The apparent infection of NA-C1300 cell cultures by nucleocapsid material of the Canadian Arctic strain of rabies virus

Murine neuroblastoma (NA-C1300) and baby hamster kidney (BHK-21/C13) cell cultures were infected with the Canadian Arctic strain of rabies virus. Subcultures were passed following incubation for 3 to 4 days at 35 degrees C. The supernatant fluids from the BHK cultures demonstrated increasing infectivity in both NA and BHK cells concomitantly with an increase in the number of parent cells staining with an anti-glycoprotein stain. On the other hand, the supernatant fluids from the NA cultures initially showed higher infectivity in NA cells than in BHK cells. This feature was related to a low production of glycoprotein-staining cells in the parent NA cultures. The reduction of infectivity in NA cells of some NA supernatant fluids (and brain suspensions) by anti-nucleoprotein antibodies suggests that nucleocapsid material is, in some manner, capable of infecting NA cells. Infectivity of this virus strain in experimental mice is also related to the production of glycoprotein and may not be correlated with the degree of infection in NA cell cultures.     

Growth, plaque assay and immunofluorescent studies on Tataguine virus in cell culture.

The growth characteristics of Tataguine virus were studied in Cercopithecus monkey kidney (Vero); rhesus monkey kidney (LLC-MK2), baby hamster kidney (BHK-21); porcine kidney (PK-15), mouse fibroblasts (L-929) and Aedes albopictus cell monolayers. The virus replicated without producing any cytopathology in Vero, BHK-21 and Aedes albopictus: but not in the other three cell culture systems. Two or three subsequent serial blind passages in those cultures supporting the growth of the virus did not produce any appreciable increase in virus titre. Immunofluorescent staining of inoculated Vero cells demonstrated the presence of Tataguine virus antigen in the cytoplasm of infected cells. Plaques 1--1.5 mm in diameter were produced only in Vero cell culture. In neutralization tests performed on Tataguine virus, immune mouse and hamster sera, higher antibody titres were obtained by plaque reduction than mouse protection tests.     

Ibaraki Virus,an Agent of Epizootic Disease of Cattle Resembling Bluetongue

Ibaraki virus multiplied and induced cytopathic effects in primary cell cultures of bovine, sheep and hamster kidney and chick embryo, and cultures of BHK21-WI2 cells of baby hamster kidney origin and mouse fibroblastic L cells, but did not in primary cultures of horse and swine kidney cells and HeLa cell cultures. The virus was readily passaged serially in 4 to 5-day-old eggs using the yolk sac inoculation and incubation at 33.5 C. The viral growth was better in eggs incubated at 33.5 C than 37 C, and in younger eggs, with high yields in yolk, yolk sac and embryo. The virus was passaged serially in newborn mice by the intracerebral route. The virus multiplied in the brain of mice of any age, but younger mice supported better viral growth and developed encephalitis. As the age of mice increased, the morbidity and mortality became lower, no deaths being observed in 2 to 3-week-old mice. These observations in cell cultures, embryonated eggs and mice emphasize the similarity of Ibaraki virus to bluetongue virus. No evidence was obtained that young adult rabbits and weanling guinea pigs are susceptible to Ibaraki virus. The virus seemed to have little if any pathogenicity but infectivity of a low grade for sheep, while the virus is capable of inducing clinical illness, even severe in some instances, in cattle. This is in contrast to bluetongue virus which is highly pathogenic for sheep and much less so for cattle. Serial passages in embryonated eggs and suckling mice resulted in attenuation for cattle of Ibaraki virus.     

Bovine Epizootic Fever

A virus, the Yamaguchi strain, was serially propagated in suckling hamsters, mice and rats, and hamster kidney BHK21-WI2 cells from a natural case in the 1966 outbreak of bovine epizootic fever, an acute febrile disease of cattle, resembling ephemeral fever, known in Japan since 1949. An acute phase blood from the natural case was first passaged in calves by intravenous inoculation, and a blood specimen at the second passage was used to initiate serial hamster passage. Infected hamsters died with nervous symptoms, and serial passage was readily accomplished by intracerebral inoculation with brain emulsions. The hamster passage line of virus was serially passaged by intracerebral inoculation in 1- or 2-day-old mice, and then in rats 1 or 2 days after birth, which developed fatal encephalitis. The hamster passage virus was also serially propagated with cytopathic effect in cultures of BHK21-WI2 cell cultures. These viral lines were shown to be neutralized by, and to produce specific complement-fixing antigen reactive with, convalescent sera of calves infected with the original Yamaguchi strain, confirming the identity of these lines as the Yamaguchi strain. The hamster passage line, when inoculated intravenously in calves, induced an acute febrile illness which was similar to bovine epizootic fever; all the inoculated calves had viremia and developed neutralizing and complement-fixing antibodies against the virus. Serological evidence for infection with this virus was obtained in natural cases in the 1966 outbreak. These findings seem to justify this virus to be the causative agent of bovine epizootic fever.     

