Growth of Trypanosoma cruzi in Cultures of Chick Embryo Cells, and Effects of Furazolidone and Tris (p-Aminophenyl)Carbonium Chloride

SYNOPSIS. Monolayers of cells of coverslips were produced by culturing known numbers of trypsinized chick cells in growth medium (solution 199 plus 20% calf serum) at 37 C for 2 days. The fluid was then replaced with maintenance medium (solution 199 plus 5% calf serum) containing various known numbers of T. cruzi and the preparations were incubated at 33 C for 5 days; fresh maintenance medium was substituted on the 2nd or 3rd day. The inocula of parasites were obtained from T. cruzi -cell cultures, supplemented with 2% sterile NNN overlay, or from NNN cultures.
The numbers of extracellular parasites, proportions of infected cells, and percentage distribution of infected cells relative to the number of intracellular leishmanial bodies were determined on days 2 or 3 and 5 of parasite cultivation in many experiments. Analyses of the data gave the following results. Extracellular parasites increased 2- to 14-fold during the first 2 or 3 days, depending upon the source and size of the inocula, and 10- to 20-fold during the last 2 or 3 days. Cell infection continued throughout incubation at daily rates of 1.4-4.5%; 8-22% of the cells became infected during the 5 days of incubation. Intracellular growth was reflected most clearly by increases in the proportion of cells having >10 leishmanial bodies. This increase was about 5% daily during the last 2 or 3 days of incubation.
A useful test procedure for assessing the antiparasitic action and chick embryo cell toxicity of drugs is illustrated by data obtained with furazolidone and tris ( p -aminophenyl)carbonium chloride.     

Erythroid stem cells in Friend-virus infected mice

The erythropoietic stem cell compartment was studied in Friend-virus (polycythemic strain, FV-P) infected DBA/2 and NMRI mice with the CFUE and BFUE technique. Early after infection there was a depression in CFUE number in bone marrow and spleen, followed by an increase of the CFUE concentration, earlier and more pronounced in the spleen than in the marrow. Three days after FV-P infection an erythropoietin (Ep) independent CFUE population started to grow and replaced the normal Ep-dependent population within 8 to 12 days. The shift to Ep independency was not gradual. CFUE colonies of FV-P infected bone marrow cells were two to three times larger than control colonies after three days in vitro incubation. BFUE colonies increased in number during the first days of infection, but were totally lost after more than ten days. After velocity sedimentation of bone marrow cells of FV-P infected animals, however, the BFUE containing fractions showed normal BFUE colony growth and normal Ep sensitivity. In unfractionated bone marrow cell cultures BFUE colony growth could be observed later than ten days post infection when the cultures were refed with medium. It was therefore concluded that the loss of BFUE colony growth after FV-P infection was an in vitro artefact due to inadequate culture conditions.     

Kinetics of murine type C virus-specific DNA synthesis newly infected cells.

Replicating transforming functions of Rauscher leukemia virus (RLV) and the RLV pseudotype of Moloney sarcoma virus in mouse embryo fibroblasts were found to be most sensitive to inhibition by cytosine arabinoside (ara-C) 30 to 90 min after infection. The initiation of intracellular RLV DNA synthesis was detected by nucleic acid hybridization within this time interval. Treatment of infected cells with cytosine arabinoside abolished RLV DNA synthesis. Peak synthesis of the DNA complementary to the infecting RLV genome, the (-) strand, occurred 40 to 60 min after infection. During this interval two s two species of DNA were observed with estimated molecular weights of 0.5 X 10(5) to 1.0 X 10(5) and 3 X 10(6). Peak synthesis of the (+) strand viral DNA occurred 50 to 70 min after infection. The initial species detected had a molecular weight of 1.5 X 10(5) to 4.0 X 10(5) which shifted as a function of time to 3 X 10(6). Both (+) strand species were initially detected in the cytoplasm followed by a rapid (10-min interval) appearance of the faster-sedimenting species in the nucleus. The virus-specific (-) and (+) strand DNA species are presumably unintegrated intermediates in provirus formation.     

