Cytopathic effect induced by rabies virus in McCoy cells.

Both fixed and street rabies virus when cultivated in McCoy cells caused cytopathic changes 24 to 72 h after infection, depending on the multiplicity of infection. The cytopathic effect (CPE) was easily recognizable and resembles that induced by other members of the Rhabdovirus group, such as vesicular stomatitis virus, in several cell cultures. Higher titers of the Pasteur strain (PV) of fixed rabies virus were found in supernatants of McCoy cells when compared to those in VERO cells. The virus titer increased with the number of passages attaining a high titer after three passages. Rabies antigens were detected by direct immunofluorescence labeling in most McCoy cells of the infected culture, and specific antibodies neutralized the virus growth and CPE. There was also inhibition by treatment of the cells with human interferon (HuIFN) -alpha or -gamma, but not by murine interferon (MuIFN) -alpha, -beta or -gamma. Rabies-infected McCoy cell cultures may provide a useful assay system, based on the induction of CPE, the high virus production and the sensitivity to IFN.     

Characterization of rabies viruses recovered from persistently infected BHK cells.

Persistent rabies virus infection with a cyclic rising and falling pattern was easily initiated in BHK cells. In striking contrast with the same chronic infection established by T. J. Wiktor and H. F. Clark ((1972). Infect. Immun.6, 988–995), this virus and cell interaction was not mediated by interferon, and the infected cells were not resistant to a heterologous virus challenge. The persistently infected cells produced small-plaque viruses (SPV) and, after a number of subcultures, these SPV replaced entirely the wild large-plaque viruses (LPV). Due to the marked cytopathic effects (CPE) on BHK cells, SPV was less efficient than LPV for the establishment of viral persistence.A fraction containing defective interfering (DI) rabies virus particles was obtained by density gradient centrifugation of virus stocks from the persistently infected cells. The DI particles could be quantitatively assayed to the degree that they suppressed the CPE by the standard rabies virus. Cyclic production of DI particles in the persistent infection was demonstrated by this method. This evidence plus the fact that the homologous virus resisted superinfection indicate that the DI particles of rabies virus play a major role in the establishment and maintenance of a persistent state of infection in vitro.     

Frequency analysis of cytolytic T lymphocyte precursors (CTL-P) generated in vivo during lethal rabies infection of mice. II. Rabies virus genus specificity of CTL-P

Cytolytic T lymphocyte precursors (CTL-P) were sensitized in vivo by intraplantar infection of C57BL/6 mice with a lethal dose of rabies virus, strain ERA (ERA). As a result of sensitization CTL-P matured to interleukin-receptive CTL-P (IL-CTL-P) that could be expanded in vitro to Thy-1+, Lyt-2+ CTL clones in the presence of IL without subjection to antigen-driven selection. After infection with ERA, IL-CTL-P-derived CTL lysed fibroblasts infected with rabies virus but not those infected with another rhabdovirus, the vesicular stomatitis virus. These CTL, however, did not discriminate between fibroblasts infected with the serologically closely related laboratory strains of classic rabies virus, ERA and HEP-Flury, and the serologically distinct rabies-related African isolate Mokola. This finding implies that in vivo sensitized IL-CTL-P recognize common genus-specific determinants expressed on cells infected with members of the lyssavirus genus.     

Abortive rabies in rabbits and white rats infected intracerebrally

Summary Non-fatal rabies was successfully reproduced in rabbtis infected intracerebrally with a highly pathogenic strain of street virus isolated from a man who had died of hydrophobia after a dog bite and in white rats infected intracerebrally with the CVS strain of fixed virus. All the animals were pretreated with a sublethal intraperitoneal dose of live rabies virus. The surviving animals showed residual neurological symptoms (except one rat) in the form of paresis (both mild and marked) and high titres fo virus-neutralizing antibody in the brain comparable to the level of serum antibody.Successful reproduction of abortive rabies in rabbits infected intracerebrally with the classical strain of street virus suggests that different forms of rabies infection may probably exist in nature.     

