Comparative susceptibility of eleven mammalian cell lines to infection with trachoma organisms.

Eleven mammalian cell lines, HeLa 229, HeLa M, Hep-2, FT, BHK-21, Vero, MK-2, MPK, L-WO5A2, McCoy, and L-929, were tested for their susceptibility to infection with Trachoma strains TW-3 (type C, ocular origin) and UW-5 (type E, genital origin). All the cell layers were pretreated with diethylaminoethyl-dextran before inoculation of the organisms, and the inocula were centrifuged onto the cell layers. HeLa 229 was found to be the most sensitive to infection as determined by inclusion counts. The next most susceptible were cell lines MK-2, Hep-2, McCoy, and HeLa M, in that order. Infectivity in these cells ranged from 89 to 12% of that observed in HeLa 229. The remaining cell lines, BHK-21, L-WO5A2, L-929, Vero, MPK, and FT, were much less susceptible with infectivity less than 10% that of HeLa 229. HeLa 229 cells and 5-iodo-2'-deoxyuridine-pretreated McCoy cells have been used most extensively with Trachoma organisms in our laboratories. Infectivity in these two cell culture systems, both pretreated with diethylaminoethyl-dextran, was compared using 13 Trachoma strains of both ocular and genital origins of different immunotypes. The two systems performed similarly except with two type C, three type I, and one type J strains, With the type C, I and J strains tested, considerably fewer inclusions were found in 5-iodo-2'-deoxyuridine-pretreated McCoy than in HeLa 229 because inclusion formation of these strains in McCoy cells was not enhanced by 5-iodo-2'-deoxyuridine pretreatment.     

The effect of fetuin and other sialoglycoproteins on the in vitro penetration of Trypanosoma cruzi trypomastigotes into fibroblastic cells

Heat-inactivated calf-, human-, and especially fetal calf serum stimulate infection of Vero cells by cell culture-derived trypomastigotes of Trypanosoma cruzi: the stimulatory effect is more marked with extracellular activated parasites or trypsinized trypomastigotes than with recently released parasites. The augmented invasion is not the consequence of a stimulation of attachment of trypomastigotes to host cells. Various sialoglycoproteins like fetuin, transferrin, fibrinogen, alpha-1-antitrypsin, mucin and goat-IgG are also effective in enhancing in vitro infectivity. Colominic acid also stimulates invasion, but other non-sialic polyanionic compounds are either ineffective (chondroitin sulfate, poly-aspartic acid) or inhibitory (heparin, phytic acid, myo-inositol hexasulfate). Fetuin, the best stimulatory compound tested, gives half-maximal activation with approximately 0.03 mg ml-1, and total activation with 0.5-1 mg ml-1. The enhancement of infectivity is time-dependent (2-3 h for maximal activation) at 37 degrees C and does not occur at 0 degrees C. Desialidated-fetuin or -fetal calf serum do not stimulate infectivity at all. Treatment with fetuin of parasites alone (or Vero cells alone), followed by removal of free fetuin and by interaction with untreated Vero cells (or parasites) indicates that the stimulation effect of fetuin occurs mainly on the trypomastigotes. No specific binding of [125I]fetuin to the parasites could be demonstrated, and incubation with exogenous neuraminidase of trypomastigotes previously activated by fetuin, reverses nearly completely the stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation by cell trypsinization of Sendai virus expression in African green monkey kidney cells: first infection and establishment of a carrier state.