Kinetics of the basic sections of the hemopoietic system in radiation chimeras]

During first 3 days after mice irradiation and syngeneic bone marrow transplantation in them the number of CFUs (about 0,5% of the injected cells) was stable, although the proliferation induction began 24 hours after transplantation. As it was shown by the method of "thymidine self-distruction". Twenty four hours later all the CFUs entered the mitotic cycle. On the contrary, the commited cells (granulopoesis precursors) compartment (CFUc) enters the logarithmic growth phase since the first day. The exponential growth of the CFUs number was observed from the 4th day simultaneously with the increasing of the proliferation rate of CFUc and the beginning of the recovery of the bone marrow cells total number. In late radiation chimeras (1 month after radiation and reconstitution) the total number of CFUs was 50--70% of the initial. The other hemopoetic parameters were in the normal limits.     

Heterologous interference in Aedes albopictus cells infected with alphaviruses.

Maximum amounts of 42S and 26S single-stranded viral RNA and viral structural proteins were synthesized in Aedes albopictus cells at 24 h after Sindbis virus infection. Thereafter, viral RNA and protein syntheses were inhibited. By 3 days postinfection, only small quantities of 42S RNA and no detectable 26S RNA or structural proteins were synthesized in infected cells. Superinfection of A. albopictus cells 3 days after Sindbis virus infection with Sindbis, Semliki Forest, Una, or Chikungunya alphavirus did not lead to the synthesis of intracellular 26S viral RNA. In contrast, infection with snowshoe hare virus, a bunyavirus, induced the synthesis of snowshoe hare virus RNA in both A. Ablpictus cells 3 days after Sindbis virus infection and previously uninfected mosquito cells. These results suggested that at 3 days after infection with Sindbis virus, mosquito cells restricted the replication of both homologous and heterologous alphaviruses but remained susceptible to infection with a bunyavirus. In superinfection experiments the the alphaviruses were differentiated on the basis of plaque morphology and the electrophoretic mobility of their intracellular 26S viral RNA species. Thus, it was shown that within 1 h after infection with eigher Sindbis or Chikungunya virus, A. albopictus cells were resistant to superinfection with Sindbis, Chikungunya, Una, and Semliki Forest viruses. Infected cultures were resistant to superinfection with the homologous virus indefinitely, but maximum resistance to superinfection with heterologous alphaviruses lasted for approximately 8 days. After that time, infected cultures supported the replication of heterologous alphaviruses to the same extent as did persistently infected cultures established months previously. However, the titer of heterologous alphavirus produced after superinfection of persistently infected cultures was 10- to 50-fold less than that produced by an equal number of previously uninfected A. albopictus cells. Only a small proportion (8 to 10%) of the cells in a persistently infected culture was capable of supporting the replication of a heterologous alphavirus.     

Kinetics of the clonal proliferation of granulocytes and macrophages in cultures of mouse bone marrow cells as supported by two distinct types of colony-stimulating factors

Two different types of colony-stimulating factors (CSF) were used to support the clonal growth of myeloid progenitor cells (CFUc) in semi-solid agar or viscous methylcellulose cultures of mouse bone marrow cells. The cultures stimulated for 5 days with RSP-2-P3 cell CSF (CSFRSP) contained mainly granulocyte colonies, whereas the cultures stimulated for 10 days with human urine CSF (CSFhu) contained mainly monocyte/macrophage colonies. Four lines of study were carried out: 1) a kinetic study using combinations of the two types of CSFs in the same culture; 2) a study of transferring CFUc from the initial 3-day cultures to recipient cultures containing the same or different types of CSF; 3) an examination of the morphology over time of colonies that were confined by glass capillaries plunged in agar; and 4) electron microscopic observations on disintegrating granulocytes. The results of all these lines of study suggest that about one third of the CFUc can be stimulated both by CSFRSP and CSFhu while the other two thirds react specifically either with CSFRSP or with CSFhu. The present study also suggests that granulocytes in the culture stop proliferation and disintegrate while macrophages are still growing there. Thus, mixed-type colonies containing both macrophages and granulocytes later become macrophage colonies.     

In vitro establishment of lytic and nonproductive infection by herpes simplex virus type 1 in three-dimensional keratinocyte culture.

