Persistently infected cultures as a source of hepatitis A virus

Primary African green monkey kidney, continuous African green monkey kidney cell line BS-C-1, and buffalo green monkey kidney cultures were infected with a uniform inoculum of hepatitis A virus (HAV). Although both the cell line BS-C-1 and primary African green monkey kidney cultures produced useful amounts of virus, HAV was detected earlier and in greater quantities in primary African green monkey kidney cultures. A persistently infected primary African green monkey kidney culture was developed. The influence of incubation time (4 to 40 days) and concentration (2 to 15%) of fetal calf serum in the maintenance medium on production of HAV by this culture was examined. An incubation period of 24 to 28 days was found to be optimal; reducing this period led to decreased yields of HAV. No significant difference in the amount of HAV produced was observed with differing concentrations of fetal calf serum. Three different methods of extraction and the effect of multiple extractions on the recovery of HAV from cell lysates were examined. Sonication was a critical factor. Two extractions yielded more than 90% recoverable virus. Yields in excess of 10(11) physical particles of HAV per 850-cm2 roller bottle were routine. The total yield could be increased by concentrating the HAV present in spent maintenance medium by using bentonite or organic flocculation.     

Mutations within the 5'' nontranslated region of hepatitis A virus RNA which enhance replication in BS-C-1 cells.

Passage of human hepatitis A virus (HAV) in cell culture results in attenuation of the virus as well as progressive increases in the efficiency of virus replication in cell culture. Because the presence of identical mutations within the 5' nontranslated regions (5'NTRs) of several independently isolated cell culture-adapted HAV variants suggests that the 5'NTR may play a role in determining this change in virus host range, we constructed chimeric infectious cDNA clones in which portions of the 5'NTR of cell culture-adapted HM175/p35 virus were replaced with cDNA from either wild-type virus (HM175/wt) or a second independently isolated, but closely related cell culture-adapted virus (HM175/p16). Substitution of the complete 5'NTR of HM175/p35 with the 5'NTR of HM175/wt resulted in virus with very small replication foci in continuous African green monkey kidney (BS-C-1) cells, indicating that 5'NTR mutations in HM175/p35 virus are required for optimal growth in these cells. A chimera with the 5'NTR sequence of HM175/p16 retained the large foci of HM175/p35 virus, while the growth properties of other viruses having chimeric 5'NTR sequences indicated that mutations at bases 152 and/or 203 to 207 enhance replication in BS-C-1 cells. These findings were confirmed in one-step growth experiments, which also indicated that radioimmunofocus size is a valid measure of virus replication competence in cell culture. An additional mutation at base 687 of HM175/p16 had only a minor role in enhancing growth. In contrast to their effect in BS-C-1 cells, these 5'NTR mutations did not enhance replication in continuous fetal rhesus monkey kidney (FRhK-4) cells. Thus, mutations at bases 152 and/or 203 to 207 enhance the replication of HAV in a highly host cell-specific fashion.     

Comparative sensitivity of various cell culture systems for isolation of viruses from wastewater and fecal samples.

In efforts to define the most sensitive cell culture systems for recovery of viruses from wastewaters, 181 samples were inoculated in parallel into tube cultures of various cell types and were plaqued in bottle and petri dish cultures of three types of monkey kidney cells. Polioviruses were recovered most frequently in the RD line of human rhabdomyosarcoma cells, group A coxsackieviruses in RD and human fetal diploid kidney (HFDK) cells, group B coxsackieviruses in the BGM line of African green monkey kidney cells, echoviruses in RD and primary rhesus monkey kidney (RhMK) cells, and reoviruses in RhMK cells. BGM cells were unsatisfactory for recovery of viruses other than polioviruses and group B coxsackieviruses, and a line of fetal rhesus monkey kidney (MFK) was not a satisfactory substitute for primary RhMK. With RhMK cells, comparable numbers of virus isolations were made in tube cultures and in plaque assays conducted in bottle cultures, but with BGM and MFK cells, fewer isolations were made by plaquing than by inoculation of tube cultures. In comparative plaque assays on fecal samples under three different overlays in bottle and plate cultures of RhMK, BGM, and MFK cells, it was found that plaquing in the most sensitive system, RhMK, was less efficient for virus recovery than was inoculation of tube cultures of RhMK or HFDK cells. Overall, plaque assays performed in petri dishes in a CO(2) incubator yielded fewer virus isolates than did parallel plaque assays performed in closed bottle cultures. Other limitations of plaque assays for recovery of human enteric viruses are discussed.     

