-1 (BHV-1) recombinants which express pseudorabies virus (PRV) glycoproteins gB, gC, gD and gE

We have constructed recombinants of bovine herpesvirus-1 (BHV-1) which express pseudorabies virus (PRV) gB, gC, gD or gE either individually or in combination. To test the protective immunity, mice were inoculated with these BHV-1 recombinants and challenged three weeks later with virulent PRV. A BHV-1 recombinant, BHV-1/TF7-1, which express PRV gC, gD, gE and gI but not PRV gB, protected all 7 mice from the challenge with 20 LD50 virulent PRV and 6 out of 7 from the challenge with 100 LD50 PRV, while one dead mice survived for 5 days after the challenge. All the control mice died in 3 days. BHV-1 recombinants which express PRV gB, gC and gD individually also gave some protection but not so effective as BHV/TF7-1. Before challenging with virulent PRV, sera were collected from immunized mice and antibody against PRV was assayed. Western blot analyses indicated that all the recombinants induced antibody in mice against PRV gB, gC or gD individually or in combination. Virus neutralizing (VN) titer against PRV was highest in mice which were inoculated with BHV-1/TF6-1, which expresses PRV gB. BHV-1/TF7-1, which was more effective to protect mice from the challenge of virulent PRV, induced lower VN titer.     

Inactivation of glycoprotein gE and thymidine kinase or the US3-encoded protein kinase synergistically decreases in vivo replication of pseudorabies virus and the induction of protective immunity

To evaluate the contribution of glycoprotein E (gE), thymidine kinase (TK), and the US3-encoded protein kinase (PK) in the induction of protective immunity to pseudorabies virus (PRV), we intranasally inoculated pigs, the natural host of this virus, with mutant PRV strains in which the genes encoding these proteins were inactivated. Both single and double mutants were constructed. Of these proteins, gE has previously been demonstrated to induce antibodies (in mice and pigs), which require complement to neutralize the virus, and helper T cell responses (in mice). PK and TK have thus far not been reported to induce B or T cell responses. All mutants had a strongly reduced virulence for pigs in comparison with wild-type (wt) PRV. After primary infection, most virus was excreted by wt PRV-inoculated animals. Animals inoculated with gE-PK- and gE-TK- double mutants excreted less virus than animals inoculated with gE-, PK-, and TK- single mutants. After challenge infection with the virulent PRV strain NIA-3, no virus was excreted by wt PRV- and PK- mutant-immunized animals, indicating complete protective immunity. Only one of seven gE- and two of seven TK- mutant-immunized animals excreted virus after the challenge inoculation. In contrast, most animals immunized with the gE-PK- or gE-TK- double mutants excreted virus after the challenge inoculation. Daily mean virus excretion after challenge infection was inversely correlated with daily mean virus excretion after primary infection. In most animals, lack of virus excretion was associated with lack of secondary antibody responses, probably attributable to inadequate stimulation of memory B cells as a consequence of early elimination of viral antigen. Thus, inactivation of gE, TK, and PK all affected the immunogenicity of PRV and the effect of gE and TK and gE and PK inactivation appeared synergistic. We found no simple correlation between in vitro growth properties of the mutants and their immunogenic capacity. Strains lacking PK reached lower end titers in vitro than the other mutants. The most likely explanation for the lower protective capacity of some of the mutants appears their reduced replicative capacity in some cells or tissues in vivo, rather than a loss of particular epitopes.     

Identification and characterization of the glycoprotein E and I genes of canine herpesvirus

We have determined the sequence of the gE and gI genes of canine herpesvirus (CHV), DFD-6 strain. The gE ORF codes for a 522 a.a. polypeptide with a signal sequence at the amino-terminus and a trans-membrane domain at the carboxy-terminus. The gI ORF codes for a 259 a.a. polypeptide with a signal sequence but no trans-membrane domain. Comparison with another line of CHV indicated that the DFD-6 gI gene underwent a frame-shift mutation which caused the loss of the trans-membrane domain. Antibodies against the gE and gI polypeptides detected a 94 kDa gE and a broad band of gI (55-62 kDa) in DFD-6 infected cells, respectively. The precursor of DFD-6 gE is modified to the mature form by N-linked glycosylation only in the presence of gI. Together with the fact that the gI- mutant of DFD-6 produced smaller plaques, it is suggested that the truncated DFD-6 gI is functional. The precursor of DFD-6 gI is modified to the mature form by N-linked glycosylation only in the presence of gE.     