Amino acid at position 95 of the matrix protein is a cytopathic determinant of rabies virus

The molecular mechanism involved in cytopathogenicity of rabies virus has not been fully elucidated yet. A fixed rabies virus Nishigahara strain does not induce clear cytopathic effect (CPE) in mouse neuroblastoma (NA) cells, whereas Ni-CE strain, which was established after 100 passages of Nishigahara strain in chicken embryo fibroblast cells, induces CPE that is characterized by rounding, shrinkage and detachment of the cells. In this study, to identify which viral gene is associated with the CPE of Ni-CE strain, we analyzed chimeric viruses between Nishigahara and Ni-CE strains generated by reverse genetics systems of both strains. We showed that the matrix gene of Ni-CE strain is responsible for the CPE in NA cells. It was also demonstrated by infection of Nishigahara and Ni-CE mutants with a single amino acid substitution in the matrix protein (M) that an amino acid at position 95 of M is a cytopathic determinant of the virus. We also demonstrated that the CPE is, at least partly, due to apoptosis. This is the first report of identification of an amino acid residue in a rabies virus protein that is important for the cytopathogenicity of the virus.     

Rabies viruses infect primary cultures of murine, feline, and human microglia and astrocytes

Summary.  Recent studies have reported the detection of rabies viral antigens and virions in astrocytes and microglia of rabies-infected animals. As a first step toward understanding whether these glial cells may be involved in rabies virus replication, persistence, and/or pathogenesis, we explored their potential to be infected in vitro. Primary cultures of murine, feline, and human microglia and astrocytes were infected with several different rabies viruses: two unpassaged street virus isolates, a cell culture-adapted strain, and a mouse brain-passaged strain. Infection, as determined by immunofluorescence, was detected in 15 of the 16 (94%) virus-glial cell combinations. Replication of infectious virus, determined by infectivity assay, was detected in 7 of the 8 (88%) virus-cell combinations. These results show that astrocytes and microglia can be infected by rabies viruses, suggesting that they may have a potential role in disease, perhaps contributing to viral spread, persistence and/or neuronal dysfunction. Accepted November 19, 1996 Received October 21, 1996     

Sources of interferon-gamma (IFN-gamma) in early immune response to Listeria monocytogenes

Early, innate production of interferon-gamma (IFN-gamma) is a critical step in immunological defense against certain pathogens such as intracellular bacteria (e.g. Listeria monocytogenes), viruses and fungi. While activated T cells and activated natural killer (NK) cells were initially thought to be the only relevant source of IFN-gamma, macrophages (Mphi) and dendritic cells can also be stimulated to produce IFN-gamma in vitro under certain conditions. However, a convincing analysis at single cell level of the source(s) of IFN-gamma in the early immune response to an acute bacterial infection is still missing. In the light of controversial literature, the work presented here aimed to clarify the role of NK cells and other components of the innate cellular immune system in the early IFN-gamma production, thereby avoiding in vitro artifacts whenever possible. Immunocompetent C57BL/6 (wild type (WT)) and T and B cell-deficient C57BL/6 rag-1(-/-) (RAG) mice were infected intravenously with a pathogenic strain of L. monocytogenes. Leukocyte populations of spleen and liver were discriminated by characteristic surface markers and analyzed for intracellular interleukin (IL)-12 and IFN-gamma using flow cytometry. These cells have not been restimulated in vitro nor sorted before analysis. In RAG mice, at least, a large NK1.1+ cell population produced IFN-gamma 19 h p.i. No MHC class II+ population co-expressed intracellular IFN-gamma at this time point. For comparison with the immunocompetent situation, syngeneic WT mice were also infected and sacrificed 9, 19, and 29 h later. At 9 h p.i., the situation resembled that of uninfected mice. At 19 and 29 h p.i. it was again the NK1.1+ population that contained most of the IFN-gamma-positive events. MHC II + CD 19- Mphi/dendritic cells and MHC II+ CD19+ B cells did not co-express intracellular IFN-gamma at these time points. CD3+ T cells were also found to contain intracellular IFN-gamma; most were also CD8+ and some CD4+. These results indicate that after infection of C57BL/6 mice with L. monocytogenes, NK1.1+ cells and, to a lesser extent, CD3+ cells are the prominent sources of innate IFN-gamma. MHC II+ cells do not play a significant role in the early IFN-gamma production following an acute primary bacterial infection.     

