Homologous interference of lymphocytic choriomeningitis virus: detection and measurement of interference focus-forming units.

Lymphocytic choriomeninigitis (LCM) virus defective interfering (DI) particles form foci of protected cells in a monolayer under an agarose-containing overlay medium. Foci originate from one cell dually infected with at least 1 interference focus-forming unit and infectious virus. As a result, an interfering factor is produced and released which interacts with neighboring cells, thereby protecting them against cytopathic lysis by challenge virus. The property of individual LCM virus DI particles to induce countable foci has been made the basis of quantitative assay that is comparable in every respect to the plaque assay of infectious virus and is much more sensitive and probably more accurate than other procedures used to measure LCM virus DI particles. LCM virus was passaged, undiluted, 10 times in cell cultures. When yields were analyzed as to concentrations of PFU and interference focus-forming units, both entities were found to fluctuate with the pattern expected from theoretical considerations.     

Comparison of Various Methods for Preparation of Viral Serological Antigens from Infected Cell Cultures

In efforts to prepare more potent and sensitive viral serological antigens, several aspects of the production of antigens from infected cell cultures were studied. Antigens derived from whole, infected culture material and from the cellular and fluid phases were compared. Freezing and thawing, sonication, and alkaline buffer extraction were compared for effectiveness in releasing antigen from host cells. The effect of the multiplicity of infection on titers of viral antigens produced in cell cultures was studied. Generally, higher titered antigens were derived from the infected cells than from the culture fluids, but for certain viruses complement-fixing (CF) antigens derived from the culture fluids gave higher antibody titers than did cell-associated antigens. With each virus-host cell system studied, treatment with alkaline buffers extracted appreciable amounts of CF antigen from the host cells, but in some instances more antigen was released by freezing and thawing or by sonication. Extraction of infected cells with alkaline buffers was not a satisfactory method for preparation of hemagglutinating (HA) antigens for any of the viruses studied. The highest-titered HA antigens were produced from infected cells disrupted by freezing and thawing or sonication. The highest titered CF and HA antigens were produced from cell cultures infected at multiplicities of one or greater. Complement-fixing antigens produced by infecting cells in suspension and then planting had lower titers than antigens produced in parallel by infecting developed monolayers. Optimal methods are summarized for preparation of serological antigens to a variety of viruses of man.     

Persistent infection of a rat kidney cell line with Rauscher murine leukemia virus

Duc-Nguyen, Huu (National Cancer Institute, Bethesda, Md.), Edith N. Rosenblum, and Robert F. Zeigel. Persistent infection of a rat kidney cell line with Rauscher murine leukemia virus. J. Bacteriol. 92:1133-1140. 1966.-The propagation of a murine leukemia virus (Rauscher) in a kidney cell line, derived from a rat with lymphoid leukemia, was studied. A complement-fixing (CF) antigen reacting with Rauscher immune sera was detected at various passage levels, which correlated with the visualization by use of electron microscopy of viral buds and viral particles in different stages of maturation in all passages. Five-month-old monolayers continued to shed virus and to yield high CF antigen titers. The cell-free supernatant fluid from cultures of the 14th passage was shown to be infectious for a normal rat kidney cell line, as evidenced by the appearance of the CF antigen in this line. Interferon production was not demonstrated in infected cultures. The overall data indicated that rat kidney cells could be used to propagate Rauscher virus in a carrier state.     

A rapid fluorescent focus-inhibition test for determining the neutralizing-antibody response to lymphocytic choriomeningitis virus.

Levels of neutralizing antibody to lymphocytic choriomeningitis (LCM) virus in the sera of 66 infected persons were assayed by a rapid fluorescent focus-inhibition test (RFFIT). The test was more sensitive than the mouse-neutralization (MN) test and could be completed in less than 24 h. The RFFIT titers were compared with titers obtained by the indirect fluorescent-antibody (IFA) and complement-fixation (CF) tests. Neutralizing antibody detected by the RFFIT remained positive after IRA, CF and MN antibodies had disappeared. The RFFIT for detection of LCM antibody is specific and reproducible and seems especially useful for determining the incidence and epidemiology of LCM virus infections.     

