Human spumavirus replication in human cells

It was previously reported that the replication of the human syncytium-forming virus (HSFV), a spumavirus, occurred only in fibroblast-like cell lines (human fetal diploid lung #645 [HFDL]) but not in epithelial-like lines (recovered amnion) [RA]. Factors that may be involved in such a phenomenon were the subject of this investigation. While both permissive (HFDL) and nonpermissive (RA) cell lines supported the replication of several representative animal viruses and adsorbed HSFV equally well, immunofluorescent staining of HSFV antigens revealed markedly fewer fluorescing cells in nonpermissive cultures. Infectious center assays of infected nonpermissive cells indicated the formation of significantly fewer infectious centers. The rate of DNA synthesis was markedly greater in the permissive cell lines. In addition, in the permissive cell line, the amount of proviral DNA revealed by the Hirt procedure and isopycnic banding in CsCl was significantly increased and was infectious as determined by the calcium phosphate-DMSO transfection assay. These results indicate that resistance of HSFV infection in nonpermissive cell cultures is probably an intracellular event.     

Infection of human amnion cells with cytomegalovirus.

Human cytomegalovirus (CMV), known to replicate in vitro in human fibroblastic cells, was found to replicate in epithelial human amnion (HA) cells. Large syncytia formed in these cells after infection with CMV; inclusion bodies were observed in the nuclei, and CMV antigens were demonstrated in both the cytoplasm and the nucleus by indirect immunofluorescence techniques. The synthesis of virus DNA was also detected, and the production of infectious virus was followed. The titers were lower (from 10(4) to 6 X 10(5) using different isolates of CMV) than those obtained in human embryo fibroblast (HEF) cells, and the replication cycle was slower in HA than in HEF cells.     

Cell cultures of spotted souslik--preliminary characterization of their growth and sensitivity to viruses

Cell cultures from the cutaneo-muscular tissue of fetuses (SE), and from lungs (SL), kidneys (SK), testes (STe) of adult spotted sousliks were obtained. Cells from lungs (SL) were viable at 4 degrees C three times longer than fibroblastic cells of human embryos (HEF). At 37 degrees C the growth rate of SL, HEF and L929 cells was similar. However, at 26 degrees C SL cells grew faster than HEF or L929 cells and their population doubling time was shorter. The cultures of SE, SL and SK cells were sensitive to vesicular stomatitis virus (VSV), Newcastle disease virus Radom (NDV-R) and Hertfordshire (NDV-H) strains, to vaccinia virus and herpes simplex virus type 1 and type 2. All these viruses were able to reproduce in souslik cell cultures and caused characteristic CPE.     

Rubella virus does not induce apoptosis in primary human embryo fibroblast cultures: a possible way of viral persistence in congenital infection

Congenital rubella is a persistent infection that contrasts with acute postnatal infection. Basis of the Rubella virus (RV) persistence still remain unknown, though several hypotheses have been postulated. RV induces apoptosis in cell lines, maybe as a way of cell-autonomous defense mechanism against virus. Considering the pattern of c-oncogenes expression during embryogenesis, which promotes proliferation while it inhibits apoptosis in specific cells, at certain times, it can be proposed that when RV infection establishes early in gestation, embryo cells that are proliferating have their apoptotic pathways shut down; then infected proliferating embryo cells cannot execute their apoptotic death program. We here report that RV induces apoptosis in human normal-term placenta chorionic villi explants (CVE) and in monolayers of cytotrophoblasts (CTB), but does not induce apoptosis in primary human embryo fibroblasts (HEF) cultures. These results suggest distinct responses to RV infection when comparing differentiated cells, as CTB, to cells with high proliferating potential, as HEF. RV shoots apoptosis in the former, whereas in fibroblastic dividing cells derived from embryo, RV appears not to be enough stimulus to activate the genetic program of cell death.     

Growth in culture of adenocarcinoma cells from the small intestine of sheep.

Explant cultures were initiated from adenocarcinoma of the small intestine in sheep and from various metastases. Several cell types grew, most being fibroblastic in nature. However, 2 cultures yielded mixed cells which arranged themselves into areas of epithelial-like cells surrounded by fibroblast-like cells and this pattern was consistent over 30 subcultures and several months of culturing. The epithelial-like cells were separated from the others by the use of a modified medium containing citrulline or by sedimentation through a bovine serum albumin solution. Various properties, including their growth rate in 5% and 0.5% serum, the absence of surface fibronectin and their ability to grow in semi-solid agar, indicated that they may represent carcinoma cells. Screening for virus production from these cells and all other explant cultures proved negative.     

