Low-passage Clinical Isolates Research Articles

Increased Frequency of Specific Locus Mutation Following Human Cytomegalovirus Infection

The effect of human cytomegalovirus (HCMV) infection on the frequency of mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus was studied in Chinese hamster lung V79 cells. When V79 cells were infected with HCMV (strain AD169) at multiplicities of 0.1 to 50 plaque forming units (PFU) per cell the presumptive mutation frequency, as determined by the number of 6-thioguanine-resistant (TGr) colonies, was increased up to 16.8-fold (P< 0.005), depending on the multiplicity of infection. Increases in the mutation frequency at thehprtlocus were also observed for other laboratory-adapted HCMV strains (C-87, Davis) and for low passage clinical isolates (82-1, 84-2). The expression time required for the maximum increase in TGrcolonies was 3 days and was consistent among the HCMV strains evaluated in this study. UV-irradiation of HCMV stock up to a dose of 9.6 × 104ergs/mm2increased the mutation frequency, but further exposure to UV light or to heat (56° for 30 min) significantly decreased the frequency of TGr-resistant colonies, suggesting that expression of HCMV genes was involved in the mutation process. HCMV-induced TGrcells demonstrated substantially reduced (>96%) incorporation of [3H]hypoxanthine. PCR analysis of thehprtlocus demonstrated deletions in 9 of 19 HCMV-induced TGrcolonies randomly selected for further study, while 2 of 17 spontaneously developed TGrcolonies demonstrated deletions. Although insertions were not detected in spontaneously developed clones, 3 of 19 HCMV-induced TGrclones had insertions in thehprtgene. Neither HCMV-specific DNA sequences nor HCMV-specific proteins were detected in the TGrclones obtained after HCMV infection. Infection of V79 cells with HCMV also increased their sensitivity to mutation withN-methyl-N′-nitro-N-nitrosoguanidine, giving a synergistic enhancement of the mutation frequency. These results indicate that HCMV infection has the capacity to induce mutations in the cellular genome and increase the sensitivity of infected cells to mutation by genotoxic chemicals. Although inactivated HCMV particles are responsible for a modest increase in the mutation frequency, expression of HCMV genes is associated with a substantial enhancement of the mutation frequency.

Porphyromonas gingivalis invasion of gingival epithelial cells.

Porphyromonas gingivalis, a periodontal pathogen, can invade primary cultures of gingival epithelial cells. Optimal invasion occurred at a relatively low multiplicity of infection (i.e., 100) and demonstrated saturation at a higher multiplicity of infection. Following the lag phase, during which bacteria invaded poorly, invasion was independent of growth phase. P. gingivalis was capable of replicating within the epithelial cells. Invasion was an active process requiring both bacterial and epithelial cell energy production. Invasion was sensitive to inhibitors of microfilaments and microtubules, demonstrating that epithelial cell cytoskeletal rearrangements are involved in bacterial entry. P. gingivalis, but not epithelial cell, protein synthesis was necessary for invasion. Invasion within the epithelial cells was not blocked by inhibitors of protein kinase activity. Invasion was inhibited by protease inhibitors, suggesting that P. gingivalis proteases may be involved in the invasion process. Low-passage clinical isolates of P. gingivalis invaded with higher efficiency than the type strain. Serum inhibited invasion of the type strain but had no effect on the invasion of a clinical isolate. Invasion of gingival epithelial cells by P. gingivalis may contribute to the pathology of periodontal diseases.

Human cytomegalovirus induces IL-6 and TNF alpha from macrophages and microglial cells: possible role in neurotoxicity.