Comparative Sensitivity of Various Cell Culture Systems for Isolation of Viruses from Wastewater and Fecal Samples

In efforts to define the most sensitive cell culture systems for recovery of viruses from wastewaters, 181 samples were inoculated in parallel into tube cultures of various cell types and were plaqued in bottle and petri dish cultures of three types of monkey kidney cells. Polioviruses were recovered most frequently in the RD line of human rhabdomyosarcoma cells, group A coxsackieviruses in RD and human fetal diploid kidney (HFDK) cells, group B coxsackieviruses in the BGM line of African green monkey kidney cells, echoviruses in RD and primary rhesus monkey kidney (RhMK) cells, and reoviruses in RhMK cells. BGM cells were unsatisfactory for recovery of viruses other than polioviruses and group B coxsackieviruses, and a line of fetal rhesus monkey kidney (MFK) was not a satisfactory substitute for primary RhMK. With RhMK cells, comparable numbers of virus isolations were made in tube cultures and in plaque assays conducted in bottle cultures, but with BGM and MFK cells, fewer isolations were made by plaquing than by inoculation of tube cultures. In comparative plaque assays on fecal samples under three different overlays in bottle and plate cultures of RhMK, BGM, and MFK cells, it was found that plaquing in the most sensitive system, RhMK, was less efficient for virus recovery than was inoculation of tube cultures of RhMK or HFDK cells. Overall, plaque assays performed in petri dishes in a CO₂ incubator yielded fewer virus isolates than did parallel plaque assays performed in closed bottle cultures. Other limitations of plaque assays for recovery of human enteric viruses are discussed.     

Trypanosoma Manulis N. Sp. From the Russian Pallas Cat Felis Manul

ABSTRACT. The morphology of Trypanosoma manulis n. sp. is described from living and stained specimens obtained from the blood of a Pallas cat, Felis manul , from Kazakhstan. the cat was also infected with a Hepatozoon sp. and feline immunodeficiency virus. the morphology of the trypanosome most closely resembles that of Trypanosoma mpapuense Reichenow and Trypanosoma heybergi Rodhain found in bats. Trypanosoma manulis does not grow well in conventional media, but co-culture with African green monkey kidney cells in Eagle's Minimum Essential Medium supplemented with 10% fetal calf serum at approximately 27° C resulted in luxuriant growth of trypanosomes. Under these growth conditions, epimastigotes adhered to the surface of the culture flask and to African green monkey kidney cells, as well as forming large rosettes. At 37° C, although growth was poor, transformation of the epimastigotes into the bloodstream forms occurred. This represents the first report of a trypanosome of the subgenus Megatrypanum in a felid.     

Uptake of Rabies Virus into Epithelial Cells by Clathrin-Mediated Endocytosis Depends upon Actin

Rabies virus (RABV) causes a fatal zoonotic encephalitis. Disease symptoms require replication and spread of the virus within neuronal cells; however, in infected animals as well as in cell culture the virus replicates in a broad range of cell types. Here we use a single-cycle RABV and a recombinant vesicular stomatitis virus (rVSV) in which the glycoprotein (G) was replaced with that of RABV (rVSV RABV G) to examine RABV uptake into the African green monkey kidney cell line BS-C-1. Combining biochemical studies and real-time spinning-disk confocal fluorescence microscopy, we show that the predominant entry pathway of RABV particles into BS-C-1 cells is clathrin dependent. Viral particles enter cells in pits with elongated structures and incomplete clathrin coats which depend upon actin to complete the internalization process. By measuring the time of internalization and the abundance of the clathrin adaptor protein AP2, we further show that the pits that internalize RABV particles are similar to those that internalize VSV particles. Pharmacological perturbations of dynamin or of actin polymerization inhibit productive infection, linking our observations on particle uptake with viral infectivity. This work extends to RABV particles the finding that clathrin-mediated endocytosis of rhabdoviruses proceeds through incompletely coated pits which depend upon actin.     

Persistent infection of cultured mammalian cells by Japanese encephalitis virus.