The preferential cytotoxicity of reovirus for certain transformed cell lines

Summary The susceptibility of a variety of cell lines of different mammalian origin to cytotoxic (CT) induction by either ultraviolet light-irradiated reovirus type 2 (UVR2) or viable reovirus type 2 plus the protein synthesis inhibitor, cycloheximide, was examined. The following groups of cells were found to be susceptible to CT-induction: certain tumor cells and spontaneously transformed cell lines of human origin and certain virally and spontaneously transformed cell lines of murine origin. The following groups of cells were found to be resistant: normal human diploid cell lines, primary and continuous cell cultures of subhuman primates, primary mouse cells, normal rat kidney cells and baby hamster kidney cells. Susceptibility to CT-induction could not be related to the adsorption of virus to cells, nor to the capacity of the cell to support virus replication.The research was supported by Public Health Service grant CA-13768 from the National Cancer Institute.     

Ultrastructural changes induced by influenza viruses in permissive and nonpermissive cells

Human diploid fibroblast (LEP) cells and hamster embryo fibroblast (HEF) cells were infected with four influenza viruses, viz., A/NWS, A/WS, and their recombinants r₁₂ and r₁₄. NWS virus could be propagated in both cell systems and the recombinants could be propagated in HEF cells only, while WS virus could not be passed in either type of cells. After 24 hr, or earlier, all of the cells in both types of culture infected with any of the four viruses produced hemagglutinin and a great majority of them also produced ribonucleo-protein antigen. Electron microscopic examination revealed all of the ultrastructural changes typical of influenza virus replication in HEF and LEP cells when these were infected with NWS virus and in HEF cells when these were infected with WS virus or either of the recombinants. In LEP cells infected with the latter three viruses, only a low percentage (less than 5%) of cells exhibited such alterations; a great majority were free of any detectable change. These results indicate that cells can be infected with influenza virus and synthesize some virus-specific products without being morphologically altered.     

Characterization of Mason-Pfizer virus induced cell transformationin vitro

Summary Several types of cell strains and established cell lines of simian and human origin failed to demonstrate foci of altered cells following infection with the Mason-Pfizer monkey virus (M-PMV). However, most diploid cultures, after infection, lived longer and displayed the ability to grow in soft agar medium. The number of cell colonies developing in the soft agar was directly proportional to the amount of virus added to the culture. Two types of cell colonies were isolated from soft agar after infection of monkey foreskin cells with M-PMV. One had characteristic fibroblastic morphology, and the other showed an epithelioid cell phenotype. The ratio of fibroblastic colonies to epithelioid colonies was in excess of 20:1. The epithelioid cultures displayed a complete lack of topoinhibition, formed three dimensional cellular dome structures, and demonstrated significant karyotypic alterations. Fibroblastic sublines, on the other hand, did not show formation of domes but presented some lack of topoinhibition. The majority of cells in fibroblastic sublines also continued to show a normal rhesus chromosome complement. Although both epithelioid and fibroblastic transformed cell types produced intracellular M-PMV antigen and virus particles, the infectious virus titers were significantly different. The noninfectious virus preparations recovered from some of the fibroblastic sublines contained a high percentage of aberrant forms of M-PMV.With 6 Figures     

Ageing and the fusion sensitivity potential of human cells in culture: Relation to tissue origin,donor age,and in vitro culture level and condition

Cell fusion was induced by inactivated Sendai virus in different human diploid cell lines. These were derived from the kidney, lung or skin originating from embryos or adult donos and representing predominantly epithelioid cells (kidney) and fibroblastic cells (lung and skin). The fusion sensitivity (FS) potentials of these cell lines were determined and related to various aspects of cell ageing. In case of the fibroblastic lines, an inverse relation was demonstrated both to the culture age in vitro and to the donor age. The FS potential of embryonal fibroblasts decreased some 40–50% during the in vitro cultivation. In comparison to exponentially growing fibroblasts, the FS potentials were higher in cells in the stationary phase of growth. This was shown to correlate well with the fact that the life-span in calendar time also increased in cultures predominantly grown in the stationary phase. In the case of the kidney cells, the FS potentials were some 50% higher than those of fibroblasts. Since the cellular manifestation of the FS potential most likely primarily resides in the cell membrane—cytoskeleton structure, the results emphasize the importance of this system in relation to ageing.     

