The herpes simplex virus type 1 (HSV-1) glycoprotein K(gK) is essential for viral corneal spread and neuroinvasiveness.

PURPOSE: To determine the role of herpes simplex virus-1 (HSV-1) glycoprotein K(gK) in corneal infection, neuroinvasion, and virus latency in trigeminal ganglia of mice. METHODS: The recombinant virus HSV-1 (McKrae) Delta gK (MKDelta gK) carrying a deletion of the gK gene was constructed by insertional/deletion mutagenesis and replaced by a gene cassette constitutively expressing the enhanced green fluorescence protein. The gK deletion of the MKDelta gK virus was rescued to produce the wild-type-like virus MKgK. Balb/c mice were infected ocularly with either virus, and the infection pattern in the eye, clinical disease progression, and establishment of viral latency was monitored. RESULTS: Mice infected with the MKDelta gK strain produced in a gK complementing cell line did not exhibit clinical signs when compared with mice infected with the MKgK virus. Direct visualization of infected eyes revealed that the MKDelta gK virus was unable to spread in mouse corneas, while the MKgK rescued virus spread efficiently. Nineteen of 20 scarified and 5/12 unscarified mice infected with the MKgK virus produced infectious virus after coculture with permissive cells, while 0/20 scarified and 0/12 unscarified mice infected with the MKDelta gK virus produced infectious virus. HSV DNA was detected in trigeminal ganglia by PCR in 19/20 scarified and 9/12 unscarified mice inoculated with MKgK, while HSV DNA was detected in the trigeminal ganglia of 3/20 scarified and 0/12 unscarified mice inoculated with MKDelta gK. CONCLUSIONS: The results show that HSV-1 gK is essential for efficient replication and spread in the corneal epithelium and trigeminal ganglia neuroinvasion in MKDelta gK inoculated mice.

Primary human herpesvirus 8 infection generates a broadly specific CD8(+) T-cell response to viral lytic cycle proteins.

Human herpesvirus 8 (HHV-8) is a recently discovered gammaherpesvirus that is the etiologic agent of Kaposi sarcoma (KS). The natural history of primary HHV-8 infection, including clinical outcome and host immune responses that may be important in preventing disease related to HHV-8, has not been elucidated. The present study characterized the clinical, immunologic, and virologic parameters of primary HHV-8 infection in 5 cases detected during a 15-year longitudinal study of 108 human immunodeficiency virus type 1 seronegative men in the Multicenter AIDS Cohort Study. Primary HHV-8 infection was associated with mild, nonspecific signs and symptoms of diarrhea, fatigue, localized rash, and lymphadenopathy. There were no alterations in numbers of CD4(+) or CD8(+) T cells or CD8(+) T-cell interferon gamma (IFN-gamma) production to mitogen or nominal antigen. CD8(+) cytotoxic T-lymphocyte precursor (CTLp) and IFN-gamma reactivity were detected during primary HHV-8 infection, with broad specificity to 5 lytic cycle proteins of HHV-8 encoded by open reading frame 8 (ORF 8; glycoprotein B homolog of Epstein-Barr virus), ORF 22 (gH homolog), ORF 25 (major capsid protein homolog), ORF 26 (a minor capsid protein homolog), or ORF 57 (an early protein homolog), in association with increases in serum antibody titers and appearance of HHV-8 DNA in blood mononuclear cells. CD8(+) T-cell responses to HHV-8 decreased by 2 to 3 years after primary infection. This antiviral T-cell response may control initial HHV-8 infection and prevent development of disease.

CD8+ cytotoxic T lymphocyte responses to lytic proteins of human herpes virus 8 in human immunodeficiency virus type 1-infected and -uninfected individuals.

T cell immunity to lytic proteins of herpesviruses is important in host control of infection. We have characterized the cytotoxic T lymphocyte (CTL) response to 5 human herpesvirus 8 (HHV-8) homologues of lytic proteins in HHV-8-seropositive individuals. HLA class I-restricted, CD8(+) CTL responses to >/=1 HHV-8 lytic protein were detected in all 14 HHV-8-seropositive study subjects tested, with or without human immunodeficiency virus type 1 (HIV-1) infection, but not in any of 5 HHV-8-seronegative individuals. Seven of these study subjects with both HHV-8 and HIV-1 infection had greater anti-CTL reactivity to glycoprotein H (open-reading frame 22) than did the 7 study subjects infected only with HHV-8. Moreover, there was a strong, inverse correlation between HIV-1 load and glycoprotein H-specific CTL lysis in the study subjects infected with both viruses. CTL reactivity to HHV-8 lytic proteins may be involved in host control of HHV-8-related diseases, such as Kaposi's sarcoma.