Interferon induction in marrow-derived macrophages: regulation by L cell conditioned medium

Mouse bone marrow cells grown in medium enriched with L cell conditioned medium (LCM) as a source of colony stimulating factor (CSF) yield populations of adherent macrophages which are quite sensitive to induction of interferon (IFN) by viral and nonviral inducers. We examined the role of LCM in the sensitivity of marrow macrophage cultures to IFN induction. Removal of LCM from the cultures for as little as 3 hours markedly reduced the IFN titers induced by a double stranded ribopolynucleotide (poly I:C) or a lipopolysaccharide (LPS), while induction by Newcastle disease virus (NDV) was unaffected. Addition of anti-CSF serum to LCM medium also reduced IFN titers in response to polyI:C but had no effect on NDV induction. The inhibitory effect of anit-CSF indicates that the LCM requirement is at least partially related to the colony stimulating activity of the medium. We postulate that CSF regulates the initial interaction of macrophages with polyI:C or LPS rather than the synthesis and secretion of interferon by the phagocytes. Nearly complete restoration of IFN induction with polyI:C was obtained when LCM deprived cultures were reincubated with LCM medium previously conditioned by marrow cultures.     

Rescue of a positive stranded RNA virus from antigen negative neuroblastoma cells.

Single cell clones of latently infected mouse neuroblastoma cells were isolated from a culture chronically infected with mouse hepatitis virus in the presence of an antiviral antibody. These cell clones did not produce infections virus or exhibit viral cytopathic effects during cultivation at 32, 37, or 39°C. Infectious virus was isolated from single cell clones via fusion with permissive cells using polyethylene glycol, but not after fusion with inactivated Sendai virus or following treatment with metabolic inhibitors. One cell clone (S-3) from which virus was rescued was negative for viral antigen by immunofluorescence. The S-3 cell clone and no demonstrable virus antigen by complement-fixation tests using cytoplasmic extracts or virus-specified proteins detectable by polyacrylamide gel electrophoresis. The rescued viruses exhibited a temperature dependent growth defect at 32°C and have been classified as cold sensitive mutants. This study suggests that a complete genome of a positive stranded RNA virus can remain latent in infected cells without the expression of detectable virus antigen.     

Antigens of Pichinde virus I. Relationship of soluble antigens derived from infected BHK-21 cells to the structural components of the virion.

Antigens detected by the complement-fixation (CF) test were prepared from BHK-21 cells infected with Pichinde virus.The preparations contained two antigens demonstrable by immunodiffusion. The antigen present in abundance was heat stable, Pronase resistant, and had a molecular weight of 20,000 to 30,000 as estimated by gel filtration. Polyacrylamide gel electrophoresis of purified antigen demonstrated two low-molecular-weight polypeptides. An identical antigenic determinant was found by disrupting purified virus with Nonidet P-40; however, none of the viral polypeptides co-migrated with the polypeptides derived from purified CF antigen. Pronase digestion of disrupted virus did not alter antigenicity but degraded the viral peptides to sizes similar to those associated with the major CF antigen. These observations suggest that the major CF antigen of Pichnide virus is a cleavage product of the structural proteins of the virus.     

Rubella Virus: Continuous and Concurrent Production of Complement-fixing Antigen and Infectious Virus in Suspension Cultures

Rubella complement-fixing (CF) antigen and infectious virus were produced continuously and concurrently for as long as 63 days in suspension cultures of BHK-21 cells prepared from uncloned monolayer stock cultures. CF titers ranged from 1:4 to 1:32, and the peak infectivity titer was greater than 8.0 (TCID(50) log(10)) per ml. Suspension cultures could be recultivated after prolonged storage in liquid nitrogen. The resulting monolayer or suspension cultures also produced CF antigen. Suspension cultures provide an effective system for the long-term continuous and concurrent production of rubella virus diagnostic reagents.     

Analysis of Aleutian disease virus infection in vitro and in vivo: demonstration of Aleutian disease virus DNA in tissues of infected mink.