The F strain of herpes simplex virus type 1 (HSV-1) was tested for its ability to produce lytic or nonproductive infection in squamous epithelial cells cultured in a three-dimensional organotypic tissue culture. For the tissue culture, we used HaCat cells (immortalized skin keratinocytes) and normal fibroblasts derived from the skin. The cultures were infected with HSV-1 (5 PFU) either when the epithelial cells had grown as a monolayer with a confluence of 80% on the collagen fibroblast gel or 30 min after lifting of the epithelial cells into the air-liquid interface. The cultures were collected 1 week after inoculation. Typical cytopathic effects of HSV infection (ballooning and reticular degeneration with multinucleate giant cells) were seen only in those cultures in which the epithelial cells were infected before lifting. The presence of HSV was confirmed by DNA and RNA in situ hybridization and PCR. No morphological changes were found in cultures infected after lifting into the air-liquid interface. No infectious virus was recovered either from cells or culture supernatant. However, these cultures were positive for HSV DNA on PCR and showed expression of the LAT gene by in situ hybridization and Northern blot (RNA) hybridization. The present results indicate that both nonproductive and lytic HSV infection can be produced in vitro and the outcome of the infection depends on the time of viral inoculation in relation to epithelial maturation.     

Radiosensitivity of pluripotent stem cells detectable by cloning in the spleen of irradiated mice

It was shown that the dose--effect curves describing the radiosensitivity of CFUc of the bone marrow irradiated in vitro (0.04-3.7 Gy) and treated with normal rabbit serum (NRS) and anti-mouse-brain serum (AMBS) has two differently sloping portions indicating that two CFUc populations differing in radiosensitivity are present in the bone marrow. D0 was 0.93 Gy after irradiation with doses of 0.04-0.75 Gy and treatment with NRS, and 0.33 Gy after incubation of the bone marrow with AMBS. The addition of thymus cells "straightened" the dose--effect curve for the bone marrow treated with AMBS: in this case D0 was 1.81 Gy exceeding considerably the values of D0 for intact bone marrow. The CFUc population is suggested to be heterogeneous in radiosensitivity.     

Differentiation of an adult neuron cell line increases susceptibility to rabies infection

A wide variety of in vitro models have been used for studying rabies infection, however, currently, no central nervous system (CNS) adult neuron cultures are available. The current study determined the susceptibility to rabies infection in an adult CNS neuron cell line (CAD-R1). Cultures of CAD-R1 cells were held for 5 days in medium containing serum (undifferentiated CAD-R1 cells) or in serum-free medium (differentiated CAD-R1 cells). They were then infected with highly neurotropic rabies virus (RV) strain (CVS), obtained from fibroblastic cells (CVS-BHK) or from adult mouse brain (CVS-MB). Undifferentiated and differentiated cells were infected with the two RV strains, but the percentage of infected cells in differentiated cultures was significantly greater (83% and 79%, respectively) than in undifferentiated cells (51% and 60%) (Student's t test<0.05). Susceptibility to infection apparently depended on cellular differentiation state, possibly due to acquisition of additional morphological and biochemical characteristics during the differentiation process that made them more susceptible to RV infection. Therefore, CAD R1 cells may represent a good model for RV infection, making them a useful tool for studying RV neurotropism, infection pathogeny, isolation of street virus or producing safer and most potent vaccines.     

Persistent infection of cultured cells with mouse hepatitis virus (MHV) results from the epigenetic expression of the MHV receptor.

The A59 strain of murine coronavirus mouse hepatitis virus (MHV) can cause persistent infection of 17C1-1 cells and other murine cell lines. Persistently infected cultures released large amounts of virus (10(7) to 10(8) PFU/ml) and were resistant to superinfection with MHV but not to infection with unrelated Semliki Forest and vesicular stomatitis viruses. The culture medium from persistently infected cultures did not contain a soluble inhibitor such as interferon that protected uninfected cells from infection by MHV or vesicular stomatitis virus. The persistent infection was cured if fewer than 100 cells were transferred during subculturing, and such cured cultures were susceptible to reinfection and the reestablishment of persistent infection. Cultures of 17C1-1 cells that had been newly cloned from single cells consisted of a mixture of MHV-resistant and -susceptible cells. 17C1-1/#97 cells, which were cured by subcloning after 97 passages of a persistently infected culture over a 1-year period, contained 5 to 10% of their population as susceptible cells, while 17C1-1/#402 cells, which were cured by subcloning after 402 passages over a 3-year period, had less than 1% susceptible cells. Susceptibility to infection correlated with the expression of MHV receptor glycoprotein (MHVR [Bgp1a]). Fluorescence-activated cell sorter analysis with antibody to MHVR showed that 17C1-1/#97 cells contained a small fraction of MHVR-expressing cells. These MHVR-expressing cells were selectively eliminated within 24 h after challenge with MHV-A59, and pretreatment of 17C1-1/#97 cells with monoclonal antibody CC1, which binds to the N-terminal domain of MHVR, blocked infection. We conclude that the subpopulation of MHVR-expressing cells were infected and killed in acutely or persistently infected cultures, while the subpopulation of MHVR-nonexpressing cells survived and proliferated. The subpopulation of MHVR-negative cells produced a small proportion of progeny cells that expressed MHVR and became infected, thereby maintaining the persistent infection as a steady-state carrier culture. Thus, in 17C1-1 cell cultures, the unstable or epigenetic expression of MHVR permitted the establishment of a persistent, chronic infection.     