Glycoprotein B of human herpesvirus 8 is a component of the virion in a cleaved form composed of amino- and carboxyl-terminal fragments.

Human herpesvirus 8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus (KSHV) is the only known human member of the Rhadinovirus genus of the gammaherpesvirus subfamily. Antibodies against peptides representing portions of the amino- and carboxyl-termini of HHV-8 gB were produced and used to detect gB expression in Vero cells transfected with the gB gene, in the HHV-8-harboring cell line, BCBL-1, and in purified virions. Expression of gB was detected in approximately 3% of uninduced BCBL-1 cells, while up to 30% of the cells expressed gB after 12-O-tetradecanoylphorbol-13-acetate (TPA) induction of virus replication. Indirect immunofluorescence assays and confocal microscopy showed that gB was distributed throughout the cytoplasm of BCBL-1 cells and transfected Vero cells. Immunoblot analyses of virion preparations revealed the presence of full-length as well as two smaller than full-length gB-derived species corresponding to the amino- and carboxy-terminal portions of gB, respectively. Biochemical analysis of the gB carbohydrate moieties using glycosylation inhibitors revealed that gB contained N-linked oligosaccharides of the high-mannose type, characteristic of precursor carbohydrate chains added in the endoplasmic reticulum.

Production of a rabbit anti-cockatiel immunoglobulin G and characterization of its cross-reactivities with immunoglobulin G of other psittacine species.

The purpose of this work was to produce rabbit anti-cockatiel immunoglobulin G (IgG) and compare its cross-reactivity with sera from eight other psittacine birds: Quaker parakeet, budgerigar, green-wing macaw, blue-fronted Amazon parrot, eclectus parrot, African grey parrot, Patagonian conure, Moluccan cockatoo. Cockatiel IgG did not bind to protein A or G; therefore, these proteins could not be used in column chromatography to isolate the IgG. A combination of serum IgG precipitation by ammonium sulfate and yolk IgG extraction from egg was loaded in sodium dodecyl sulfate-polyacrylamide gel upon which the IgG was resolved by electrophoresis. The resolved IgG in sodium dodecyl sulfate-polyacrylamide gel was stained with Coomassie blue, cut, crushed in phosphate-buffered saline, and injected into rabbits. The rabbit anti-cockatiel IgG produced in this way reacted with a single protein in gel immunodiffusion assay with all nine psittacine bird sera but not with those of chicken and ostrich. Immunoelectrophoresis confirmed the cross-reactivity of different psittacine sera with the anti-cockatiel IgG serum but not with ostrich and chicken sera. This antiserum detected antibody responses in sera from cockatiels vaccinated against chlamydial major outer membrane protein in an immunoblot assay.

Herpes simplex virus type 1 glycoprotein K is not essential for infectious virus production in actively replicating cells but is required for efficient envelopment and translocation of infectious virions from the cytoplasm to the extracellular space.