Two African green monkey kidney (AGMK) cell lines, 37RC (interferon-producing) and Vero (non-interferon-producing), were infected by egg-grown Sendai virus passaged in eggs at high and low m.o.i. The appearance of haem-adsorption, and cytopathic effect (c.p.e.) as well as the presence of haemagglutinating virions in the supernates were much more pronounced with a virus seed obtained with 10(-3) diluted passages than with a seed obtained with undiluted inoculum. They were also independent of interferon production (data obtained in 37RC and Vero cells were almost superimposable). In studies carried out with the virus seed prepared at high dilution the establishment of infection was maximal when monolayers were infected as soon as 5 h after trypsinization and cell seeding, regardless of cell density. Virus in supernates obtained from cultures infected 5, 20 or 50 h after seeding exhibited a greatly reduced infectivity for monkey cells, but not apparently for chick embryos. Trypsin treatment of the virus supernates restored their infectivity for AGMK cells more efficiently for virus released from cells infected 5 h after seeding than for virus released from cells infected later after seeding. In keeping with these observations, virus in supernates from cultures infected 5, 20 or 50 h after seeding contained increasing amounts of auto-interfering virions. The decreased infectivity obtained in cell supernates was not accounted for by major differences in virus RNA synthesis. Similarly, the optimum infection established in cells seeded only for 5 h was not due to increased virus adsorption. Several lines of cells surviving first infection with egg-grown Sendai virus have been obtained and kept in cultures for 3 to 18 months. Virus release and c.p.e. apparently become reduced in the cells surviving the first infection until the newly repopulated monolayers must undergo trypsinization. Weekly protease treatments of the cells reactivate all parameters of virus infection which again will tend to disappear slowly and then reappear following each trypsinization ('intermittent' carrier state). The establishment and the 'intermittent' reactivation of these lines were not prevented by the inclusion in the medium of anti-Sendai virus serum. Temperature-sensitive ts functions do not seem to play an important role in this virus-host relationship.     

Serological Diagnosis of Trypanosoma rangeli Infected Patients. A Comparison of Different Methods and its Implications for the Diagnosis of Chagas' Disease

Venous blood from 65 Panamanian schoolchildren living in an area endemic for both Trypanosoma cruzi and T. rangeli were screened for the presence of these parasites. Trypanosoma rangeli were isolated and cultured from four individuals. Serological tests of all 65 sera were performed, including immunohaemagglutination (IHA), indirect immunofluorescence assay (IF) and ELISA using both T. rangeli and T. cruzi as antigens, as well as T. cruzi synthetic peptides in an ELISA assay. Results indicated a higher immunoreactivity to T. rangeli preparations than to T. cruzi within the studied population, which could be divided into four 'serological responder' groups. Interestingly, the panel of SAPA and other T. cruzi synthetic peptides were not useful in the discrimination of patients. Furthermore, patients from whom parasites had been isolated could not be distinguished from those of two other groups. Significant immunoreactivity to T. cruzi preparations was displayed in all responder sera, despite total lack of evidence of infection with this parasite. The immunobiological significance of T. rangeli infection is unclear, but these data indicate that it is a compounding problem in the accurate diagnosis of pathological T. cruzi infection by serological analysis. The relationship of these cohabiting species, in respect to infection outcome and immunological activation, is discussed.     

Expression of fluorescent genes in Trypanosoma cruzi and Trypanosoma rangeli (Kinetoplastida: Trypanosomatidae): its application to parasite-vector biology

Two Trypanosoma cruzi-derived cloning vectors, pTREX-n and pBs:CalB1/CUB01, were used to drive the expression of green fluorescent protein (GFP) and DsRed in Trypanosoma rangeli Tejera, 1920, and Trypanosoma cruzi Chagas, 1909, isolates, respectively. Regardless of the species, group, or strain, parasites harboring the transfected constructs as either episomes or stable chromosomal integrations showed high-level expression of fluorescent proteins. Tagged flagellates of both species were used to experimentally infect Rhodnius prolixus Stal, 1953. In infected bugs, single or mixed infections of T. cruzi and T. rangeli displayed the typical cycle of each species, with no apparent interspecies interactions. In addition, infection of kidney monkey cells (LLC-MK2) with GFP-T. cruzi showed that the parasite retained its fluorescent tag while carrying out its life cycle within cultured cells. The use of GFP-tagged parasites as a tool for biological studies in experimental hosts is discussed, as is the application of this method for copopulation studies of same-host parasites.     