Flow cytometric assessment of transduction efficiency and cytotoxicity of herpes simplex virus type 1-based amplicon vectors

BACKGROUND: In this study, we compared herpes simplex virus type 1 (HSV-1) amplicon vector stocks prepared by transient cotransfection with two different BAC-cloned packaging-defective HSV-1 helper genomes, fHSVDeltapacDelta27 and fHSVDeltapac, with respect to transduction efficiency and cytotoxicity. Both fHSVDeltapacDelta27 and fHSVDeltapac are packaging defective because the pac signals have been deleted; fHSVDeltapacDelta27 contains an additional deletion in the HSV-1 ICP27 gene, which increases the safety of the system. METHODS: HSV-1 amplicon pHSVGFP under the control of the HSV-1 immediate-early (IE) 4/5 promotor was packaged into virus particles by transient cotransfection with either fHSVDeltapacDelta27 or fHSVDeltapac DNA. Cultures were infected with the two different vector stocks and examined under the fluorescence microscope and analyzed by flow cytometry over a 5-day period to assess transduction efficiency and cytotoxicity. RESULTS: Both vector stocks, pHSVGFP[fHSVDeltapacDelta27] and pHSVGFP[fHSVDeltapac], efficiently transduced the target cells. Interestingly, the highest mean fluorescence intensities were measured at 1 day after infection, whereas the number of GFP-fluorescent cells reached a peak at day 3 after infection. At day 3 after infection, a slight increase in the number of dead cells was observed in those cultures transduced with high doses of vector stock. Between days 3 and 4 after infection, the number of dead cells increased dramatically in all the cultures, transduced and nontransduced. Only the cultures infected with a high dose of pHSVGFP[fHSVDeltapac] displayed a significant further increase in the number of dead cells between days 4 and 5 postinfection. CONCLUSIONS: Flow cytometry allowed comparison of transduction efficiency and cytotoxicity mediated by the two different amplicon vector stocks. Cultures infected with pHSVGFP[fHSVDeltapacDelta27] were more viable than those infected with pHSVGFP[fHSVDeltapac](P < 0.05). The practical implications of this study are at the level of vector design. Flow cytometry has proven a fast and reliable approach to assess the quality of potential gene transfer vectors prior to their use in (pre) clinical trials.     

T-dependent suppression of the primary antibody response to sheep erythrocytes in mice infected with Trichinella spiralis.

Mice infected for 20 days with the parasitic mematode Trichinella spiralis had significantly reduced numbers of splenic antibody-forming cells (AFC) and decreased serum hemagglutinin titers following intraperitoneal immunization with sheep erythrocytes (SE). Similarly, when immunized in vitro to SE, cultures of splenocytes from infected mice developed fewer AFC than cultures of normal cells. Splenocytes from infected mice actively suppressed the in vitro response of normal cells to SE, and this in vitro suppression was abolished by lysis with anti-thy 1 antiserum and enhanced by lysis with anti-immunoglobulin antiserum. The addition of supernatant fluids from cultures of splenocytes from infected mice to cultures of normal cells on Day 0 of culture reduced by 70% the number of AFC produced by these cultures. These results indicate the presence of T-suppressor cells and suggest that antigen-induced suppression (antigenic competition) is one mechanism of Trichinella-induced suppression.     

Erythropoietin responses and physical characterization of erythroid progenitor cells in Rauscher virus infected BALB/c mice.