Persistent infections were established by serial undiluted passage of flavivirus Japanese encephalitis virus in a line of rabbit kidney cells (MA-111). The persistently infected cells resembled uninfected cells in most respects. Low levels of infectious virions were released from a small percentage of cells, and a larger and more variable percentage was shown to possess viral antigen by fluorescent-antibody staining. Released viruses were shown to interfere with replication of wild-type Japanese encephalitis virus. Persistently infected MA-111 cells could not be superinfected with homologous wild-type Japanese encephalitis virus but could be superinfected with two heterologous viruses. Transfer of cell culture medium from persistently infected MA-111 cells to a line of African green monkey kidney cells (Vero) resulted in similar persistent infections in the latter cells. Temperature sensitivity and host-cell interferon production were not involved in establishment or maintenance of persistence. Determination of ratios of physical particles to infectious particles revealed that many defective, noninfectious viruses were present, suggesting that defective interfering particles may be responsible for persistency.     

Response of Children to Inactivated Measles Vaccine Prepared in Bovine Renal Cell Culture

Formalin-inactivated, alum-adsorbed measles vaccine was readily prepared from virus grown in calf kidney cell culture infected with the Sugiyama strain of measles virus which had been adapted to this cell culture. The vaccine induced no side reaction of any consequence in vaccinated children, but demonstrated antigenic capacity in children as well as guinea pigs, comparable to that of currently used killed measles vaccines prepared from virus grown in monkey kidney or chick embryo tissue cultures. The host system employed for the preparation of this vaccine has an advantage over monkey kidney or chick embryo tissue cultures which are currently used for manufacture of killed measles vaccine. Bovine kidneys are much easier to obtain and cultivate. Of importance is the fact that calf kidneys are practically free of latent virus, whereas monkey kidneys and chick embryos frequently harbor latent viruses.     

Comparative Immunogenicities of Chikungunya Vaccines Propagated in Monkey Kidney Monolayers and Chick Embryo Suspension Cultures

A comparative study was made of Formalin-inactivated Chikungunya vaccines prepared from the virus propagated in African green monkey kidney monolayers and concentrated chick embryo suspension cultures. The vaccine prepared in the chick embryo suspension cultures was significantly more protective to mice against a live homologous virus challenge and stimulated the production of 4 to 5 times more circulating antibodies than the vaccine prepared with virus grown in African green monkey kidney monolayer cultures.     

Growth of Murine Cytomegalovirus in Various Cell Lines

Murine cytomegalovirus (MCMV) was capable of infecting and replicating in both primary and continuous cell lines obtained from various species. In African green monkey kidney (BSC-1) cells, primary rabbit kidney cells, and baby hamster kidney (BHK-21) cells, there were cytopathic effects (CPE) and virus replication upon initial exposure of cells to virus. In primary fetal sheep brain (FSB) cells, L cells, and rabbit kidney (RK-13) cells, it was necessary to subculture the infected cells one or more times before appearance of CPE and replication of virus. In the case of the infected FSB cultures, it was found that the virus effect could be induced if subculturing were accomplished by trypsinization but did not occur if cells were subcultured by scraping. FSB-grown virus replicated better in FSB than in mouse embryo fibroblast (MEF) cells. The CPE produced in all of the above cell lines was similar to that observed in MEF infected with MCMV. The virus grown in different cell lines was completely neutralized when mixed with several reference sera prepared in rabbits or mice. The populations of virions released from infected MEF and FSB cells were compared by isopycnic centrifugation in potassium tartrate, and no differences were revealed in the buoyant densities of the populations. Human embryonic brain cells, human embryonic kidney cells, a human lung fibroblast cell strain (WI-38), HeLa, and Hep-2 were not susceptible to MCMV.     

Purification and properties of epithelial growth inhibitor (EGI) from human platelets: its separation from type beta transforming growth factor (TGF-beta)

We previously reported that sera from various kinds of animals contain a protein(s) capable of inhibiting the growth of the non-malignant epithelial cell line derived from Buffalo rat liver (BRL). In the present study, a similar epithelial cell-specific growth inhibitor (EGI) was purified to homogeneity from an acid-ethanol extract of human platelets. During purification, EGI was separated from the major component of type beta transforming growth factor (TGF-beta), which can stimulate the colony formation of the non-malignant fibroblastic cell line derived from rat kidney (NRK) in soft agar in the presence of epidermal growth factor (EGF). The purified EGI had an Mr of 27,000, and was composed of two subunits identical in Mr. It significantly inhibited the growth in monolayer cultures of three non-malignant epithelial cell lines, BRL, MDCK (from Madin-Darby canine kidney) and BSC-1 (from African green monkey kidney), at doses lower than 40 pg/ml in medium containing 10% fetal calf serum. Its inhibitory activity was stable against heating at 90 degrees C for 3 min, but not against treatment with 50 mM dithiothreitol. In addition, TGF-beta was also partially purified from the same extract. The purified TGF-beta did not show any inhibitory activity toward the growth of BRL, MDCK, BSC-1, or NRK.     