Improved technique for transient expression and negative strand virus rescue using fowlpox T7 recombinant virus in mammalian cells

The suitability of recombinant T7 polymerase produced using either the highly attenuated MVA strain of vaccinia (MVA-T7) or fowlpox virus (FP-T7) for transient expression and negative strand virus rescue was compared in two mammalian cell lines (MDBK and Vero) and in primary cells of bovine, ovine and caprine origin. Such primary cells are more permissive for the growth of wild type strains of morbilliviruses, such as Rinderpest virus and Peste des petits ruminants virus. MVA-T7 was found to be highly cytopathic in the primary cells, multiplying rapidly and killing the cells within 3-5 days of infection, even when very low multiplicities of infection (MOI) were used. In contrast, FP-T7, which appeared to express similar amounts of T7 polymerase, was found to be non-cytopathic in a variety of primary and established cell lines of mammalian origin and was suitable for use in virus rescue experiments. MDBK cells and primary cells, unlike Vero cells, could not be efficiently transfected and so were unsuitable for virus rescue. Optimal conditions for rinderpest virus rescue in Vero cells were established using FP-T7 in place of MVA-T7. This system will be suitable for rescuing other viruses which grow in Vero cells.     

Mosquito cells bind and replicate hepatitis C virus

Several studies have demonstrated some hepatitis C virus (HCV) replication in lymphocyte and hepatocyte cell lines such as in African green monkey Vero cells. The aim of the present study was to select other cell lines able to bind and replicate HCV. Human hepatoma PLC/PRF/5 cells, human lymphoma Namalwa cells, Vero and mosquito AP61 cells were inoculated with HCV-positive plasma, washed six times and examined for the presence of the viral genome at different times post infection, using an RT-PCR method. Binding of HCV to cells was estimated by HCV RNA detection in cells 2 hr after inoculation and in the last wash of these cells. Successive virus passages in cells were carried out. All the cells studied were able to bind HCV but only AP61 and Vero cells provided evidence of replication and production of infectious virus: virus RNA was detected during 28 days post-infection in four successive virus passages. CD81 molecules, a putative HCV receptor, were detected by cytofluorometric analysis. Vero cells express CD81 molecules whereas these molecules were not detected on AP61 cells. It is suggested that other receptors are involved in HCV binding to Vero and AP61 cells.     

Cell invasiveness of Proteus mirabilis and Proteus vulgaris strains

Cell penetration ability of haemolytic and non haemolytic Proteus rods was compared. Among four Proteus strains all were able to invade the tested cells (Vero 135, HeLa, L-929 and human blood lymphocytes) but the expression of this feature by haemolytic strains was markedly higher. The survival and multiplication of intracellular bacteria, especially in the case of fresh human blood lymphocytes may be of importance for the development of infection in higher organisms.     

A block in glycoprotein processing correlates with small plaque morphology and virion targetting to cell-cell junctions for an oral and an anal strain of herpes simplex virus type-1

Summary The characteristics of two clinical isolates of HSV-1 obtained from an oral (424) and an anal (490) lesion were compared with the highly passaged KOS strain. In contrast to KOS, the clinical isolates produced small plaques, were more cell-associated and the predominant viral glycoprotein species for gC and gD in infected cell lysates was the precursor, high mannose glycoform. Total virus production in Vero cells was equivalent for the three virus strains in one-step growths. Pulse-chase studies of glycoprotein C processing showed a reduction in rate at 7.5h post infection and a significant block in processing at 10.5h post infection for 424 and 490 but not KOS. Similar results were obtained for gD. The significant reduction in glycoprotein processing for 424 and 490 suggests a block in transport of viral glycoproteins or virions to and through the Golgi apparatus. Extracellular virions and the cell surface, prior to cell lysis, contained the processed gC glycoform suggesting a competent cellular glycan processing system. Upon co-infection of 424 or 490 with KOS or a gC^– KOS strain, gC was processed to levels equivalent to KOS indicating that 424 and 490 are not inhibitory but that an activity(s) encoded by KOS facilitates maturation of gC from 424 and 490. Unlike KOS infected Vero cells, virion-containing vacuoles were observed in the cytoplasm at 12h p.i. and extracellular virions were concentrated at cell-cell junctions of 424 or 490 infected cells but not in the perinuclear region. These results suggest that intracellular transport of viral glycoproteins and virions in 424 and 490 infected cells is different from KOS infected cells. The reduced level of viral glycoprotein maturation, virus release, cell surface presence and presence of virions at cell-cell junctions are consistent with small plaque production in tissue culture cells.Portions of this work were presented in the 17th International Herpesvirus Workshop, Pittsburgh, PA, 1993.     