Restriction to human cytomegalovirus replication in vitro removed by physiological levels of cortisol

Since viral infection is in most cases contrary to the survival of the host cell, it is reasonable to assume that cells possess innate viral replication inhibitory mechanisms. Even between strains of permissive cells, degrees of permissiveness are observed. Restriction to human cytomegalovirus (CMV) replication in vitro is well known, especially in epithelioid cells or cells derived from certain organs. We have studied restriction in a fibroblastic strain of human embryonic kidney cells. By treatment of cell cultures with maximum physiologic concentration of the hormone cortisol (25 micrograms%) both pre and post virus inoculation, susceptibility to laboratory strain Ad169 CMV and low-passaged clinical isolate JSS CMV was enhanced by factors of 6.4 +/- 0.7 and 11.1 +/- 0.4, respectively; effectively converting these cells to a totally permissive state. A linear dose response, which peaks at 25 micrograms% and declines thereafter up to 300 micrograms%, is evident for both virus strains in this enhancement system. Breakdown of restriction increases in linear fashion with increasing time of cortisol pretreatment of cells. The characterization of cortisol effects converting restrictive human fetal cells in vitro to the permissive state further indicates that human hormones may play a significant role in CMV susceptibility in vivo.     

Enhanced parainfluenza I (6/94) virus detection in latently infected human brain cell cultures by treatment with cytochalasin D and dimethyl sulfoxide.

The ability of cytochalasin D (CD) and dimethyl sulfoxide (DMSO) to enhance parainfluenza I (6/94) virus replication was studied in various cell culture systems. Treatment of CV1 cells with CD (1 microgram/ml) dissolved in DMSO prior to primary 6/94 virus exposure at 10(0)--10(5) multiplicities of infection did not substantially enhance virus replication. However, there was a transient increase in cell associated virus one day after infection of DMSO-treated cultures. CD treatment of cultures of human brain cells latently infected with 6/94 virus (LIHB cells) did not enhance 6/94 virus detection. Cocultivation of CV1 cells with CD-treated LIHB cell cultures, and cocultivation of LIHB cell cultures with CD-treated CV1 cells, resulted in the production of both cell-associated and cell-free 6/94 virus three and five days after cocultivation. No virus was detected after similar cocultivation of untreated LIHB cell cultures with untreated CV1 cells. The usefulness of CD-DMSO treatment in the rescue of virus from 6/94 LIHB cell cultures appears limited to a cocultivation system. The use of these techniques to enhance virus rescue from human tissues suspected of harboring latent viral genomes is discussed.     

Replication of cytomegalovirus in human epitheloid diploid cell line

Summary Human diploid BAMB cells with epitheloid morphology, which had been derived from amniotic fluid cells, were capable of supporting the replication of human cytomegalovirus (CMV), without prior treatment of the cells with halogenated pyrimidines. The growth of this virus in BAMB cells and in human diploid fibroblastoid (LEP) cells was compared in parallel tests. Virus replication was slower and less efficient in the former than in the latter system. The most characteristic morphological feature of the CMV-infected BAMB cells was the formation of multinucleated giant cells which frequently contained more than a hundred nuclei; such cells were not seen in LEP cultures. The development of ultrastructural changes was slower in BAMB cells than in LEP cells. The additional most marked differences concerned the place of viral envelopment and the production of cytoplasmic dense bodies. While in LEP cells most nucleocapsids were enveloped from the inner leaflet of the nuclear membrane, in the other system a great majority of the particles acquired their envelopes by budding into vacuoles. Cytoplasmic dense bodies were rare in infected LEP cells but very frequent in BAMB cells. Budding of these structures into vacuoles was also observed.With 9 Figures     

Genetic expression of human adenoviruses in simian cells. Evidence for interserotypic inhibition of viral DNA synthesis