Antibody-induced and cytoskeleton-mediated redistribution and shedding of viral glycoproteins, expressed on pseudorabies virus-infected cells

Fluorescein isothiocyanate-labeled porcine pseudorabies virus (PrV) polyclonal antibodies were added to PrV-infected swine kidney cells in vitro at 37 degrees C. In approximately 47% of the infected cells, the addition induced passive patching and subsequent energy- and microtubule-dependent capping of all viral envelope glycoproteins, expressed on the plasma membranes of the infected cells. Further contraction and extrusion of the capped viral glycoproteins occurred in approximately 30% of the capped cells 2 h after the addition of antibodies and was accompanied by a concentration of F-actin beneath the caps. At that time, about 18% of the extruded caps were shed spontaneously into the surrounding medium. Mechanical force released 85% of the extruded caps, leaving viable cells with no microscopically detectable levels of viral glycoproteins on their plasma membranes. Experiments with PrV deletion mutants showed that viral glycoproteins gE and gI are important in triggering viral glycoprotein redistribution. Since the PrV gE-gI complex exhibits Fc receptor activity which facilitates capping, the importance of gE and gI may be partially explained by antibody bipolar bridging.     

Immunization of cattle with a BHV1 vector vaccine or a DNA vaccine both coding for the G protein of BRSV

A gE-negative bovine herpesvirus 1 (BHV1) vector vaccine carrying a gene coding for the G protein of bovine respiratory syncytial virus (BRSV) (BHV1/BRSV-G) induced the same high degree of protection in calves against BRSV infection and BHV1 infection as a multivalent commercial vaccine. A DNA plasmid vaccine, carrying the same gene as the BHV1/BRSV-G vaccine, significantly reduced BRSV shedding after BRSV infection compared with that in control calves, but less well than the BHV1/BRSV-G vaccine. Flow cytometric analysis showed a significant relative increase of gamma/delta+ T cells in peripheral blood after BRSV challenge-infection of the calves of the control group but not in the vaccinated groups. These results indicate that the G protein of BRSV can induce significant protection against BRSV infection in cattle, and that the BHV1/BRSV-G vaccine protects effectively against a subsequent BRSV and BHV1 infection.     

Current status and trends of guided tissue regeneration materials

The partial nucleotide sequence of two BamHI fragments that span the unique short region (US), terminal repeat region (TR) and internal repeat region (IR) of canine herpesvirus (CHV) has been determined. Data obtained revealed several open reading frames (ORF's) identified as the US2, US3, gI, gE and US9 homologues of herpes simplex virus type 1 (HSV1). The CHV homologues also show significant identity in amino acid sequence with those encoded by feline herpesvirus type 1 (FHV1), bovine herpesvirus (BHV1) and equine herpesvirus (EHV1). Translation of another ORF showed little amino acid identity with the gene products of other alpha-herpesviruses. Its genomic position relative to the other CHV homologues would suggest it is the US8.5 gene of CHV.     

Shigellosis in Moscow: new trends in the epidemiological process and their social determinants]

Herpes simplex virus-1 (HSV-1) glycoprotein E (gE) is a multifunctional protein capable of both binding the Fc portion of IgG and mediating cell-to-cell spread of HSV-1. Here we report that the domain on gE involved in IgG binding is distinct from the domain involved in mediating cell-to-cell spread. To do this we have used five mutants of the HSV-1 strain NS: NS-gE(null), a gE deletion virus; rNS-gE(null), a gE rescued virus; NS-gE339, a gE mutant virus with a four amino acid insert at position 339; rNS-gE339, a gE rescue of NS-gE339; and NS-gE406, a gE mutant virus with the same four amino acids inserted at position 406. Using IgG coated sheep red blood cells in rosetting assays, we show that the NS-gE339 does not bind IgG, yet retains the ability to mediate normal cell-to-cell spread. These results demonstrate that the gE domain involved in IgG binding differs from the domain involved in cell-to-cell spread.     

