The Titration of Trachoma and Inclusion Blennorrhoea Viruses in Cell Cultures

Furness, G.; Graham, Doris M.; Reeve, P.

doi:10.1099/00221287-23-3-613

Cited by 125 publications

(69 citation statements)

References 7 publications

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“…1). These findings are in line with recent descriptions of trachoma-infected tissue cultures (Gordon, Quan & Trimmer, 1960; Furness, Graham, Reeve & Collier, 1960;Bernkopf et al 1960), and comparable with the earlier accounts of changes that accompany multiplication of other members of the psittacosis group (Bedson & Bland, 1932). The cytopathic changes developed synchronously throughout the cell sheets ; counts indicated that more than 90 yo of the cells were infected a t the outset.…”

Section: Phase-cmtrast Microscopysupporting

confidence: 81%

Structure and Replication of the Trachoma Agent in Cell Cultures, as shown by Electron Microscopy

Armstrong

Valentine

Fildes

1963

Journal of General Microbiology

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SUMMARYPropagation of the trachoma agent in human cell cultures has made possible the observation by electron microscopy of sequential stages in the replication of the micro-organism. Scattered reticular foci with incomplete limiting membranes were present in the cytoplasm of HeLa cells 12 hr. after exposure to the infectious agent in high multiplicity. After 24 hr. the foci had coalesced to give a single reticular mass. Within it there appeared large and discrete developmental forms from which, by progressive subdivision and diminution in size, !mica1 elementary bodies were formed. The elementary bodies had a prominent limiting membrane, and a dense core within which a fibrillar nucleoid could be recognized. The controversial taxonomic status of the trachoma agent is considered briefly in the light of ultrastructural evidence.

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Section: Phase-cmtrast Microscopysupporting

confidence: 81%

Structure and Replication of the Trachoma Agent in Cell Cultures, as shown by Electron Microscopy

Armstrong

Valentine

Fildes

1963

Journal of General Microbiology

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“…HeLa cells were used to propagate Chlamydia trachomatis serovar L2 (LGV 434) for purification using established protocols (35,36). Chlamydial titers were determined using conventional protocols to establish multiplicities of infection (MOI), which were based on inclusion forming units (IFU) and determined in HeLa cells (36,37) using a polyclonal rabbit or guinea pig anti-C. trachomatis serovar L2 EB antibody and secondary antibodies conjugated to DyLight fluors (Jackson ImmunoResearch Laboratories, West Grove, PA). siRNA knock down of syntaxin 6 and VAMP4.…”

Section: Methodsmentioning

confidence: 99%

“…As we noted no differences in the numbers of inclusions formed (specifically, no abrogation of initial entry and infection) in the absence of VAMP4 or syntaxin 6 compared to the numbers of inclusions in controls (data not shown), we wished to assess the impact of these proteins on chlamydial development by examining infectious progeny. To determine infectious progeny, control (nontargeting), VAMP4, or syntaxin 6 siRNA-treated HeLa cells (VAMP4) or C2BBe1 cells (syntaxin 6) were infected with C. trachomatis serovar L2 for 40 to 42 h and lysed, and titers of lysates containing infectious Chlamydia organisms were used to inoculate a fresh monolayer of HeLa cells as previously described (37,45). At 30 h postinoculation, the secondary infections were fixed and processed for indirect immunofluorescence assay.…”

Section: Vamp4 Localization To the Chlamydial Inclusionmentioning

confidence: 99%

Vesicle-Associated Membrane Protein 4 and Syntaxin 6 Interactions at the Chlamydial Inclusion

et al. 2013

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The predominant players in membrane fusion events are the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family of proteins. We hypothesize that SNARE proteins mediate fusion events at the chlamydial inclusion and are important for chlamydial lipid acquisition. We have previously demonstrated that trans-Golgi SNARE syntaxin 6 localizes to the chlamydial inclusion. To investigate the role of syntaxin 6 at the chlamydial inclusion, we examined the localization and function of another trans-Golgi SNARE and syntaxin 6-binding partner, vesicle-associated membrane protein 4 (VAMP4), at the chlamydial inclusion. In this study, we demonstrate that syntaxin 6 and VAMP4 colocalize to the chlamydial inclusion and interact at the chlamydial inclusion. Furthermore, in the absence of VAMP4, syntaxin 6 is not retained at the chlamydial inclusion. Small interfering RNA (siRNA) knockdown of VAMP4 inhibited chlamydial sphingomyelin acquisition, correlating with a log decrease in infectious progeny. VAMP4 retention at the inclusion was shown to be dependent on de novo chlamydial protein synthesis, but unlike syntaxin 6, VAMP4 recruitment is observed in a species-dependent manner. Notably, VAMP4 knockdown inhibits sphingomyelin trafficking only to inclusions in which it localizes. These data support the hypothesis that VAMP proteins play a central role in mediating eukaryotic vesicular interactions at the chlamydial inclusion and, thus, support chlamydial lipid acquisition and chlamydial development.

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“…The infected cells from six batches of 4 1 of infected L cells were homogenized in a Teflon grinder and yielded a total of 3-4 x 1 0~~ inclusion-forming units (i.f.u.) (Furness, Graham & Reeve, 1960;Taverne & Blyth, 1971). The agent was purified by a slight modification of the method of Tamura & Higashi (1963); before and after treatment with nucleases and trypsin, the preparation was centrifuged at 75008 for 60 min at 4 "C through a 6 cm column of 25 yi , * Present address : National Institute for Biological Standards and Control, Holly Hill, Hampstead, London N W 3 6RB.…”

Section: Methodsmentioning

confidence: 99%