Human cytomegalovirus (HCMV) can frequently infect the central nervous system (CNS) in the setting of immunosuppression such as transplantation and infection with the human immunodeficiency virus (HIV). Our laboratory previously reported that HCMV infection of human brain aggregates preferentially infected a microglial/macrophage (M/M) and caused a neuropathology that differed between strains and could occur in the absence of antigen expression. We extended these studies by infecting a human brain cell aggregate model with four low passage clinical isolates of HCMV. Two patterns of cytopathology emerged after infection; a lacy eosinophilic appearance or a glial nodular formation concomitant with a decreased aggregate size. None of the infections were positive for HCMV antigen; however, all were positive for HCMV DNA. We also infected primary macrophages and microglial cells with the same HCMV isolates. Microglial cells were more susceptible to HCMV infection resulting in a lytic infection. Production of potentially neurotoxic cytokines, IL-1, IL-6 and TNF alpha, from HCMV-infected macrophages and microglial cells were evaluated to explain brain aggregate cytopathology. Supernatants from HCMV-infected macrophages and microglial cells produced similar levels of TNF alpha (< 30 pg ml-1) but showed strain and cell source variation in the production of IL-6; microglial cultures produced > 4 fold higher levels. None of the supernatants contained IL-1. Treatment of brain aggregates with either IL-6 or TNF alpha resulted in morphologic alterations and/or a decrease in size consistent with HCMV infection or supernatant treatment.

CGP 53437, an orally bioavailable inhibitor of human immunodeficiency virus type 1 protease with potent antiviral activity

CGP 53437 is a peptidomimetic inhibitor of human immunodeficiency virus type 1 (HIV-1) protease containing a hydroxyethylene isostere. The compound inhibited recombinant HIV-1 protease with a Ki of 0.2 nM. The inhibition constant versus human cathepsin D and human cathepsin E was 4 nM. Human pepsin and gastricsin were inhibited with Kis of 8 and 500 nM, respectively, and human renin was inhibited with a Ki of 190 microM. The replication of HIV-1/LAV, HIV-1/Z-84, and HIV-1/pLAI was inhibited with a 90% effective dose of 0.1 microM in acutely infected MT-2 cells. The 50% cytotoxic dose was 100 microM. Similar antiviral activity was observed when the compound was added up to 10 h after infection. At the effective concentration, processing of Gag precursor protein p55 was greatly reduced, confirming an action on the late stage of the virus life cycle, as expected. The efficacy of the inhibitor was also demonstrated by using primary human peripheral blood lymphocytes infected with the HIV-1/LAV strain, low-passage clinical isolates obtained from HIV-1-seropositive individuals (including a zidovudine-resistant strain), and HIV-2/ROD. In these cells, CGP 53437 delayed the onset of HIV replication in a dose-dependent fashion (substantial effects with concentrations of > or = 0.1 microM) as long as the inhibitor was maintained in the culture. CGP 53437 was orally bioavailable in mice. Concentrations in plasma 10-fold in excess of the in vitro antiviral 90% effective dose could be sustained for several hours after oral application of 120 mg/kg. Therefore, CGP 53437 has the potential to be a therapeutically useful anti-HIV agent for the treatment of AIDS.

Zidovudine susceptibility testing of human immunodeficiency virus type 1 (HIV) clinical isolates

Traditional antiviral susceptibility testing methods using cell lines can be applied to no more than about 30% of clinical HIV isolates (Larder et al., 1989a; Fenyo et al., 1989). We tested the cell-free supernatant from low passage clinical HIV isolates using donor peripheral blood mononuclear cells (PBMC). Drug susceptibility was assessed by measuring the effect of increasing zidovudine (ZDV) concentrations on HIV P24 antigen production. Susceptibility results were obtained on 24 27 consecutive clinical isolates and 6 6 laboratory isolates. The mean IC 90 of isolates from untreated patients was 0.008 μM ZDV (range: 0.002–0.038). The IC 90s of isolates from ZDV-treated patients ranged from 0.007 to greater than 10 μM ZDV. All isolates with an IC 90 < 0.1 μM ZDV had a wild type sequence at codon 215 of the HIV pol gene; 11 12 isolates with an IC 90 > 0.1 μM ZDV had a mutation at codon 215 ( P <0.001). Among 16 ZDV-treated patients, there was a modest correlation between the change in CD4 count from the start of ZDV treatment and the IC 90 of the patient's isolate following treatment ( r = 0.51). Susceptibility testing using donor PBMC can be a sensitive means of testing a broad range of clinical HIV isolates.

Emergence of mutant HIV reverse transcriptase conferring resistance to AZT.