We characterized the glycoprotein K (gK)-null herpes simplex virus type 1 [HSV-1] (KOS) delta gK and compared it to the gK-null virus HSV-1 F-gKbeta (L. Hutchinson et al., J. Virol. 69:5401-5413, 1995). delta gK and F-gKbeta mutant viruses produced small plaques on Vero cell monolayers at 48 h postinfection. F-gKbeta caused extensive fusion of 143TK cells that was sensitive to melittin, a specific inhibitor of gK-induced cell fusion, while delta gK virus did not fuse 143TK cells. A recombinant plasmid containing the truncated gK gene specified by F-gKbeta failed to rescue the ICP27-null virus KOS (d27-1), while a plasmid with the delta gK deletion rescued the d27-1 virus efficiently. delta gK virus yield was approximately 100,000-fold lower in stationary cells than in actively replicating Vero cells. The plaquing efficiencies of delta gK and F-gKbeta virus stocks on VK302 cells were similar, while the plaquing efficiency of F-gKbeta virus stocks on Vero cells was reduced nearly 10,000-fold in comparison to that of delta gK virus. Mutant delta gK and F-gKbeta infectious virions accumulated within Vero and HEp-2 cells but failed to translocate to extracellular spaces. delta gK capsids accumulated in the nuclei of Vero but not HEp-2 cells. Enveloped delta gK virions were visualized in the cytoplasms of both Vero and HEp-2 cells, and viral capsids were found in the cytoplasm of HEp-2 cells within vesicles. Glycoproteins B, C, D, and H were expressed on the surface of delta gK-infected Vero cells in amounts similar to those for KOS-infected Vero cells. These results indicate that gK is involved in nucleocapsid envelopment, and more importantly in the translocation of infectious virions from the cytoplasm to the extracellular spaces, and that actively replicating cells can partially compensate for the envelopment but not for the cellular egress deficiency of the delta gK virus. Comparison of delta gK and F-gKbeta viruses suggests that the inefficient viral replication and plaquing efficiency of F-gKbeta virus in Vero cells and its syncytial phenotype in 143TK- cells are most likely due to expression of a truncated gK.

An amphipathic alpha-helical synthetic peptide analogue of melittin inhibits herpes simplex virus-1 (HSV-1)-induced cell fusion and virus spread.

The effects of hecate, a 23-amino acid synthetic peptide analogue of melittin, on HSV-1-induced cell fusion and virus multiplication was investigated. Hecate completely inhibited cell fusion induced by HSV-1 syncytial (syn) mutants HSV-1 HFEM (tsB5) and HSV-1 mp (MP) at a concentration of 5.0 microM. Metabolic labeling experiments indicated that hecate did not adversely affect cellular growth and protein synthesis. The synthesis of virus-specified glycoproteins B, C, D, and H was reduced in the presence of hecate; however, the transport of these glycoproteins to the surface of infected cells was not affected. Production of infectious virions for wild-type and syn mutants tsB5 and MP was reduced in the presence of hecate. The effect of hecate on virus titer was dependent on the multiplicity of infection. Virus titers were reduced 2-28-fold at an M.O.I. of 0.1, 3-6-fold at an M.O.I. of 0.5, and 0-2.5-fold at an M.O.I. of 2.5. Direct treatment of semipurified virions with hecate reduced titers by approximately 4-fold for KOS, 2-fold for tsB5, and over 30-fold for MP.

Specific antigens of Chlamydia pecorum and their homologues in C psittaci and C trachomatis.

OBJECTIVE: To analyze the antigenic cross reactivity of various proteins of strains of Chlamydia pecorum, C psitaci, and C trachomatis. SAMPLES AND PROCEDURES: Strains FC-Stra and LW-613 of C pecorum, strains B577, Fitz-9, and 68C of C psitaci, and strain LGV-2 of C trachomatis were studied. Strains of C pecorum were propagated in Georgia bovine kidney cells, and other chlamydial strains were propagated in L cells or Georgia bovine kidney cells. Partially purified chlamydial elementary bodies propagated in RAG cells, a BALB/c cell line cloned from a renal adenocarcinoma of BALB/c mice, were used to immunize BALB/c mice for production of monoclonal antibodies. Rabbits were inoculated with yolk sack-propagated, purified elementary bodies to produced polyclonal antisera. The reaction of monoclonal antibodies and polyclonal antisera with chlamydial proteins was analyzed by use of immunoblot techniques. RESULTS: Two monoclonal antibodies reacted with a 90-kd protein of C psittaci and a 94-kd protein of C pecorum strains. One monoclonal antibody reacted strongly with a 67-kd protein of C pecorum and strain B577 of C psittaci, but weakly with proteins of strains 6BC and LGV-2. Another monoclonal antibody reacted with a 46-kd protein of 2 C pecorum strains and of strain B577 of psittaci, but not with those of strains 6BC and LGV-2. Two monoclonal antibodies reacted with a 20-kd protein of C pecorum and a 22-kd protein of C psittaci and LGV-2 strains. Polyclonal antisera reacted similarly with the proteins identified by monoclonal antibodies in the various chlamydial strains. CONCLUSIONS: Reactions of several monoclonal antibodies with chlamydial antigens indicated that 67- and 46-kd proteins contain genus- and species-specific epitopes, respectively; a 94-kd protein of C pecorum is homologous to a 90-kd protein of C psittaci and C trachomatis strains; and a 20-kd protein of C pecorum corresponds to a 22-kd protein of C psittaci and C trachomatis.