The morphology of cell lines suitable for vaccine production studied by scanning electron microscopy]

Five nontumorogenic cell lines suitable for vaccine production were studied by SEM. It was shown that diploid cell line 41 originated from sheep embryo kidney and also two heteroploid cell lines, line 4921 originated from embryo skin and muscles of the African green young monkey and line 4647 from kidney of the adult monkey, maintained normal cell morphology and normal growth pattern in early and in later passages in cultures. Some alterations in epithelial dense monolayer formation were revealed in the heteroploid cell lines: in line 455 originated from spleen of the adult African green monkey, and in line 4184 originated from line 41. The revealed alterations can be considered as the early morphological signs of the transformation of epithelial cells in culture. These cell lines also retained the stability of their morphological characteristics at the earlier and later passages. All the studied cell lines were free of contaminating agents.     

Defectiveness of Interferon Production and of Rubella Virus Interference in a Line of African Green Monkey Kidney Cells (Vero)

Vero cells, a line of African green monkey kidney cells, failed to produce interferon when infected with Newcastle disease, Sendai, Sindbis, and rubella viruses, although the cells were sensitive to interferon. Further, infection of Vero cells with rubella virus did not result in interference with the replication of echovirus 11, Newcastle disease virus, or vesicular stomatitis virus, even in cultures where virtually every cell was infected with rubella virus. Under the same conditions, BSC-1 cells and other cells of primate origin produced interferon and showed rubella virus interference. The results indicate that the presence of rubella virus in the cell does not in itself exclude multiplication of other viruses and that rubella virus interference appears to be linked to the capability of the cell to produce interferon.     

Possibilities of vaccine manufacture in human diploid cell strains with a serum replacement factor.

Cell lines MDCK (canine kidney), BGM (Buffalo green monkey kidney) and human embryonic lung fibroblast will support viral growth efficiently in medium without serum. Both MRC-5 and WI-38 cell strains have been approved by the Food and Drug Administration for manufacturing viral vaccines against cytomegalovirus and varicella-zoster virus. In this study we examine these two cell lines and viruses for their ability to grow in medium containing a serum replacement. The serum substitute used is LPSR-1 (low protein serum replacement). Using LPSR-1 in defined medium, we demonstrate multipassage cell growth and viral cultivation and replication equivalent to those obtained in medium containing fetal bovine serum (FBS). Viral growth after complete elimination of FBS varies and depends on cell line and virus. Serum substitutes can eliminate FBS in the viral growth phase of vaccine production and reduce costs.     

Plating and maintenance of epithelial tumor cells in primary culture: Interacting roles of serum and insulin

We have attempted to define the best conditions for primary culture of epithelial cells from mouse mammary tumors. Both fetal calf serum and insulin increase the rate of cell attachment to the culture substratum in dose-dependent ways. Under suboptimal conditions, epithelial cells begin to detach again from culture dishes after 2 days; this allows us to calculate the maintenance of cultures by comparing the amount of DNA in the monolayer at an early and a late time (‘maintenance ratio’). The cells are better maintained when more serum is used, up to about 4%. At low serum doses, maintenance is also responsive to the amount of insulin in the medium; insulin has little effect when more than 3% serum is present. However, the maintenance of the cells is responsive to the serum dose, regardless of the insulin dose. Furthermore, the optimum insulin concentration decreases as more serum is added to the medium. We conclude: that epithelial cells of mammary tumors require insulin to function properly in culture; that as little as 4% serum supplies an adequate dose of insulin-like activity for cell maintenance; and that fetal calf serum also supplies additional compounds which the cells need to survive.     

Preservation at -195 degrees C of trypsinized organs for primary cultures

Trypsinized cells from rabbit kidney can be kept at -195, 8 degrees C indefinitely. When necessary, propagation in monolayer cultures is readily obtained as primary cultures, provided that some conditions be respected: 1) only glass culture flask must be used. Falcon plastic vessels do not allow cell attachment in this type of primary culture 2) the lag period is particularly long: it taken about 20 days before the beginning of cell multiplication 3) fetal calf serum is toxic for the cells in the 2nd and 3rd change of the culture medium, during the lag period.     