Efficiencies and kinetics of infection in different cell types/lines by African and Asian strains of Zika virus

After recent outbreaks, Zika virus (ZIKV) was linked to severe neurological diseases including Guillain-Barré syndrome in adults and microcephaly in newborns. The severities of pathological manifestations have been associated with different ZIKV strains. To better understand the tropism of ZIKV, we infected 10 human and four nonhuman cell lines (types) with two African (IbH30656 and MR766) and two Asian (PRVABC59 and H/FP/2013) ZIKV strains. Cell susceptibility to ZIKV infection was determined by examining viral titers, synthesis of viral proteins, and replication of positive and negative strands of viral genome. Among nonhuman cell lines, only Vero cells were efficiently infected by ZIKV. Among human cell lines, all were permissive to ZIKV infection. However, 293T and HeLa cells showed differential susceptibility towards African strains. In 293T cells, the NS1 protein was expressed at the high level by African strains but was almost not expressed by Asian strains though there was no obvious difference in viral genome replication, suggesting that the differential susceptibility might be controlled at the stage of viral protein translation. This study provides comprehensive results of the permissiveness of different cell types to both African and Asian ZIKV strains, which might help clarify their different pathogenesis.     

HeLa cell infection by Yersinia enterocolitica: evidence for lack of intracellular multiplication and development of a new procedure for quantitative expression of infectivity.

The in vitro invasive properties of bacteria have frequently been studied by the use of HeLa cell cultures in chamber slides, using microscopic examination to enumerate intracellular bacteria. When this system was used to examine invasive properties of Yersinia enterocolitica, it resulted in rapid internalization of high numbers of bacteria during the infection phase which prevented subsequent discrimination of intracellular multiplication. A modified procedure was developed which standardized the ratio of bacteria to HeLa cells (i.e., multiplicity), the time for the infection phase, and the addition of specific antiserum with gentamicin for restricting bacterial uptake during the intracellular growth phase. Studies with this modified chamber slide system found that strains of human isolates of Y. enterocolitica (serotypes O:3, O:8, O:5,27, and O:6,30) exhibited different degrees of cell infection but did not multiply intracellularly. A second test system was developed that used roller tubes and viable cell counts for the enumeration of intracellular bacteria. This roller tube system confirmed that internalized bacteria did not multiply inside HeLa cells over a 24-h period. The roller tube system with viable cell counts for enumeration is a simplified technique for quantitative comparison of in vitro infectivity of HeLa cells by Y. enterocolitica.     

Variable infection of Vero cells and homologous interference after co-cultivation with HeLa cells with persistent defective infection by Edmonston measles virus.

The HeLa subline K11A-HG-1 (line of HeLa cells persistently infected with Edomonston measles virus but containing little or no transmissible infectious virus) was co-cultivated with Vero cells. Focal syncytia were formed containing measles antigen and accumulations of nucleocapsid-like structures with no detectable production of transmissible infectious virus or positive hemadsorption. The infection aborted between 2 and 3 weeks after preparation of co-cultures. Upon subculture of co-cultures, occasionally complete infections (progressive syncytial degeneration, hemadsorption, and production of transmissible infectious virus) appeared. A linear dose response curve for nontransmissible infection was obtained along with evidence that measles antigen had to be present on the surface of K11A-HG-1 cells for their infectivity for Vero cells. The basis for initiation of Vero cell infection by living K11A-HG-1 cells, but not by nonviable intact K11A-HG-1 cells killed by a virus-preserving technique, nor by disrupted K11A-HG-1 cells, is, at present, a matter of speculation. However, several lines of evidence were obtained which suggested that subsequent development of delayed variable transmissible Vero cell infection occurred because of a type of viral interference, including the presence of an inhibitor in K11A-HG-1 cultures, the bulk of which was cell-associated.     