Aleutian disease virus (ADV) infection was analyzed in vivo and in vitro to compare virus replication in cell culture and in mink. Initial experiments compared cultures of Crandell feline kidney (CRFK) cells infected with the avirulent ADV-G strain or the highly virulent Utah I ADV. The number of ADV-infected cells was estimated by calculating the percentage of cells displaying ADV antigen by immunofluorescence (IFA), and several parameters of infection were determined. Infected cells contained large quantities of viral DNA (more than 10(5) genomes per infected cell) as estimated by dot-blot DNA-DNA hybridization, and much of the viral DNA, when analyzed by Southern blot hybridization, was found to be of a 4.8-kilobase-pair duplex monomeric replicative form (DM DNA). Furthermore, the cultures contained 7 to 67 fluorescence-forming units (FFU) per infected cell, and the ADV genome per FFU ratio ranged between 2 X 10(3) and 164 X 10(3). Finally, the pattern of viral antigen detected by IFA was characteristically nuclear, although cytoplasmic fluorescence was often found in the same cells. Because no difference was noted between the two virus strains when cultures containing similar numbers of infected cells were compared, it seemed that both viruses behaved similarly in infected cell culture. These data were used as a basis for the analysis of infection of mink by virulent Utah I ADV. Ten days after infection, the highest levels of viral DNA were detected in spleen (373 genomes per cell), mesenteric lymph node (MLN; 750 genomes per cell), and liver (373 genomes per cell). In marked contrast to infected CRFK cells, the predominant species of ADV DNA in all tissues was single-stranded virion DNA; however, 4.8-kilobase-pair DM DNA was found in MLN and spleen. This observation suggested that MLN and spleen were sites of virus replication, but that the DNA found in liver reflected sequestration of virus produced elsewhere. A final set of experiments examined MLN taken from nine mink 10 days after Utah I ADV infection. All of the nodes contained ADV DNA (46 to 750 genomes per cell), and although single-stranded virion DNA was always the most abundant species, DM DNA was observed. All of the lymph nodes contained virus infectious for CRFK cells, but when the genome per FFU ratio was calculated, virus from the lymph nodes required almost 1,000 times more genomes to produce an FFU than did virus prepared from infected cell cultures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Respiratory syncytial virus infection of human airway epithelial cells is polarized, specific to ciliated cells, and without obvious cytopathology

Gene therapy for cystic fibrosis (CF) lung disease requires efficient gene transfer to airway epithelial cells after intralumenal delivery. Most gene transfer vectors so far tested have not provided the efficiency required. Although human respiratory syncytial virus (RSV), a common respiratory virus, is known to infect the respiratory epithelium, the mechanism of infection and the epithelial cell type targeted by RSV have not been determined. We have utilized human primary airway epithelial cell cultures that generate a well-differentiated pseudostratified mucociliary epithelium to investigate whether RSV infects airway epithelium via the lumenal (apical) surface. A recombinant RSV expressing green fluorescent protein (rgRSV) infected epithelial cell cultures with high gene transfer efficiency when applied to the apical surface but not after basolateral inoculation. Analyses of the cell types infected by RSV revealed that lumenal columnar cells, specifically ciliated epithelial cells, were targeted by RSV and that cultures became susceptible to infection as they differentiated into a ciliated phenotype. In addition to infection of ciliated cells via the apical membrane, RSV was shed exclusively from the apical surface and spread to neighboring ciliated cells by the motion of the cilial beat. Gross histological examination of cultures infected with RSV revealed no evidence of obvious cytopathology, suggesting that RSV infection in the absence of an immune response can be tolerated for >3 months. Therefore, rgRSV efficiently transduced the airway epithelium via the lumenal surface and specifically targeted ciliated airway epithelial cells. Since rgRSV appears to breach the lumenal barriers encountered by other gene transfer vectors in the airway, this virus may be a good candidate for the development of a gene transfer vector for CF lung disease.     