Most simian cells are permissive for SV40 and adenovirus-SV40 hybrids but nonpermissive for human adenoviruses, and the defect has been shown to take place at the level of processing of late viral mRNAs (Klessig and Grodzicker, 1979). Viral DNA synthesis and virus progeny production were studied in simian cells infected with different adenovirus serotypes. Adenoviruses belonging to oncogenic subgroups A and B (Ad31 and Ad3) failed to replicate their DNA in CV1 cells, whereas DNA replication occurred for all the other serotypes. Co-infection of CV1 cells with SV40 and Ad3 (or Ad31) resulted in the inhibition of SV40 DNA synthesis, as well as cellular DNA synthesis. The inhibition was not related to adenovirus DNA replication, since SV40 did not complement the Ad3/Ad31 replication defective function. Similar results were obtained in coinfected BSC and MK2 simian cell lines. Inhibition of Ad2ND1 DNA synthesis and gene expression also occurred in co-infection of simian cells with nondefective Ad2ND1 hybrid and defective Ad3/Ad31. In permissive human cell lines (HeLa or KB) co-infected with Ad2 and Ad3 (or Ad31), a dominant, inhibitory effect of Ad3 (or Ad31) over Ad2 was also observed. The inhibition appeared to function stoichiometrically and not catalytically, and to involve early adenovirus gene products. In both simian and human cells a hierarchy of dominance appeared between serotypes belonging to different subgroups. The degree of inhibitory effect occurred in the following decreasing order: Ad3 and Ad7 (subgroup B), Ad9 (D), Ad4 (E), Ad31 (A), Ad2 and Ad5 (C).     

Efficiency of isolation of human rotavirus in primary African green monkey kidney cells

Out of 212 human rotavirus (HRV) containing fecal specimens, 173 (81.6%) yielded virus on first passage in primary African Green monkey kidney cells (AGMK), while additional 34 specimens, did not yield virus on first passage. However, following blind passages, 18 of the 34 yielded virus in passage levels 2-8, thus raising the overall isolation rate to 90.1%. The isolation rate of HRV strains obtained in embryonic Rhesus monkey kidney cell line (MA-104), was only 41.4%. ELISA tests performed on fluids from infected cell cultures proved to be an efficient tool to measure virus replication. No differences were encountered in the isolation rates between subgroup I and II strains, while viruses lacking the antigenic determinants of both subgroups did not grow at all. However, one of those unusual group A strains was isolated and grew well in AGMK cells. Primary AGMK and MA-104 cells supported the growth of tissue culture adapted virus most efficiently when compared with six human and primate cell types.     

Susceptibility of human and non-human cell lines to HCV infection as determined by the centrifugation-facilitated method

The centrifugation-facilitated inoculation method was used to test 51 human and non-human cell lines for ability to support HCV replication. As determined by nested RT-PCR, one fifth of the cell lines tested were virus positive 15 days post inoculation suggesting that the centrifugation-facilitated inoculation is an efficient method for cell infection with HCV. However, virus production by infected cultures remained of low grade, thus showing that the unknown factors which limit HCV replication in vitro are not overcome by the procedure.     

Increased resistance of trisomic-21 cells to virus replication: role of interferon.

Fibroblast cell cultures derived from individuals with Down's syndrome (trisomy 21) show a markedly increased resistance to the replication of a variety of viruses (including vesicular stomatitis virus, polio, and herpes simplex virus), as compared to either normal diploid fibroblasts or fibroblasts which are monosomic for chromosome 21. The increased resistance of trisomic-21 cells to virus replication can be attributed to the increased sensitivity of these cells to the antiviral action of interferon, produced during the course of the virus infection.     

Restricted replication of herpes simplex virus in human monocyte cultures: role of interferon

Exposure of fresh human monocytes in culture to high or moderate concentrations of herpes simplex virus (HSV) resulted in an abortive infection or in a highly restricted replication of the virus. Infectious progeny virus yields were obtained either by diluting the inoculum virus to multiplicities of 0.1-0.0001 PFU/cell, or by cultivation of the cells for a few days before exposure to the virus. Interferon was synthesized and released into the medium of monocyte cultures infected with the higher multiplicities, while the lower productive multiplicities or inoculations of HSV into mature macrophage cultures resulted in a production of only small amounts, if any, of interferon. Inhibition of the morphological differentiation of monocytes was seen in cultures infected with the higher multiplicities of HSV (m.o.i. 1.0-0.1 PFU/cell) and was not unlike that caused by exogenous interferon added into uninfected monocyte cultures. Antisera against the leukocyte interferon were able to enhance the production of infectious HSV in monocyte-macrophage cultures. These results suggest that interferon induced in monocytes by high multiplicities of HSV can prevent the productive replication of HSV. Apart from its possible direct antiviral effect interferon may cause this restriction of HSV replication by inhibiting the differentiation of host monocytes.