(Krc,eived 2 Ortobrr IYYI) The likelihood of emergence of drug resistance is a function of mutation rate and the number of replicative events. The mutation rate of HIV is presumed to be at least as high as any virus with a single stranded RNA genome. The number of replicative events in a host infected with HIV are incalculably high as a result of years of persistent replication that is poorly restricted by immune surveillance. Viral chemo- therapy in humans has selected for drug resistant variants of influenza A virus, herpes simplex virus. varicella zoster virus, cytomegalovirus and rhinovirus. ' The prospect of drug resistant AZT therefore did not appear as a surprise to many. In the initial study of HIV susceptibility to AZT (zidovudine),' isolation of HIV was attempted with 186 peripheral blood mononuclear cell specimens from subjects at different stages of infection who h,ad received therapy with AZT for various periods. A total of 46 isolates were obtained from 33 individuals. The use of MT2 lympho- blastoid cells for the isolation procedure permitted the preparation of large stocks of virus that could be frozen in riliquots and titrated. Susceptibility to AZT with these low-passage clinical isolates of HIV could not be tested reliably with the described assays of inhibition of cytopathology or production of p24 antigen.j This dilemma was resolved by the USIZ of a CD4-expressing HeLa cell line (obtained from B. Chesebro, National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratories, Hamilton, Mont.) that readily permitted plaque reduction assays with all isolates te~ted.~ In addition, this system permitted assay of a wide range of compounds, including nucleosides, interferons, and recombinant soluble CD4. Study of these original isolates permitted several conclusions: (I) Isolates from subjects not treated with AZT display a narrow range of susceptibility to AZT, with

Chemical and biological activities of a 64-kilodalton outer sheath protein from Treponema denticola strains

This study examined the distribution of the major outer sheath proteins (MOSP) in several Treponema denticola strains and reports the isolation of a 64-kDa protein from the outer sheath of human clinical isolate T. denticola GM-1. The outer sheath was isolated by freeze-thaw procedures, and the distribution of outer sheath proteins was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). T. denticola GM-1, MS25, SR-5, and three low-passage clinical isolates possessed an MOSP with a relative molecular mass of 60 to 64 kDa. This MOSP was absent in T. denticola ATCC 35404 (TD-4) and clinical isolate SR-4. The latter possessed an MOSP of 58 kDa. 125I labeling revealed both MOSP to be dissociated forms of higher-molecular-mass oligomeric units between 116 and 162 kDa. Two-dimensional SDS-PAGE confirmed the modifiability of these MOSP. Isoelectric focusing of the 64-kDa MOSP indicated a pI of 6.7. Immunoblots with antiserum to GM-1 whole cells revealed the 64-kDa protein to be immunogenic and not cross-reactive with the MOSP of TD-4 or SR-4, and monospecific antibody to the 64-kDa protein recognized common epitopes on the high-molecular-weight oligomeric protein. These antibodies did not react with any component of TD-4 whole cells in immunoblots or in immunogold electron microscopy. Fab fragments inhibited the adherence of T. denticola GM-1 to human gingival fibroblasts by 78% (1:1,600; 0.72 micrograms of protein per ml), while TD-4 adherence was not inhibited. Amino acid analysis revealed a slightly acidic protein, devoid of cysteine, with 36% hydrophobic residues. Cyanogen bromide fragmentation of the 64-kDa protein revealed that a 42-kDa fragment contained a T-L-D-L-A-L-D segment which was 100% homologous with an integrin alpha subunit of a human leukocyte adhesion glycoprotein p 150,95.

Use of the Polymerase Chain Reaction to Analyse Sequence Variation Within a Major Neutralizing Epitope of Glycoprotein B (gp58) in Clinical Isolates of Human Cytomegalovirus

The heterogeneity of low passage human cytomegalovirus (HCMV) strains was determined by HindIII typing of 28 clinical isolates from transplant patients. These data have shown that, in general, each patient&apos;s strain has a unique restriction profile, usually comprising combinations of HindIII sites present in one or more of the tissue culture-adapted strains AD169, Towne and Davis. To map sequence changes in a more refined manner we performed detailed analyses of 33 low passage clinical isolates, including those aforementioned, analysing a sequence within glycoprotein B containing a major neutralizing epitope. A 149 bp sequence containing the epitope (amino acids 608 to 625) was amplified using the polymerase chain reaction, the products were cloned and their DNA sequence was determined. Comparison of the DNA and deduced amino acid sequences with those of HCMV strain AD169 revealed that there was a high degree of conservation of the epitope between the 33 clinical isolates. However 10 of the isolates possessed silent mutations and three isolates contained mutations producing amino acid changes within the neutralizing epitope. The possible functional significance of these changes is discussed.