Virus Pneumonia of Pigs; Attempts at Propagation of the Causative Agent in Cell Cultures and Chicken Embryos

Two strains of the agent of virus pneumonia, were tested for the ability to propagate in 12 types of cell cultures and in chicken embryos. The 5 primary cell cultures used were: swine kidney, lung, bone marrow, testicle, and chicken embryo kidney; and the 7 serial passage cell cultures were: swine kidney, kidney-tumor, testicle, bone-marrow, bovine kidney, and human cervical carcinoma (HeLa). The agent of virus pneumonia was propagated in primary swine kidney and in HeLa cell cultures as shown by the production of typical gross and microscopic lesions in pigs inoculated with cell future fluids. Third passage cell culture fluids, produced typical gross lesions in pigs, but fourth passage cell culture fluids produced only microscopic lesions, and no lesions were produced by sixth and eleventh passage fluids. Control pigs receiving fluids from uninoculated cell cultures remained free of gross or microscopic lesions, as did uninoculated controls. Cytopathic effects were not detected in any of the inoculated cell cultures and no cellular changes were detected by staining with Giemsa stain or acridine orange. Neither lesions nor deaths occurred in chicken embryos inoculated with both strains of virus pneumonia virus. Pneumonia was not produced in pigs inoculated with suspensions from second chicken embryo passage of the 2 strains inoculated by the chorioallantioic sac, the amniotic sac, and the yolk sac routes. Identical gross and microscopic lesions were produced in pigs inoculated with either pneumonic lung suspensions or with virulent cell culture fluids. Gross lesions consisted of areas of light to reddish-purple consolidation usually limited to the anterior, cardiac, and intermediate lobes of the lungs. Pleuritis and pericarditis were never present in experimentally produced virus pneumonia. The microscopic lesions were characterized by: 1. perivascular and peribronchiolar lymphoid infiltration and hyperplasia, 2. alveolar interstitial thickening and infiltration, and 3. alveolar exudates consisting of alveolar cells, lymphocytes, plasma cells, and neutrophiles.     

In Vitro Selection of High-Infectious, Leukemogenic Virus from Low-Infectious, Non-Leukemogenic Type C Virus from a Malignant ST/a Mouse Cell Line

Low-infectious, nontransforming type C virus was isolated from an in vitro spontaneously transformed ST/a mouse cell line, ST-L1. The virus released by ST-L1 cells was NB-tropic and XC(-). It gave rise to very small peroxidase antibody plaques (PAP) in cultures which initially were nonproducing. Sodium dodecyl sulfate (SDS)-polyacrylamide gels of the structural proteins of the ST-L1 virus showed an envelope glycoprotein with an apparent mass of 65 kilodaltons (kdal). The mouse cells SC-1, BALB/3T3, and NIH/3T3 could be productively infected with cell-free supernatants from the ST-L1 cell line; however, virus was detected in supernatant fluids only after two to four subcultures of the infected cells. The virus thus produced was XC(+) and a large plaque former. The virus released from infected SC-1 cells was N-tropic, whereas the viruses from infected NIH/3T3 and BALB/3T3 cells were NB-tropic. The structural proteins of the N- and NB-tropic viruses could be distinguished on SDS polyacrylamide gels, the major dissimilarity being a difference in the mobility of the p30. All these viruses had an envelope glycoprotein with an apparent mass of 70 kdal. The infectivity of the viruses, measured as PAP per nanogram of p30, was 30- to 60-fold lower for the virus released from the ST-L1 cell line than that of the viruses after passage in SC-1, NIH/3T3, and BALB/3T3 cells. None of the viruses could infect rabbit or mink cells. Inoculation of the viruses into newborn mice showed that the ST-L1 virus was non-leukemogenic, whereas the NB-tropic virus selected from this after passage in BALB/3T3 or NIH/3T3 cells was highly leukemogenic. Viruses isolated from leukemic animals were indistinguishable with respect to host range and protein mobilities in SDS gels from the ones with which the mice were inoculated. Although the SC-1-selected virus was highly infectious in vitro, it was only weakly, if at all, leukemogenic.     