Infection of BALB/c mice with Rauscher leukemia virus (RLV) gives rise to pronounced erythrocytopoiesis manifesting in splenomegaly and is associated with progressive development of anemia. In the spleen erythroid colony forming units (CFU-E) increase exponentially up to 800-fold that of normal levels by the third week of infection. In vitro these CFU-E are dependent on erythropoietin for colony formation, their erythropoietin requirements being higher than that of CFU-E from normal mice. Numbers of CFU-E in spleen and degree of splenomegaly in anemic RLV infected mice were also shown to be modified by red blood cell transfusion, but progression of the disease was not stopped. Erythroid burst forming units (BFU-E) were also responsive to erythropoietin. However, a small proportion of cells also formed BFU-E colonies at concentrations which did not support growth of normal marrow BFU-E. When compared to normal, CFU-E found in RLV-infected spleen have similar velocity sedimentation rates. However, buoyant density separation of leukemic spleen cells indicated that CFU-E were more homogeneous (modal density 1.0695 g/cm3) than CFU-E from normal spleen. Analysis of physical properties of CFU-E and the nonhemoglobinized erythroblast-like cells, which accumulate in the spleen showed that they differed mainly in their distribution of cell diameter. Our findings show that erythroid progenitor cells in RLV infected mice are responsive to erythropoietin in vitro. Also in vivo erythropoiesis appears to be under control of erythropoietin but other factors which lead to progression of RLV disease apparently exist. Most proerythroblast-like cells, which are characteristic of this disease, apparently lack the potential to form colonies and may be more mature than CFU-E.     

Quantitative aspects of granulocytic progenitor cell (CFUc) mobilization from extravascular sites in dogs using dextran sulphate (DS)

The relationship between the increase in the blood CFUc concentration after intravenous injection of dextran sulphate (DS) and the pre-existing levels of spontaneously circulating CFUc was studied in dogs. After 15 mg DS/kg body weight the CFUc numbers per ml blood rose by a factor of 3.7 over the pre-injection values, from 78 +/- 11 (SEM) to 359 +/- 50, in normal dogs, and increased by a factor of 3.9 in 0.84-Gy-r-irradiated animals which had a reduced initial CFUc concentration per ml, from 35 +/- 8 to 116 +/- 43. The injection of 20 mg DS/kg body weight into unirradiated dogs caused an increase, by a factor of 11.5, of the pre-injection CFUc concentration, from 101 +/- 20 to 921 +/- 106. On the basis of the mobilization curves for individual dogs, a significant correlation was found between the normal blood CFUc value and the number of CFUc mobilized by DS for both dose levels. From the descending part of the mobilization curves obtained after 15 mg DS/kg body weight, kinetic parameters of canine circulating CFUc were derived. The mean blood transit time (t) was 1.4 +/- 0.5 hr and the half time (T/2) was 1.0 +/- 0.4 hr.     

Immunoregulation in experimental disseminated histoplasmosis: flow microfluorometry (FMF) studies of the Thy and Lyt phenotypes of T lymphocytes from infected mice

Previous studies have shown that mice infected i.v. with 6 X 10(5) yeast phase Histoplasma capsulatum (Hc) develop suppressed immune responses during weeks 1 to 4 of infection but that by weeks 8 to 12 of infection these responses return to normal. In this study total and differential cell counts showed that as early as the third day of infection there was a marked reduction in the number of lymphocytes recovered from the peripheral blood, bone marrow, and thymus of infected animals. Concomitantly, there was an increase in the number of splenic lymphocytes. By day 28 both the total and differential cell counts were similar in both infected and normal animals. Flow microfluorometric (FMF) studies comparing the Thy-1.2, Lyt-1, Lyt-2, and surface immunoglobulin (slg) phenotypes of lymphocytes from normal and infected mice were performed. Between days 5 and 7 the thymocytes from infected mice displayed a higher relative fluorescence intensity (RFI) of the Thy-1.2 marker than normal thymocytes, whereas at day 10, the RFI was less than that of normal thymic lymphocytes. Between days 7 and 10 of infection the RFI of the Lyt-2 marker was less on thymocytes from Hc-infected mice; however, there was no change in the Lyt-1 marker. Examination of these lymphocyte markers in blood, spleen, and mesenteric lymph nodes showed that there were decreases in the RFI of both the Thy-1.2 and Lyt-2 between days 5 and 10 of infection. No changes were observed in the Lyt-1 or slg markers. By day 28 there were no differences between the normal and infected mice with respect to any surface marker in any of the organs studied. In other experiments, the effect of adrenalectomy before infection on these surface markers was studied. Absolute numbers of Thy-1.2+, Lyt-1+, and Lyt-2+ cells were significantly increased in the spleen and significantly decreased in the thymus and peripheral blood of infected mice relative to normal controls. These studies suggest that there is a migration of cells from the thymus, blood, and bone marrow to the spleens of mice with disseminated Hc infection.     

Stimulation by granulocyte-macrophage colony-stimulating factor of Leishmania tropica killing by macrophages.