Detection of strain-specific antigenic epitopes on the lipo-oligosaccharide of Haemophilus parasuis by use of monoclonal and polyclonal antibodies.

OBJECTIVE: To investigate the antigenic diversity of lipo-oligosaccharides of Haemophilus parasuis. PROCEDURES: Immunoblot assays were done with monoclonal and polyclonal antibodies on whole-cell lysates. Individual colonies of H parasuis strains H 54, H 53, and H 128 were tested for reactivity with lipo-oligosaccharide-specific monoclonal antibodies after a single passage on chocolate agar, and colonies of strain H 54 were analyzed after 10 passages. Colony blot tests were used to screen H parasuis strains for spontaneously occurring antigenic variation in their lipo-oligosaccharides. RESULTS: Eight H parasuis strains were separated into 4 lipo-oligosaccharide serovars on the basis of immunoblot reactions with 3 polyclonal rabbit antisera. Nine monoclonal antibodies against lipo-oligosaccharides of a lipo-oligosaccharide-serovar I strain reacted with all tested serovar I strains but failed to react with other H parasuis strains. CONCLUSIONS: Variations in the antigenic reactivity after 1 or 10 passages on chocolate agar were not observed. The serovar I lipo-oligosaccharide strains included virulent as well as avirulent H parasuis strains, indicating that these epitopes do not correlate directly with virulence properties of H parasuis.

Resolution of genotypic and phenotypic properties of herpes simplex virus type 1 temperature-sensitive mutant (KOS) tsZ47: evidence for allelic complementation in the UL28 gene.

Herpes simplex virus type 1 (HSV-1) mutant tsZ47 was reported to be temperature sensitive for virus growth and transport of viral glycoproteins to the cell surface and to contain two different mutations (B. A. Pancake, D. P. Aschman, and P. A. Schaffer, (1983) J. Virol. 47,568-585). However, we found that similar amounts of glycoproteins B, C and H were expressed at the cell surface at the permissive and non-permissive temperatures and in addition, tsZ47 virus contained only a single mutation. UL28-null virus, gC delta 7B, failed to complement tsZ47 in mixed infections and tsZ47 replicated in UL28 but not gB transformed cell lines. A ts lesion of tsZ47 was mapped within a 1333 bp region of the UL28 gene by marker-rescue using overlapping DNA fragments. DNA sequencing identified a C to T transversion resulting in an R to W amino acid change at UL28 amino acid position 531. Southern Blot analysis and transmission electron microscopy demonstrated that tsZ47, is defective in cleavage and encapsidation of viral DNA. Mutant virus ts1203 (C. Addison, F.J. Rixon, and V.G. Preston, (1990) J. Gen. Virol. 71, 2377-2384) that contains a mutation in the 5' end of UL28, complemented tsZ47 in mixed infections. This suggests that allelic complementation may be occurring and UL28 may encode a protein with independently functioning domains, or that it participates in a multimer.

Role of the Na+,K+ pump in herpes simplex type 1-induced cell fusion: melittin causes specific reversion of syncytial mutants with the syn1 mutation to Syn+ (wild-type) phenotype.

To evaluate the importance of the Na+,K+ pump and ionic gradients in virus-induced cell fusion, we investigated the effects of melittin, a 26 amino acid bioactive peptide found in honey bee venom, on cell fusion caused by HSV-1 syncytial mutants. Melittin inhibited fusion of Vero cells caused by HSV-1 mutant viruses mP(MP), KOS (syn20) and KOS (FFV3) containing the syncytial mutation syn1 in glycoprotein K. However, it did not affect cell fusion caused by mutants HFEM(tsB5) or KOS amb1511-7 with mutations in glycoprotein B. Melittin caused specific reversion of syn1 mutant virus plaques to syn+ (wild-type) plaque morphology, and inhibited virus adsorption and penetration. It also inhibited the Na+,K+ pump activity, and the binding of 3H-ouabain to the Na+,K+ pump of infected Vero cells. The Na+,K+ pump activity of infected Vero cells in comparison to mock-infected cells was significantly decreased. Ouabain, a specific inhibitor of the Na+,K+ pump, inhibited fusion of Vero cells caused by all syncytial virus strains.