Replication of herpes simplex virus type 1 on hydroxyurea-resistant baby hamster kidney cells.

Hydroxyurea-resistant (HUr) baby hamster kidney cells were isolated, subcloned, and characterized. One clonal line, which contained elevated levels of ribonucleotide reductase, lost its HU resistance during passage in the absence of the inhibitor, whereas another clonal line was stably resistant. The replication of herpes simplex virus type 1 on these cells was compared with that of the parvovirus minute virus of mice. Herpes simplex virus type 1 was found to be as sensitive to HU on both lines of HUr baby hamster kidney cells as it was on parental (HU-sensitive) cells, whereas parvovirus replication was about eight times more resistant on HUr baby hamster kidney cells compared with the parental cells. The results suggest that herpes simplex virus type 1 cannot use the cellular reductase and may code for its own.     

Continuous cell propagation using low-charge microcarriers

Microcarrier cell culture technology has been extended by the finding that two mammalian epithelial cell lines can be continuously subcultured by simple bead-to-bead transfer in normal medium in which calcium concentrations have been reduced. Data are reported which show that the hamster ovary line CHO-Kl and the monkey kidney line LLC-MK₂ can be subcultured simply by adding fresh microcarriers to the stirred suspension culture. Thirteen generations of continuous exponential growth are demonstrated with two such subcultures for the CHO-Kl cells and with four such subcultures for the LLC-MK₂ cells. Cell generation times were unchanged by this subculturing approach compared to standard subculturing procedure using trypsin to remove cells from surfaces. We have applied this technique to the production of vesicular stomatitis virus (VSV) from CHO-Kl cells. Viral yields were comparable (less than twofold difference) in microcarrier cultures which were subcultured via bead-to-bead transfer or by the standard means of removing cells from microcarriers with trypsin.     

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.     

Changes in hydroxy and carboxylic acid compositions in the supernatant fluids of mosquito cell cultures infected with dengue viruses

Hydroxy and carboxylic acids in the supernatant fluids of mosquito cell cultures infected with four serotypes of dengue viruses (DEN) were analyzed by frequency-pulsed electron capture gasliquid chromatography. The hydroxy acid profiles of all virus-infected cell cultures differed qualitatively and quantitatively from the profile of normal cell culture. Furthermore, the profiles of hydroxy acids in the DEN 1- and DEN 4-infected cultures were type specific. Although quantitative differences of a few peaks could be found between the hydroxy acid profiles of DEN 2- and DEN 3-infected cultures, in the absence of clear qualitative differences the two profiles were considered to be essentially indistinguishable. The carboxylic acid profiles of virus-infected cultures differed from the profile of a normal cell culture, but none of the four serotypes of DEN viruses induced type-specific profiles. Thus, these findings contrasted to previous results with rhesus monkey kidney cell cultures (LLC-MK₂), in which serotype-specific sets of hydroxy acids and a DEN 1-specific set of carboxylic acids were released in the supernatant fluids by the infection with dengue viruses.     

Effect of enforced expression of human bcl-2 on Japanese encephalitis virus-induced apoptosis in cultured cells.

Infection by Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes acute encephalitis in humans and induces severe cytopathic effects in different types of cultured cells. This study attempted to determine whether apoptosis contributes to virus-induced cell death in a culture system by characterizing JEV lytic infection in baby hamster kidney BHK-21 cells, murine neuroblastoma N18 cells, and human neuronal progenitor NT2 cells. According to our results, the replication of JEV, and not the UV-inactivated virions per se, triggered apoptosis in these cell lines, as evidenced by nuclear condensation, DNA fragmentation ladder, and in situ end labeling of DNA strand breaks with terminal transferase (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay). Different strains of JEV, regardless of whether they are neurovirulent to mice, could induce apoptosis of the infected cells. In addition, enforced expression of the human protooncogene bcl-2 in BHK-21 cells, which did not influence virus production, appeared to delay the process of JEV-induced apoptosis, despite the fact that most infected cells were inevitably killed after prolonged cultures. However, Bcl-2 proteins expressed in N18 cells failed to block JEV-induced apoptosis, although they did prevent Sindbis virus-induced apoptosis from occurring in the same cells. This finding suggests that these two viruses may utilize similar but not identical mechanisms to kill their infected cells. The results presented here thus demonstrate that apoptosis can be a general mechanism for JEV-induced cell death and that enforced bcl-2 expression may be inadequate in protecting all cell types from JEV-induced apoptosis in cell cultures.     