Varicella-zoster virus Fc receptor component gI is phosphorylated on its endodomain by a cyclin-dependent kinase

Varicella-zoster virus (VZV) glycoprotein gI is a type 1 transmembrane glycoprotein which is one component of the heterodimeric gE:gI Fc receptor complex. Like VZV gE, VZV gI was phosphorylated in both VZV-infected cells and gI-transfected cells. Preliminary studies demonstrated that a serine 343-proline 344 sequence located within the gI cytoplasmic tail was the most likely phosphorylation site. To determine which protein kinase catalyzed the gI phosphorylation event, we constructed a fusion protein, consisting of glutathione-S-transferase (GST) and the gI cytoplasmic tail, called GST-gI-wt. When this fusion protein was used as a substrate for gI phosphorylation in vitro, the results demonstrated that GST-gI-wt fusion protein was phosphorylated by a representative cyclin-dependent kinase (CDK) called P-TEFb, a homologue of CDK1 (cdc2). When serine 343 within the serine-proline phosphorylation site was replaced with an alanine residue, the level of phosphorylation of the gI fusion protein was greatly reduced. Subsequent experiments with individually immunoprecipitated mammalian CDKs revealed that the VZV gI fusion protein was phosphorylated best by CDK1, to a lesser degree by CDK2, and not at all by CDK6. Transient-transfection assays carried out in the presence of the specific CDK inhibitor roscovitine strongly supported the prior results by demonstrating a marked decrease in gI phosphorylation while gI protein expression was unaffected. Finally, the possibility that VZV gI contained a CDK phosphorylation site in its endodomain was of further interest because its partner, gE, contains a casein kinase II phosphorylation site in its endodomain; prior studies have established that CDK1 can phosphorylate casein kinase II.     

Construction of a viable BHV1 mutant lacking most of the short unique region

The 8.2 kb HindIII K-fragment of bovine herpesvirus 1 (BHV1) lies entirely within the short unique region of the genome and contains all or parts of 7 coding regions. We have probed this fragment for the presence of nonessential genes by a simple, rapid method of insertional mutagenesis. Our analysis indicates that all the genes present in the K-fragment, except the glycoprotein D gene, are nonessential for replication of BHV1 in tissue culture. After inserting a copy of the glycoprotein D gene into the thymidine kinase locus of BHV1 it was possible to delete the entire HindIII K-fragment and the contiguous 0.36 kb O-fragment in one step. The deletion mutant, which contains 7 kb less BHV1 DNA than wt virus and lacks ORF1, US2, the genes for protein kinase, glycoprotein G, glycoprotein I, and most of glycoprotein E was still replication-competent.     

A feline herpesvirus-1 recombinant with a deletion in the genes for glycoproteins gI and gE is effective as a vaccine for feline rhinotracheitis

Using a site-directed mutagenesis technique we constructed a new feline herpesvirus-1 recombinant strain containing a deletion in two genes encoding glycoproteins gI and gE. These proteins may have a role in virulence, the establishment of latency, and viral recurrence as shown in other herpesviruses of the varicella and simplex types. This recombinant was characterized and used to immunize juvenile cats against virulent virus challenge. Significant protection resulted from vaccination of cats by the subcutaneous route.     