Growth and immunogenicity of foot-and-mouth disease virus in baby hamster kidney cells adapted to and continuously grown in a serum-free chemically defined media

The production of foot-and-mouth disease (FMD) virus in baby hamster kidney (BHK) suspension cells grown in serum-free media for subsequent use in vaccines was attempted because of the limited availability of serum in quantities sufficient for propagation of large amounts of cells, as well as the possible presence of mycoplasma, viral contaminants, and interfering antibodies in sera. Suspension cultures (50 to 600 ml) of BHK-21 cells adapted to and continually passed in a glutamine-free autoclavable, chemically defined medium (BHK-S system) were infected with all seven types of FMD virus. Cells were infected at multiplicities of infection (MOI) ranging from 10^?1 to 10^?7 plaque-forming units per cell (PFU/cell). The time course of infectious virus release and the amount of complement-fixing (CF) antigen produced were then followed. Peak harvest infectivities of approximately 10^8.5 PFU/ml were obtained from 12 to 24 hr after inoculation, depending on input MOI, and were apparently independent of cell concentration over the range 1.5 to 4.0 million cells/ml; the CF endpoint dilutions increased from 1:12 at the lower cell concentrations to 1:48 at the highest cell concentration. Monovalent and trivalent vaccines have been produced using viruses from the BHK-S system, inactivated with acetylethyleneimine and emulsified in oil, and the results of tests in steers and guinea pigs are presented.     

Observations of Measles Virus Infection of Cultured Human Cells : I. A Study of Development and Spread of Virus Antigen by Means of Immunofluorescence

The development of measles virus in cultures of both primary human amnion cells and H.Ep.-2 cells has been followed by means of the indirect fluorescent antibody technic and concurrent light and electron microscope observations. The immunofluorescence studies revealed that there is a latent period for development of demonstrable measles virus antigen. In amnion cells the latent period lasted for at least 3 days. In contrast, virus antigen could be detected in H.Ep.-2 cells as early as 12 hours following inoculation. In each cell system virus antigen was seen in either nucleus or cytoplasm of infected cells, or both. Early localization tended to be perinuclear. Intranuclear fluorescence was generally less bright and less widespread than cytoplasmic fluorescence. Giant cells and long cytoplasmic spindle-shaped processes appeared regularly in infected cultures. Infectious virus was liberated into the nutrient fluid but when extracellular virus was inhibited by antibody, spread of infection from cell to cell in the monolayer still continued. Results obtained in concurrent electron microscope studies will be presented separately. Correlation of the results of the immunofluorescence and electron microscope studies suggests the possibility that much of the immunofluorescence observed might be due to antigen in virus precursors or components.     

Comparison of Transformation and T Antigen Induction in Human Cell Lines

Skin fibroblast cultures were established from eight individuals. These cell cultures, together with WI-38 cells, were examined for susceptibility to transformation by SV40 virus. Four transformation-susceptible cell lines (TS), established from patients with Down's syndrome, were found to be three to four times more susceptible to transformation than transformation-resistant cell lines (TR) from normal individuals. TR and TS cell lines were compared for their susceptibility to induction of SV40 T antigen. For dividing cells T antigen was detected in a higher percentage of TS cells than TR cells. For nondividing cells, the reverse was found; T antigen was detected in 10-fold more cells of the TR lines than in cells of the TS lines. Similar results were obtained after infection of cells with CELO virus. Titration of vaccinia virus and influenza virus A2/Scotland/49/57 indicated that TR and TS cells were equally sensitive to the former virus, but TR cells were three to five times more sensitive to influenza virus A2/Scotland/49/57 than were TS cells.     