Cell morphology in long-term cultures of normal and abnormal amniotic fluids

Summary The cell morphology of long-term cultures of amniotic fluid cells from 10 fetuses with a neural tube defect (NTD) and three with omphalocele was examined and compared to 30 long-term cultures of normal amniotic fluids as well as a long-term culture of human fetal brain. Cultures from the amniotic fluids of the fetuses with NTD and omphalocele showed cells with the same general characteristics as normal amniotic fluid cells. However, the cultures of amniotic fluid cells from NTD pregnancies had an additional cell type also seen in fetal brain culture. This was a neuroblast-like cell, with small rounded refractile morphology and long branching processes forming clusters of varying sizes which lay on top of large flat cells. These neuroblast-like cells diminished in number with time in culture and were not present in subcultures. Their possible neuronal origin is discussed.     

Comparative Sensitivity of Tissue Cultures to Rubella Virus: Use of Guinea Pig Cells for Virus Titration

A series of 19 different primary and serial tissue cultures were investigated for their sensitivity to virulent or attenuated rubella virus (RV). Primary guinea pig tissues, a serial passage of baby hamster kidney, and primary human amnion were comparable to African green monkey kidney tissue cultures in their sensitivity. In general, primary human tissues were relatively insusceptible to the Gilchrist strain of RV. RV interfered with the growth of vesicular stomatitis virus. Based on this finding, it was possible to develop an assay method in guinea pig tissue cultures by using VSV as the challenge virus. This system appeared to be comparable in sensitivity to the use of primary monkey kidney tissue cultures for the detection of small amounts of RV and offers the advantages of economy, rapidity, and safety.     

Biophysical, Biological, and Cytochemical Features of a Virus Associated with Transplantable Hamster Tumors

A virus resembling type C murine leukemia viruses, which is associated with transplantable hamster tumors, was partially characterized with respect to certain biological, biophysical, and cytochemical features. As determined by electron microscopy, high concentrations of the virus appeared in the blood plasmas of tumor-bearing hamsters. Hamsters inoculated with virus concentrates did not show gross evidence of disease, and preliminary attempts to infect various hamster cells in tissue culture were unsuccessful. A line of cells from a virus-containing tumor which had been established in tissue culture released large numbers of the virus into the supernatant fluids by budding. The buoyant density peak of virus concentrates in potassium tartrate density gradients was 1.13 g/cm(3) by ultraviolet absorption and electron microscopic analysis. Acridine orange staining and nuclease digestion methods have indicated that the virus is probably a ribonucleic acid virus.     

Production of Rubella Hemagglutinating Antigen in Suspension Culture

Rubella virus specific hemagglutinating antigen was prepared in the fluid phase of suspension cultures. Systems employing baby hamster kidney culture adapted inocula, nonadapted inocula, and cells derived from long-term infected suspension cultures were evaluated. Optimal specific hemagglutinating titers were obtained when kaolin-treated fetal bovine serum was used in the media and when the incoulum had previously been adapted in a suspension culture system. When cultures derived from long-term infected suspension systems were studied, the number of daily harvests in which acceptable titers were present was prolonged. However, titers were generally higher in suspension systems employing cells which had recently been infected.     

Powassan Virus: Morphology and Cytopathology

Powassan virus, a North American tickborne group B arbovirus, multiplied after simultaneous inoculation into bottles or tubes of virus and trypsinized suspension of continuous-line cultures of rhesus monkey kidney cells, strain LLC-MK2. Cytopathic effects comprising cell rounding and cytoplasmic vacuolation were first observed five days after inoculation. Mixture of Powassan antiserum with virus before inoculation into tissue cultures inhibited the appearance of cytopathic effects. Hemagglutinins for rooster erythrocytes, optimally at pH 6.4 and 22° C., first appeared in tissue culture supernatant fluids four days after inoculation.Electron microscopic observation of thin sections of infected tissue culture cells showed virus particles 360-380 A.U. along outer cell membranes and edges of cytoplasmic vacuoles. In phosphotungstic acid negatively stained preparations, intact virus particles, 400-450 A.U. total diameter, were observed inside infected cells. In particles in which the peripheral layer became discontinuous, geometrically arranged subunits compatible with cubic symmetry were observed.     