Truncation of the carboxy-terminal 28 amino acids of glycoprotein B specified by herpes simplex virus type 1 mutant amb1511-7 causes extensive cell fusion.

Three amber mutations were introduced proximal to the syn3 locus of the herpes simplex virus type 1 glycoprotein B (gB) gene specifying gB derivatives lacking the carboxy-terminal 28, 49, or 64 amino acids. A complementation system that utilized gBs expressed in COS cells to complement gB-null virus K delta T was established. The 49- or 64-amino-acid-truncated gBs failed to complement gB-null virus K delta T, while the 28-amino-acid-truncated gB complemented K delta T efficiently. Mutant herpes simplex virus type 1 KOS (amb1511-7) specifying the 28-amino-acid-truncated gB fused Vero cells extensively.

Detection and antigenicity of chlamydial proteins that bind eukaryotic cell membrane proteins.

Chlamydia psittaci proteins capable of binding eukaryotic cell membranes were identified and antigenically characterized. Cell membrane proteins (CMP) of noninfected cells were labeled with biotin (B-CMP), then were extracted with 1% Triton X-100. Nitrocellulose membrane strips containing sodium dodecyl sulfate-polyacrylamide gel electrophoresis-resolved proteins of chlamydial elementary bodies (EB) were reacted with the B-CMP extract, followed by addition of streptavidin-conjugated horse radish peroxidase. Among the various strains of chlamydiae examined, a protein of approximately 16 to 18 kDa consistently bound B-CMP. A second larger protein, ranging in molecular mass from 24 to 32 kDa, also bound B-CMP. Immunoblotting techniques were used to analyze the reactions of antisera from immunized and experimentally infected animals to these proteins. A rabbit polyclonal antiserum produced against the 18-kDa adhesin of a serovar-1 strain of C psittaci (B577) reacted strongly with 18-kDa proteins of all C psittaci strains, but weakly with that of C trachomatis. Mouse antisera raised against the serovar-2 (FC-Stra) 28-kDa protein reacted only with proteins of the homologous serovar. Sera from experimentally infected animals did not react with the C trachomatis 18-kDa adhesion protein, but did react in 2 patterns with related and nonrelated C psittaci isolates. Two rabbits inoculated with infective serovar-1 EB and 1 rabbit inoculated with a serovar-2 strain reacted specifically with the 18-kDa proteins of their homologous serovars. In contrast, 2 other rabbits inoculated with the same serovar-2 strain produced antisera that reacted with all C psittaci 18-kDa proteins, as did serum from a similarly inoculated bull.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell fusion caused by herpes simplex virus-1 (HSV-1) strains tsB5 and MP is inhibited at pH 6.7 and pH 7.0.

We investigated the effect of different pH conditions on Vero cell cultures infected with herpes simplex virus-1 (HSV-1) wild-type strain KOS, and syncytial mutants HSV-1 HFEM (tsB5) and HSV-1 mp (MP). Cell fusion was inhibited when infected cells were continuously incubated with culture media adjusted to pH 6.7 or pH 7.0. Inhibition of cell fusion was rapidly reversible when infected cell cultures were returned to pH 7.5. The rate of synthesis and cell-surface expression of virus-specified glycoproteins gB, gC, gD, and gH were not affected during continuous incubation at pH 7.0, but they were reduced at pH 6.7 in comparison to pH 7.5. At later hours p.i. however, these glycoproteins continued to accumulate at all tested pH levels. Accumulation of infectious virions was substantially reduced for MP, KOS, and tsB5 at pH 6.7. At pH 7.0, KOS and tsB5 titers were greatly reduced but MP titers were not affected.

Elimination of Candida albicans from kidneys of mice during short-term systemic infections.