Detection of Coronavirus 229E Antibody by Indirect Hemagglutination

Tannic-acid treated sheep erythrocytes (fresh or glutaraldehyde preserved) were sensitized with 229E antigens from human embryonic lung (RU-1) cell cultures. Indirect hemagglutination (IHA) antigen titers in 229E-infected cell cultures paralleled virus infectivity and complement fixation (CF) antigen titers. The identity of the IHA antigen was confirmed by testing extracts from inoculated and control cell cultures for ability to inhibit IHA. Also, significant increases in IHA antibody were demonstrated with acute and convalescent serum pairs from patients with proven 229E infections. A comparison of IHA, neutralization and CF titers for 229E antibodies was made on human sera drawn from different populations. The IHA and neutralization results were in agreement on 93% of the 129 sera found to be positive by at least one of three tests. The number of antibody titers detected by the CF test was insufficient to permit comparison. Hyperimmune sera from animals immunized with OC 43 did not react with 229E by IHA. Also no increase in IHA antibody was demonstrated with acute and convalescent serum pairs from patients with seroconversions to OC 43. These findings suggest that the IHA test provides (i) a rapid and sensitive method for serodiagnosis of 229E infections and (ii) a simple and inexpensive method for seroepidemiological studies.     

Immunofluorescent Cell Assay of Infectious Pancreatic Necrosis Virus

An immunofluorescent cell (IFC) assay technique was developed for the quantification of infectious pancreatic necrosis (IPN) virus of salmonid fishes. Cover slip cultures of rainbow trout gonad (RTG-2) cells were infected with diluted virus preparations. After incubation to permit antigen development, the cells were stained with antiviral fluorescent antibody, and the number of fluorescing (infected) cells was counted. Optimal conditions for the IFC assay procedure are: (i) the use of RTG-2 cells cultured for at least 3 days at 20 C; (ii) 1-h absorption of IPN virus to RTG-2 cells at 20 C or alternatively, 4 h at 4 C; (iii) staining the infected cell cultures at 10 to 12 h postinfection. A linear relationship between the relative concentration of virus in the inoculum and the number of fluorescent cells in the first cycle of infection was observed. The IFC assay method is more sensitive than the plaque method for the assay of IPN virus.     

Stability of rubella complement-fixing antigens

Various types of rubella complement-fixing (CF) antigen preparations derived from infected BHK-21 cell cultures were examined for stability to certain chemical and physical agents. Antigens studied included: infected culture fluids concentrated 100-fold, ether-treated fluid concentrates, alkaline buffer extracts of infected cells, and large- and small-particle CF antigens separated by Sephadex gel filtration. All preparations were stable at -20 C for months, and were unaffected by three cycles of freezing and thawing. Ether treatment rendered antigens highly susceptible to thermal inactivation at 56 C; untreated antigens were inactivated slowly and showed a persistent fraction of activity even after 2 hr. Large-particle antigen was found to be slightly more susceptible than small-particle antigen to thermal inactivation and ether treatment. CF activity in alkaline buffer extracts was more labile than that in untreated or ether-treated fluid concentrates upon dialysis, Sephadex gel filtration, and sucrose density gradient centrifugation.     

Lack of correlation between cytotoxic T lymphocytes and lethal murine lymphocytic choriomeningitis

Adoptive transfer of lymph node and spleen cells from mice infected with LCM virus to similarly infected immunocompromised recipients has been the classic way to demonstrate the lethal role of T cells in the CNS disease caused by this virus. Isolation and adoptive transfer techniques are presented here which show that Thy-1+ cells isolated from the meningeal infiltrates (MI) of LCM virus-infected mice possess this property. We compared various T cell functions of MI cells taken from mice infected with two strains of LCM virus differing markedly in their pathogenicities. One of these strains, termed aggressive, caused a typical, invariably fatal, CNS disease within 7 to 10 days after infection. The other virus, termed docile, killed few mice after the standard intracerebral inoculation, and could persist in the mice for 6 mo or more. The yields of MI leukocytes from mice infected with docile virus varied from 50 to 100% of those found in mice infected with aggressive virus (3 X 10(6) cells/brain). On a cell-to-cell basis, the CTL activity in the MI of mice infected with docile virus ranged from 50 to 100% of that found in the MI of mice infected with aggressive virus. MI cells from mice infected with aggressive virus consistently caused lethal disease by adoptive transfer into immunocompromised (irradiated) recipients infected with either strain of virus. All attempts to induce lethal disease by adoptive transfer of MI cells (or splenocytes) from mice infected with docile virus into irradiated recipients failed. The latter experiments with the docile-MI cells were performed with six times the number of aggressive-MI cells needed to kill irradiated recipients by adoptive transfer. The possible reasons for this discordance between CTL and in vivo killer function are discussed.     