Activation of a Latent Measles Virus Infection in Hamster Cells

The characteristics of infectious measles virus released from latently infected hamster embryo fibroblast cells are described. Low levels of virus were released spontaneously when the cultures were incubated at 37 C; this phenomenon was observed 19 passages after the cells had been exposed to the virus and has continued through cell passage 45. The virus yield could be significantly increased by cocultivation of the hamster cells with BSC-1 cells or incubation of the latently infected cells at 33.5 C rather than at 37 C. Measles virus released after cocultivation demonstrated increased cytopathology in cell culture and reduced temperature sensitivity when compared to the virus released at 33.5 C. After cell passage 45, there was an increase in spontaneous release of virus. However, the viruses recovered by cocultivation or temperature release after cell passage 45 were nearly identical. These observations suggest a possible mechanism for measles virus activation in cells latently infected with this virus.     

Previously unrecognized virus from submaxillary glands of gnotobiotic and conventional rats

Ashe, Warren K. (National Institute of Dental Research, Bethesda, Md.), Henry W. Scherp, and Robert J. Fitzgerald. Previously unrecognized virus from submaxillary glands of gnotobiotic and conventional rats. J. Bacteriol. 90:1719-1729. 1965.-A serially transmissible cytopathic agent was isolated from histologically normal submaxillary glands, but not from various other tissues or specimens, from 74 of 97 gnotobiotic and conventional rats. Triturates of the glands or subsequent culture supernatant fluids induced specific cytopathic effects (CPE) in monolayer cultures of primary rabbit kidney cells (14 passages), a line of human skin cells (8 passages), and HeLa cells (17 passages). Transfer of supernatant fluids containing infected cells enhanced transmissibility. Neutralization of the CPE was demonstrated with sera of gnotobiotic and conventional rats and with homologous rabbit antiserum. A cold hemagglutinin specific for rabbit erythrocytes is associated with, but separable from, the infectious particle. Cultures of the agent induced no discernible effect on inoculation by various routes into suckling, weanling, or adult conventional mice, rats, and hamsters. Weanling and adult rabbits were also unaffected. Cultures for bacteria in gland extracts and infected cell supernatant fluids were uniformly negative. Negative cultures on PPLO media and negative arginine deiminase tests indicated that this agent is not a Mycoplasma. The data indicate that it is a virus whose biological and physical properties distinguish it from the known cytomegaloviruses. Because it has been found so far only in rat submaxillary glands, this agent is designated provisionally as RSMG virus.     

Toxoplasma gondii: purification of trophozoites propagated in cell culture.

A new procedure is described for the purification of trophozoites from the virulent RH strain of Toxoplasma gondii propagated in baby hamster kidney (BHK-21) cell cultures. The culture medium containing host cell debris and trophozoites was filtered through glass-wool filtering fiber, which removed most host cell material. The filtrate containing trophozoites was centrifuged, and the trophozoite pellet was resuspended and washed in phosphate-buffered saline. An average of about 75% of the original number of trophozoites was recovered. No loss of trophozoite viability was observed as determined by the rate of host cell culture monolayer destruction. The amount of host cell material contamination in the final trophozoite fraction was negligible as determined by measuring radioactivity in the trophozoite fraction after cofiltration with noninfected host cell material which had been prelabeled with radioactive precursors.     

Herpes simplex virus type 1 strain HSV1716 grown in baby hamster kidney cells has altered tropism for nonpermissive Chinese hamster ovary cells compared to HSV1716 grown in vero cells

Chinese hamster ovary (CHO) cells are traditionally regarded as nonpermissive cells for herpes simplex virus type 1 (HSV-1) infection as they lack the specific entry receptors, and modified CHO cells have been instrumental in the identification of HSV-1 receptors in numerous studies. In this report we demonstrate that the HSV-1 strain 17+ variant HSV1716 is able to infect unmodified CHO cells but only if the virus is propagated in baby hamster kidney (BHK) cells. Infection of CHO cells by BHK-propagated HSV1716 results in expression of immediate-early, early, and late viral genes, and infectious progeny virions are produced. In normally cultured CHO cells, up to a maximum of 50% of cells were permissive for BHK-propagated HSV1716 infection, with 24 h of serum starvation increasing this to 100% of CHO cells, suggesting that the mechanism used by BHK-propagated virus to infect CHO cells was cell cycle dependent. The altered tropism of HSV1716 was also evident in another nonpermissive mouse melanoma cell line and is an exclusive property resulting from propagation of the virus using BHK cells, as viruses propagated on Vero, C8161 (a human melanoma cell line), or indeed, CHO cells were completely unable to infect either CHO or mouse melanoma cells.