The candidacidal activity of kidneys, liver, and spleen's phagocytic systems was studied in mouse. Different strains of mice were inoculated intravenously (i.v.) with 1 to 2.6 x 10(4) viable Candida albicans. Elimination of the microorganisms from the kidneys, liver, and spleen were evaluated by enumeration of colony forming units (C.F.U.) recovered from homogenates of organs dissected within a short period of time (0 to 5 hr). The results indicated that the kidneys possess a capable phagocytic system which was able to eliminate the microorganisms as efficiently as those of liver and spleen. Furthermore, the ability of the liver and spleen phagocytic system as well as that of kidneys were significantly enhanced when animals were treated with Bacillus Calmette-Guerin (BCG) of mycobacterium bovis four weeks prior to induction of systemic infection with C. albicans.

Antibody response to epitopes of chlamydial major outer membrane proteins on infectious elementary bodies and of the reduced polyacrylamide gel electrophoresis-separated form.

Approximately 60% of the outer membrane of chlamydial elementary bodies (EBs) consists of the major outer membrane protein (MOMP) that has structural and metabolic functions. The antigenic properties of MOMPs from mammalian strains of serovars 1 and 2 and an avian strain of Chlamydia psittaci were analyzed. Polyclonal-monospecific antisera (PMAs), one monoclonal antibody (MAb), and polyclonal antisera (PAs) were produced against reduced polyacrylamide gel electrophoresis-separated MOMPs and against infectious EBs. Three PMAs and the MAb, which were induced by reduced polyacrylamide gel electrophoresis-separated MOMPs, reacted strongly in Western blot (immunoblot) assays with MOMPs of serovar 1 and 2 strains as well as with that of the avian strain 6BC, and two of these PMAs reacted weakly (dilution, 1:20) with the MOMP of strain LGV-2. The third PMA and the MAb against the MOMP of the serovar 2 strain did not react with the MOMP of LGV-2. Four PAs were produced against infectious EBs of the serovar 1 strain. One of these PAs reacted with the homologous MOMP and that of the avian strain 6BC but did not recognize MOMPs of other chlamydial strains. Three of the PAs reacted with MOMPs of homologous strains only and failed to recognize MOMPs of avian, serovar 2, and LGV-2 strains. Five PAs induced against infectious EBs of the serovar strain 2 reacted only with the MOMPs of the homologous strains and failed to recognize MOMPs of other strains of chlamydiae. Consequently, MOMPs of C. psittaci strains possess genus-, species-, and serovar-specific epitopes whereby the immune response to serovar-specific epitopes of MOMP predominate when infectious EBs are used for immunization.

Systemic candidosis in beige mice.

Systemic spread of Candida albicans after intravenous inoculation was compared in beige mice and their functionally normal littermates. The number of colony forming units (c.f.u.) recovered from the kidneys, livers, and spleens of beige mice was substantially greater (100 to 1000-fold) than the number cultured from the respective organs of their functionally normal littermates. Pre-treatment with silica did not alter the number of c.f.u. recovered from the organs of either beige mice or their normal littermates. These results are consistent with the hypothesis that normally functioning polymorphonuclear leukocytes are crucial to the innate defenses that control the proliferation of this fungus.

Role of activated macrophages in resistance to systemic candidosis.

To evaluate further the contribution of activated macrophages in resistance, the course of systemic candidosis was assayed in beige and NLM mice that had been previously infected with Mycobacterium bovis (BCG). Four weeks following BCG infection, mice were inoculated intravenously with 1 x 10(4) viable Candida albicans. At various times thereafter, the number of C. albicans colony-forming units in the livers, spleens, and kidneys was determined. The average number of CFU recovered from the kidneys of NLM mice decreased throughout the assay and was comparable in both BCG-treated and control mice. In contrast, the number of CFU cultured from the kidneys of untreated control beige mice progressively increased throughout the assay period. This profile of renal susceptibility was not appreciably altered in BCG-treated beige mice. However, fewer (10- to 100-fold) CFU were cultured from the livers and spleens of BCG-treated beige and NLM mice than from untreated controls. These results support the hypothesis that in the absence of functional polymorphonuclear leukocytes, activated macrophages represent a means to control the proliferation of C. albicans.