LYT1 protein is required for efficient in vitro infection by Trypanosoma cruzi

Trypanosoma cruzi invasion of host cells involves several discrete steps: attachment, parasite internalization mediated by recruitment and fusion of host cell lysosomes, and escape from the parasitophorous vacuole to liberate amastigotes to multiply freely in the cytosol. This report describes the initial characterization of the LYT1 gene and the demonstration that the gene product is involved in cell lysis and infectivity. Mutational analysis demonstrated that deletion of LYT1 resulted in attenuation of infection, which was associated with diminished hemolytic activity. Reintroduction of LYT1 restored infectivity in null mutants, confirming the critical role of LYT1 in infection. Additionally, in vitro stage transition experiments with LYT1-deficient lines showed that these parasites converted to extracellular amastigote-like cells and metacyclic trypomastigotes more rapidly than wild-type parasites, suggesting that the diminished infectivity was not a result of the LYT1 deficiency that affected the parasite's ability to complete the life cycle.     

Trypanosoma cruzi cell invasion and traffic: influence of Coxiella burnetii and pH in a comparative study between distinct infective forms

Previous studies have shown that Coxiella burnetii, an intracellular bacterium that resides within acidified vacuoles with secondary lysosomal characteristics, is an effective modulator of the intracellular traffic of trypomastigote forms of Trypanosoma cruzi. In addition, vacuolar and cellular pH are related to fusion events that result in doubly infected phagosomes. T. cruzi, the etiological agent of Chagas' disease, occurs as different strains grouped in two major phylogenetic lineages: T. cruzi I, associated with the sylvatic cycle, and T. cruzi II, linked to the human disease. In this work we compared extracellular amastigotes (EA), metacyclic trypomastigotes (MT) and tissue culture derived trypomastigotes (TCT) belonging to T. cruzi I or T. cruzi II for their ability to invade and escape from their parasitophorous vacuole (PV), in Vero cells or Vero cells harboring the bacterium, C. burnetti. Distinct invasion patterns were observed between different infective stages and between infective forms of different strains. Studies on the transference kinetics revealed that pH modulates the intracellular traffic of each infective stage, but this influence is not exclusive for each phylogenetic group. Endosomal to lysosomal sequential labeling with EEA-1 and LAMP-1 of the PV formed during the entry of each infective form revealed that the phagosome maturation processes are distinct but not strain-dependent. Due to their low hemolysin and trans-sialidase activities, MTs are retained for longer periods in LAMP-1 positive vacuoles. Our results thus suggest that despite the contrasting invasion capabilities, parasites of distinct phylogenetic group behave in similar fashion once inside the host cell.     

Development of a cell culture system susceptible to measles,canine distemper,and rinderpest viruses

Summary Three strains of chick embryo adapted canine distemper virus (Lederle, Wisconsin, and Onderstepoort strains) and chick embryo adapted LA strain of rinderpest virus were easily adapted to an established line of African green monkey kidney cells (Vero cells), which has been routinely employed for the titration of measles virus. By using these Vero cell adapted strains of canine distemper and rinderpest viruses, techniques of infectivity titration and virus neutralization in Vero cells were established. Comparative studies of cytopathology and growth characteristics of canine distemper, rinderpest, and measles viruses indicated that the behavior of the three viruses in Vero cells is almost the same. The Vero cell system was suggested as a suitable host for the comparative study of the serologic and biologic relationships among measles, canine distemper, and rinderpest viruses.     

Invasive potential of noncytotoxic enteropathogenic Escherichia coli in an in vitro Henle 407 cell model.

The invasive capacity of 13 enteropathogenic Escherichia coli strains was assessed in vitro in Henle 407 cell culture. Both fluorescent microscopy of infected monolayers stained with acridine orange and electron microscopy revealed the presence of intracellular bacteria. As shown by acridine orange-stained infected monolayers, the number of internalized bacteria increased with time. Monolayers infected for 3 h were treated with antibiotics and either [14C]glutamine or [3H]leucine and incubated for various time intervals, after which the amount of radioactivity present in the washed monolayers was measured. A significant (P less than 0.005) increase in uptake was evident for up to 4 h after the addition of radiolabeled amino acid. This finding was confirmed by an increase in bacterial number in cultured cells and in protein concentration of infected cells with time. None of the South African enteropathogenic E. coli isolates used in these studies produced Vero cytotoxin. These findings demonstrate that, in addition to adherence, cell penetration and intracellular multiplication take place in epithelial cell-derived tissue culture cells infected by enteropathogenic E. coli.     