Polyamine biosynthesis in cells infected with different clinical isolates of human cytomegalovirus.

Previous work in this laboratory showed that polyamine biosynthesis was stimulated in fibroblasts following infection with the AD169 strain of human cytomegalovirus (HCMV) or with murine cytomegalovirus (MCMV) (Tyms et al: Biophysics Research Communications 86:312-318, 1979; Advances in Polyamine Research 4:507-517, 1983). Here we compare the affect of AD169 on polyamine production in infected fibroblasts with that of the unusual Colburn strain of HCMV. The Colburn virus is unusual in that it was isolated from a 7 year old boy with encephalitis and molecular studies indicated the virus was simian like (Huang et al: Journal of Virology 26:718-723, 1978). As a consequence of CMV infection a two to ten fold increase in the spermine content of fibroblast cells is observed. Radiolabel transfer experiments show that spermine is synthesized throughout virus infection. Indeed, spermidine and spermine are specifically incorporated into the purified virions of the AD169 and Colburn strains of HCMV. Furthermore, polyamine biosynthesis is stimulated in fibroblast cells infected with a number of low passage clinical isolates of HCMV. Inhibition of polyamine biosynthesis in HCMV infection may provide a specific and novel target for antiviral chemotherapy.

Complement‐Independent neutralising monoclonal antibody with differential reactivity for strains of human cytomegalovirus

A mouse monoclonal antibody with complement-independent neutralising activity against cytomegalovirus (CMV) and reactive with the 86 kilodalton (kDa) viral glycoprotein H is described. Neutralisation tests against a range of different strains of CMV showed significant crossreactivity, but clear differences were evident between the two prototype viruses AD169 and Davis, and particularly between AD169 and several low-passage recent clinical isolates; CMV present in urine was neutralised weakly if at all.

Mild Acidic pH Inhibition of the Major Pathway of Herpes Simplex Virus Entry into HEp-2 Cells

Penetration of the KOS strain of herpes simplex virus type 1 (HSV-1) and the MS and 333 strains of herpes simplex virus type 2 (HSV-2) into HEp-2 cells at pH 6.3 was at least 100-fold less efficient than at pH 7.4. Penetration of two low passage clinical isolates was completely blocked at pH 6.3. The syncytium-forming HSV-1 strains GC and MP were less sensitive than KOS to the mild acidic conditions. The inhibition was completely reversed upon neutralization of the medium. Penetration was assayed by plaque production following protection from acid inactivation upon virus entry. Penetration of HSV-1 KOS into Vero and HEL diploid fibroblast cells was similarly inhibited. HSV-1 KOS grown in 2-deoxy-D-glucose and monensin was also extensively inhibited at pH 6.3 but virus grown in 2-deoxy-D-glucose penetrated more slowly than normal virus at pH 7.4. Electron microscopy of HSV-1 KOS infection indicated that fusion and endocytosis occur at both pH 7.4 and 6.3 but that fusion predominates at pH 7.4 and endocytosis predominates at pH 6.3. These results indicate that fusion at the plasma membrane is the major route of productive entry for HSV, that strains of HSV can differ in their pH dependence for penetration and this may determine whether virus infection can occur following endocytic uptake.

Human Cytomegalovirus and Monocytes: Limited Infection and Negligible Immunosuppression in Normal Mononuclear Cells Infected in vitro with Mycoplasma-free Virus Strains