The intracellular protozoan parasite Leishmania tropica was found to survive unharmed and to multiply for several days in normal mouse peritoneal macrophages. In contrast, when infected monolayers were treated with GM-CSF, there was a continuous decrease in the percentage of infected cells, reaching less than 10% on day 4 in culture, compared to about 30% in normal controls. Microscopic observations showed an increased number of dead parasites in GM-CSF treated infected cells. Within 5 hr of incubation with GM-CSF, almost 40% of intracellular parasites showed morphologic damage, compared to less than 10% in untreated cells. Pretreatment of macrophage monolayers with pure GM-CSF before infection led to an increased level of phagocytosis of L. tropica parasites as reflected by the percentage of infected cells and the increased number of parasites in each infected cell. GM-CSF treated cultures showed 73% infected cells containing a mean of five parasites per cell, as compared to controls in which only about 50% of macrophages were infected with only two parasites per cell. The number of dead parasites per cell was 5-fold higher in the GM-CSF treated cultures at 2 hr. After 24 hr the percentage of infected GM-CSF treated cells was less than one-third that in the control cultures.     

Adenovirus E1A 12S protein induces DNA synthesis and proliferation in primary epithelial cells in both the presence and absence of serum.

Infection of primary baby rat kidney (BRK) cells with an adenovirus that carries an E1A 12S cDNA in place of the normal E1A region (adenovirus 5 [Ad5] 12S) resulted in the induction of cellular DNA synthesis and proliferation of the epithelial cells in the population, even in the absence of serum. Increased cellular DNA synthesis was first detectable by 12 h after infection and was maintained at a 10- to 20-fold higher level than in mock-infected cells. By 5 days after infection there was a 10-fold-greater number of 12S virus-infected BRK cells. These infected BRK cells retained many of their normal epithelial cell characteristics and were not transformed. The expression of the E1A 12S protein(s) occurred early after infection. There was no induction of adenoviral gene expression or viral DNA replication in these cells. The early effects of a fully transforming gene product(s) were also examined. The Ad5-simian virus 40 hybrid virus, Ad5.SVR4, in which the early region of simian virus 40 has replaced the E1 region of Ad5, was used to infect BRK cells. The kinetics of expression of the T antigens were similar to those of the 12S polypeptides. Infection with Ad5.SV4 also resulted in the induction of cellular DNA synthesis and cell proliferation at levels similar to those observed with the 12S virus. However, infection with Ad5.SVR4 resulted in cells that had lost some of their epithelial cell characteristics and were fully transformed. Thus, although the early cellular events induced by the two genes were similar, they did not yield the same final cellular phenotype.     

The role of intrahepatic gammadelta-T cells for liver injury induced by Salmonella infection in mouse.

Liver injury was induced after infection with Salmonella choleraesuis 31N-1. In T-cell receptor-delta knockout mice, serum alanine transferase level was significantly decreased in comparison with normal control mice after Salmonella infection. On the contrary, in vivo administration of anti-gammadelta T-cell receptor monoclonal antibody (UC7-13D5) to stimulate gammadelta-T cells in infected mice significantly increased serum alanine transferase level but decreased bacterial growth compared with infected mice given control antibody (UC8; hamster IgG). These data suggest that gammadelta-T cells have effector activities not only for protection but also for liver injury during Salmonella infection.     

Diagnosis of bovine respiratory syncytial virus infection by complement fixation test.

The complement fixation test by the microtiter method was applied to the serological diagnosis of bovine respiratory syncytial (RS) virus infection. When used as complement fixing antigens, untreated infected cell culture fluid, fluorocarbon-treated, and ether-treated materials showed no differences in antigenicity among them. The complement fixing antigenicity of bovine RS virus appeared in bovine kidney and Vero cell cultures for the first time 4 days after inoculation. Both the infectivity and complement fixing antigenicity reached a maximum 6 days after inoculation. In detecting complement fixing antibody from infected cattle, the most outstanding specific reaction was obtained when 5% fresh normal calf serum had been added to the diluent of complement. Neutralizing and complement fixing antibodies were examined in serum samples from two cattle in the course of experimental infection. It was found that both antibodies turned to be positive 2 weeks after inoculation. There was a linear correlation between neutralizing and complement fixing antibody titers, when serum samples from 40 natural cases were tested in the acute and convalescent stages. In addition, common antigenicity was demonstrated between the virus of bovine origin and the Long strain of human RS virus by complement fixation test.