Persistent infection of cells in culture by measles virus. II. Effect of measles antibody on persistently infected HeLa sublines and recovery of a HeLa clonal line persistently infected with incomplete virus

Rustigian, Robert (Tufts University School of Medicine, Boston, Mass.). Persistent infection of cells in culture by measles virus. II. Effect of measles antibody on persistently infected HeLa clonal line persistently infected with incomplete virus. J. Bacteriol. 92:1805-1811. 1966.-The effect of viral antibody on persistent infection of HeLa cells by the Edmonston strain of measles virus was investigated by culturing cells from three persistently infected clones in medium supplemented with human immune globulin. The three infected HeLa clones were isolated from a persistently infected parent line. Two sublines which were grown in the presence of measles antibody developed a nonyielder state, wherein there is no detectable virus infectious for normal HeLa cultures. There is, however, continued synthesis of intracellular viral antigen and formation of viral intracytoplasmic inclusion bodies. The development of a nonyielder state was associated with a marked decrease in the degree of hemadsorption in cultures of both sublines. Further studies of the viral properties of non-yielder HeLa cell populations were made with a clone obtained from one of these sublines by plating under antibody. Persistent infection in this line was characterized by synthesis of incomplete virus even when the cells were cultured thereafter in anti-body-free medium. This was evidenced by (i) failure to recover infectious virus from the clonal population despite continued formation of intracellular viral antigen and viral intracytoplasmic inclusion bodies in a majority of the cells, (ii) the presence of only a few cells with surface viral antigen(s) including hemagglutinin, and (iii) the relatively weak antibody response to viral envelope antigen(s) after injection of cells into guinea pigs.     

Herpesvirus saimiri antigens and virus recovery from cultured cells and antibody levels and virus isolations from squirrel monkeys

In cultures of vero cells infected with a low multiplicity of Herpesvirus saimiri (HVS), the development of internal antigen, as demonstrated by indirect immunofluorescence, was detected early and preceded the release of infectious virus. Membrane antigen, determined on unfixed cells, developed shortly thereafter. A positive correlation was found between the percentage of antigen-positive cells and the amount of recoverable infectious virus in cocultivation cultures of cells of a low virus-producing line of HVS-induced tumor and vero cells. Cytosine arabinoside (araC) prevented the development of infectious HVS, typical internal antigen, and membrane antigen when vero cells were treated with the drug two hours after virus inoculation. Another type of internal antigen appearing predominantly in the nucleus was demonstrable in infected but araC-treated vero cells. The presence of antibody to this antigen was rare in naturally-infected squirrel monkeys. Colony-housed squirrel monkeys showed a high frequency of antibody to HVS internal antigen produced in infected vero cells in the absence of araC, and evidence for seroconversion in some of these monkeys was obtained. Virus was isolated from lymphocytes of six of seven squirrel monkeys tested.     

Jatobal virus antigenic characterization by ELISA and neutralization test using EIA as indicator, on tissue culture

A virus antigenic characterization methodology using an indirect method of antibody detection ELISA with virus-infected cultured cells as antigen and a micro virus neutralisation test using EIA (NT-EIA) as an aid to reading were used for antigenic characterization of Jatobal (BeAn 423380). Jatobal virus was characterized as a Bunyaviridae, Bunyavirus genus, Simbu serogroup virus. ELISA using infected cultured cells as antigen is a sensitive and reliable method for identification of viruses and has many advantages over conventional antibody capture ELISA's and other tests: it eliminates solid phase coating with virus and laborious antigen preparation; it permits screening of large numbers of virus antisera faster and more easily than by CF, HAI, or plaque reduction NT. ELISA and NT using EIA as an aid to reading can be applicable to viruses which do not produce cytopathogenic effect. Both techniques are applicable to identification of viruses which grow in mosquito cells.