Human cytomegalovirus (HCMV) infection has previously been associated with the production of immunosuppression. The mechanism by which any such immunosuppressive effect might be mediated is unclear but previous work has implicated an effect of the virus on monocytes. We have attempted to characterize the immunosuppressive activity produced by in vitro infection of normal monocytes with HCMV strain AD169. We first examined the ability of HCMV AD169 and recent clinical isolates to infect normal peripheral blood mononuclear cells in vitro. We have found by immunofluorescence analysis that only a very limited number of peripheral blood mononuclear cells (0.2 to 0.5%) showed evidence of virus infection as demonstrated by expression of the major immediate early protein. We found that the inhibitory activity of supernatants of monocytes exposed to HCMV which suppressed mitogen-driven T cell responsiveness was associated with a protein of about 95K. Experiments to investigate the mechanism of action of this inhibitor suggested the possibility of mycoplasma contamination and we were subsequently able to isolate Mycoplasma hyorhinis from our AD169 virus stock. When a series of low passage clinical isolates of HCMV were examined for their ability to cause immunosuppression, there was a direct correlation between suppression and the presence of contaminating mycoplasmas. Using mycoplasma-free isolates of HCMV we could demonstrate no immunosuppressive effect on mitogen-mediated T cell proliferation of both unseparated human peripheral blood lymphocytes and nylon wool non-adherent T cells; these virus isolates also did not suppress accessory cell function or interleukin 1 production by monocytes infected in vitro. We conclude that the previously reported immunosuppressive effects of HCMV in vitro may be attributable to the presence of mycoplasmas and are unlikely to be due to expression of HCMV in monocytes. We suggest that mycoplasma contamination of isolates of HCMV may be a more extensive problem than is currently recognized.

Detection and preliminary characterization of herpes simplex virus type 1 transcripts in latently infected human trigeminal ganglia.

RNA extracted from human trigeminal ganglia was examined for the presence of herpes simplex virus type 1 (HSV-1) transcripts by Northern hybridization (RNA blot) analysis. By using cloned DNA and single-stranded RNA probes, two abundant colinear HSV-1 transcripts (1.85 and 1.35 kilobases) were detected in ganglia from 9 of 17 individuals. These RNAs overlap the 3' end of the transcript for immediate-early gene ICP0 but are transcribed from the opposite strand; thus, they are antisense relative to the ICP0 mRNA. We also report evidence by in situ hybridization that these latently infected ganglia contain HSV-1 RNA homologous to the BamHI SP region of the genome which is transcribed in the same direction as the other latency transcripts. In Vero cells productively infected with laboratory strain HSV-1 KOS and in cultures infected with different low-passage clinical isolates, only the 1.85-kilobase transcript was detected, and there was variation in the size of this larger latency transcript. These novel transcripts may play a role in maintaining HSV-1 latency in human ganglia.

Binding of complement component C3b to glycoprotein gC of herpes simplex virus type 1: mapping of gC-binding sites and demonstration of conserved C3b binding in low-passage clinical isolates.

The sites on glycoprotein gC of herpes simplex virus type 1 (HSV-1) which bind complement component C3b were evaluated by using anti-gC monoclonal antibodies and mutants which have alterations at defined regions of the glycoprotein. Monoclonal antibodies were incubated with HSV-1-infected cells in a competitive assay to block C3b binding. Each of 12 different monoclonals, which recognize the four major antigenic sites of gC, completely inhibited C3b binding. With this approach, no one antigenic group on gC could be assigned as the C3b-binding region. Next, 21 gC mutants were evaluated for C3b binding, including 1 which failed to synthesize gC, 4 which synthesized truncated forms of the glycoprotein such that gC did not insert into the cell's membrane, and 16 which expressed gC on the cell's surface but which had mutations in various antigenic groups. Eleven strains did not bind C3b. This included the 1 strain which did not synthesize gC, the 4 strains which secreted gC without inserting the glycoprotein into the cell membrane, and 6 of 16 strains which expressed gC on the cell surface. In these six strains, the mutations were at three different antigenic sites. One hypothesis to explain these findings is that C3b binding is modified by changes in the conformation of gC which develop either after antibodies bind to gC or as a result of mutations in the gC gene. Attachment of C3b to gC was also evaluated in 31 low-passage clinical isolates of HSV-1. Binding was detected with each HSV-1 isolate, but not with nine HSV-2 isolates. Therefore, although mutants that lack C3b binding are readily selected in vitro, the C3b-binding function of gC is maintained in vivo. These results indicate that the sites on gC that bind C3b are different from those that bind monoclonal antibodies, that antibodies directed against all sites on gC block C3b binding, and that C3b binding is a conserved function of gC in vivo.