Paracrystal Formation in Cell Cultures Infected with Adenovirus Type 2

Large rod-shaped structures corresponding to paracrystals were seen in the nucleus, cytoplasm, or both of adenovirus type 2 (Ad2)-infected cells by immunofluorescence staining with antibody prepared against purified Ad2. In exception to this, Ad2-induced crystals did not stain with either hexon or fiber antibody. The crystalline structures were first observed in Ad2-infected Vero cells at 28 hr with a maximum number at 70 hr postinoculation. The kinetics of paracrystalline formation closely paralleled the experimental synthesis of infectious progeny virus. Acridine-orange staining revealed the lack of nucleic acids associated with the crystal. Also, the paracrystals stained intensely with phenanthrenequinone, suggesting that they are composed of basic proteins. Interferon induced by Newcastle disease virus from African green monkey kidney cell cultures was used to pretreat Vero cells prior to Ad2 infection. This resulted in inhibiting the formation of viral-induced paracrystals in 97% of the cells and reduced virus yields by 95%. The African green monkey kidney cell culture interferon did not reduce Ad2 yields in HeLa cell cultures or display any virus inhibitory activity in rabbit kidney cell cultures. Staining procedures, fluorescent-antibody tests with whole virus, hexon or fiber antibody, and interferon studies suggested that the paracrystals were viral-directed and composed of basic proteins (possibly core proteins).     

Translation of adenovirus 2 late mRNAs microinjected into cultured African green monkey kidney cells.

Adenovirus 2-infected monkey cells fail to synthesize fiber, a 62,000 Mr virion polypeptide expressed at late times in productively infected cells. Yet these cells contain fiber mRNA that, after isolation, can be translated in vitro. The reason for the failure of monkey cells to translate fiber mRNA has been approached by microinjecting adenovirus mRNA into the cytoplasm of cultured monkey cells. Late adenovirus 2 mRNA, isolated from infected HeLa cells, was efficiently expressed when microinjected into the African green monkey kidney cell line CV-C. Expressed viral proteins identified by immunoprecipitation included the adenovirus fiber polypeptide. This result demonstrates that the monkey cell translational apparatus is capable of recognizing and expressing functional adenovirus fiber mRNA. Microinjection of late virus mRNA into cells previously infected with wild-type adenovirus 2 failed to increase significantly the yield of infectious virus.     

Stimulation of Ornithine Decarboxylase Activity in Neural Cell Culture: Potential Role of Insulin

Abstract: Age-dependent decreases in the levels of ornithine decarboxylase activity were observed in the optic lobes, cerebral hemispheres, and midbrain-diencephalon of 6–17-day-old chick embryos. In dissociated cell cultures from chick embryonic brains a similar pattern of declining ornithine decarboxylase activity with time in culture was observed. Ornithine decarboxylase activity in the dissociated brain cell cultures was stimulated by changing the culture medium. The peak stimulatory effect was shown to occur 12 h after changing the medium. Although serum-free medium stimulated ornithine decarboxylase activity slightly, the presence of serum in the medium was the primary stimulatory factor. Both fetal calf serum and heat-inactivated fetal calf serum produced dose-dependent stimulation of ornithine decarboxylase activity. Dialyzed fetal calf sera stimulated ornithine decarboxylase, but to a lower level than that produced by nondialyzed sera. Insulin (0.5–10 μg/ml) stimulated ornithine decarboxylase activity in a dose-dependent manner in serum free medium. In addition, 10² M-L-asparagine stimulated ornithine decarboxylase activity in serum-free medium.     

Isolation and in vitro characterization of a herpesvirus from ground squirrels (Citellus sp).

A viral agent was isolated from ground squirrel primary kidney cultures which presented a spontaneous cell layer alteration. The cultural, morphologic, and physico-chemical characteristics of the agent indicated that it belongs in the herpesvirus group. The isolate presented a narrow in vitro cell host range, growing best in marmoset monkey, owl monkey, rabbit, and hamster kidney cultures and poorly in Vero and dog fetal lung cells. The agent is readily released from infected cells, a fact which indicates that it is a cell-free virus. Good plaque formation was observed only in marmoset monkey kidney monolayers. The virus was not neutralized by antisera against other herpesviruses with the exception of a partial 1-way cross-neutralization between ground squirrel agent and Herpesvirus saguinus antisera. All these characteristics indicate that this virus is likely a new member of the herpesvirus family.