Evaluation of Aedes albopictus tissue culture for use in association with arbovirus isolation.

The susceptibility and sensitivity of Aedes albopictus cell cultures to five different primary and four different low-passage arboviruses were tested. Yellow fever, West Nile, Ilesha, eastern equine encephalitis, and Flanders viruses replicated in A albopictus tissue cultures. Replication was determined by the ability of selected tissue culture fluids to infect suckling mice, and by recovery from tissue culture fluid of progressively increasing amounts of complement-fixing (CF) antigen with time. Virus persistence was demonstrated with Nodamura, western equine encephalitis, and Mayaro viruses, but multiplication was not proven; neither persistence nor multiplication was demonstrated with a Kemerovo group virus. When yellow fever and Ilesha viruses were simultaneously inoculated into A albopictus culture, CF antigen for each was consistently detected. In a more detailed comparative study of field specimens, 12 unpassaged strains of yellow fever virus were tested for infectivity in A albopictus tissue culture, Vero cells, and baby mice. Higher titers of virus were detected (0.8--2.3 log ID50 per ml) in Vero cell culture than in A albopictus tissue culture or baby mouse systems. These results suggest the feasibility of using A albopictus cells in association with the primary isolation of arboviruses.     

Equine herpes virus type 1 (EHV-1) infection induces alterations in the cytoskeleton of Vero cells but not apoptosis

Summary.  Effects of infection with two different strains of equine herpes virus type 1 (EHV-1; Piber 178/83, Kentucky D) on the cytoskeleton of Vero cells were investigated immunohistochemically, and evaluated by confocal laser scanning microscopy. Twenty four hours post EHV-1 infection the assembly of the microtubulus system of Vero cells was heavily disturbed. The Golgi region was dispersed into vesicles spread throughout the cytoplasm as demonstrated by WGA lectin binding. Other cytoskeletal elements such as cytokeratin, vimentin, and filamentous actin (F-actin) were not affected by EHV-1 infection. The nature of Vero cell death after EHV-1 infection was investigated by three different methods to include all possible stages of apoptosis. All methods failed to demonstrate characteristic apoptotic features, therefore, it seems likely that necrosis is the predominant way of cell death in EHV-1 infected Vero cells. Received February 23, 1999 Accepted April 26, 1999     

Changes in morphology and infectivity of cell culture-derived trypomastigotes of Trypanosoma cruzi

Trypomastigotes of Trypanosoma cruzi, obtained from the first burst of infected Vero cells, have only a limited invasive capability for fibroblastic cells. Intracellular amastigotes and epimastigotes do not infect these cells at all. Preincubation of the isolated trypomastigotes with Eagle's minimal essential medium/10% fetal calf serum increases 5- to 15-fold their in vitro infectivity. This increased invasive capability is accompanied, in the case of the EP strain of T. cruzi, by a morphological transformation into an amastigote-like or spheromastigote form, which is similar, but not identical to replicating intracellular amastigotes. Trypomastigotes from another isolate (BEC) also increase their infectivity several fold upon preincubation, but before any morphological differentiation occurs, suggesting that these two events are independent. The phenomenon of increased infective capability of the parasite is expressed similarly in different host cells. Parasite adhesion is stimulated 4- to 12-fold upon preincubation of the trypomastigotes. The type of serum used (fetal calf, calf, human) affects the development of infectivity, as well as the process of cell infection in itself, but not the morphological differentiation. These processes are also temperature-dependent. The highly infective parasitic forms do not synthetize DNA, but are active in RNA and protein synthesis. The results obtained indicate the existence in T. cruzi trypomastigotes of an active system for infecting fibroblastic cells, which is only partially expressed in trypomastigotes recently released from host fibroblasts but which can undergo a further extracellular maturation, thus allowing studies on the mechanism of infection in cell-free media.