Collagen injection therapy for post-prostatectomy incontinence

Serial deletions of the N-terminal 319 amino acids of rPLD1 expressed in COS-7 cells resulted in increased basal PLD activity. Incubation of the cells with phorbol myristate acetate increased the activity of endogenous and wild-type rPLD1. The mutant rPLD1 with deletion of the first 50 amino acids responded to the phorbol ester, however, rPLD1 with deletions of 115 amino acids or more did not. In cells in which constitutively active V14RhoA was co-expressed with the mutant PLDs, stimulation of PLD activity was observed with all deletion mutants. In membranes from COS-7 cells in which the mutant PLDs were expressed, only the mutant with deletion of 50 N-terminal amino acids responded to added protein kinase C-alpha and phorbol ester, in agreement with the in vivo studies. When myristoylated ADP-ribosylation factor 3 (mARF3) was added together with guanosine 5'-3-O-(thio)triphosphate, all mutants showed stimulation of PLD activity. It is concluded that the site of interaction of protein kinase C with rPLD1 is located in the N-terminal region and that Rho and ARF interact at other sites.     

Bovine T cell responses to Cryptosporidium parvum infection

To better understand the immune mechanisms important for clearing of the primary infection and the subsequent development of resistance to Cryptosporidium parvum infection, several groups have recently characterised changes within the lymphoid cell population of the intestinal mucosa and associated lymphoid tissue in calves with cryptosporidiosis. In naive animals, infection results in a significant increase in the number of CD4+ and CD8+ T cells present within the intraepithelial lymphocyte population, lamina propria and Peyer's patch of the ileum. This is accompanied by a rapid and transient increase in the number of gamma/delta T cells present within the intestinal villi. In response to a challenge infection in immune calves, there is a substantial increase in the number of CD4+ T cells present in the Peyer's patch of the ileum and a specific localization of CD8+ T cells to the epithelium of the intestinal villi. Together, these data demonstrate that C, parvum elicits a strong cell-mediated response following both primary and secondary infections in calves, and that CD8+ T cells may play an important role in the bovine immune response to C. parvum infection.     

Treatment with human growth hormone in patients with Prader-Labhart-Willi syndrome reduces body fat and increases muscle mass and physical performance

Using a monoclonal antibody based ELISA, 600 pAN7-1 plasmid-tagged mutants of Penicillium paxilli were screened for paxilline accumulation and one paxilline-negative mutant, YI-20, was identified. A molecular analysis of this mutant showed that pAN7-1 was inserted at a single site but was present as 4-6 copies arranged in a head-to-tail tandem array. Rescue of flanking sequences and analysis of the corresponding genomic region revealed that YI-20 has an extensive deletion at the site of pAN7-1 integration. Probing of a CHEF gel with the same sequences showed that associated with the deletion is a rearrangement of chromosome Va. Targeted gene disruption of wild-type sequences adjacent to the site where pAN7-1 inserted, resulted in the generation of two additional paxilline-negative mutants; both were single crossovers with deletions extending outside the region mapped. Neither of these new mutants had a rearrangement of chromosome Va, suggesting that deletion of genes on this chromosome is responsible for the paxilline-negative phenotype. Telomeric fingerprinting of genomic digests of P. paxilli, combined with pulsed-field gel electrophoresis of chromosomal DNA, established that there are a minimum of eight chromosomes in this fungus.     

Characterisation of immune responses in pancreatic carcinoma patients after mutant p21 ras peptide vaccination

This is a study of immune responses generated by mutant ras peptide vaccination of patients with pancreatic adenocarcinoma. Responding T cells from one patient were cloned and two CD4+ T-lymphocyte clones (TLC) specific for the 12 Val peptide and restricted by HLA-DR6 or DQ2 were obtained. These class II molecules have not previously been found to bind or present mutant ras peptides to T cells. The DR6-restricted TLC showed marked cytotoxicity against autologous target cells pulsed with the 12 Val peptide. Target cells pulsed with the control peptide were not killed. Responding T cells from another patient showed cross-reactivity towards the homologous ras peptides. Investigation by limiting dilution analysis (LDA) revealed different T-cell precursor frequencies for the immunising, mutant ras peptide (1:28000), compared with the normal ras peptide (1:110000).