Use of monoclonal antibodies in diagnosis of rabies virus infection and differentiation of rabies and rabies-related viruses

A panel of selected monoclonal antibodies directed against the nucleocapsid antigens of rabies and rabies-related viruses allowed the rapid identification both of antigens of rabies virus origin present in impression smears of brains from infected animals and of three rabies-related viruses (Duvenhage, Lagos bat and Mokola). Within the rabies group, the CVS and Flury HEP strains could also be identified. Analysis of several fixed and street rabies viruses reveals great diversity in the reactivity of the viruses with a panel of monoclonal antibodies directed against glycoprotein antigens. Strains of the same geographical area and species origin displayed similar patterns of reactivity, suggesting an antigenic specificity related to animal species or area. The use of hybridoma monoclonal antibodies should provide a more sophisticated method than presently available for the diagnosis of rabies virus infection.     

Intensity of virus multiplication in abortive rabies]

Experiments in random-bred and BALB/c white mice inoculated intraperitoneally with street virus, in Syrian hamsters inoculated intramuscularly with fixed viruses as well as experiments reproducing abortive rabies in 99-100% in random-bred white mice inoculated intramuscularly with fixed virus (ERA strain) showed that in the abortive infection the intensity of virus multiplication in the brain is lower than that in fatal infection, the differences being statistically significant. It was found out that from onset of signs of the disease interferon appeared to have no influence on the outcome of infection.     

H1N1亚型流感病毒在A549和BEAS-2B细胞中复制情况的初步研究

目的 探讨不同宿主来源的H1N1亚型流感病毒在A549和BEAS-2B细胞的复制情况.方法 用分离自人、禽、猪三种宿主的7株H1N1甲型流感病毒分别接种A549和BEAS-2B细胞,分析病毒感染细胞后不同时段的特点;应用受体类型不同的红细胞进行微量血凝试验,检测流感病毒的受体结合特性;同时检测了A549和BEAS-2B细胞表面的受体分布情况.结果 三种宿主来源的H1N1亚型流感病毒感染A549细胞,24 h后CPE十分明显,36 h病毒滴度达到最高值;而感染BEAS-2B细胞后,从24 h-120 h CPE都不是很明显,且所有病毒的病毒滴度都很低.对6株H1N1流感病毒的受体结合特性进行了筛查,发现部分测试病毒具有SA a-2,6Gal受体结合特异性.而A549和BEAS-2B细胞表面均含有SA a-2,3Gal及SA a-2,6Gal受体,且A549细胞表面糖受体含量明显高于BEAS-2B细胞.结论 不同宿主来源的H1N1亚型流感病毒对A549细胞都易感并能有效增殖复制,而对具有相似受体特性、上皮组织来源的BEAS-2B细胞不易感,提示支持流感病毒有效感染、复制存在宿主内的调节机制.     

Rabies virus infection in mouse neuroblastoma cells.

As part of an inquiry into factors that determine the virulence of fixed rabies virus, mouse neuroblastoma cells were infected in culture with high virulence and low virulence strains of Flury HEP virus. Low virulence virus infection differed from high virulence virus infection in (1) its more rapid production of progeny virus in the early cycles of virus infection as shown by the number of extracellular virus particles and the infectivity of the supernatant fluid; (2) its earlier development of viral antigens on the cell surface; and (3) its earlier and more severe morphologic alteration of the cell surface. Where applicable, the differences were corroborated by scanning and transmission electron microscopy of the infected cells using the critical point drying technique on whole cells. The number of cells susceptible to complement-dependent immunolysis was almost proportional to the number of cells that were surface antigen-positive regardless of the strain of the virus used. Implications of the difference in the kinetics of virus production and of the development of surface antigens between low and high virulence strains are discussed.     

Rabies Antibody Determination by Immunofluorescence in Tissue Culture

A tissue culture (TC)-fluorescent antibody (FA) technique for the measurement of rabies-neutralizing antibody was found to be reliable and comparable to the standard mouse serum neutralization test. This test was performed with BHK-21 cells infected with the ERA vaccine virus strain on Lab-Tek TC chamber slides. A Flury high egg passage (HEP) rabies virus strain grown on continuous line of African green monkey (Vero) and on BHK-21 cells was investigated to determine its utilization in a TC-FA neutralization procedure. Although both the HEP and ERA viruses infected Vero and BHK-21 cells, the amount of fluorescent antigen observed was most consistent with ERA virus and BHK-21 cells.     

Cytotoxicity reactions against target cells infected with rabies virus

Some aspects of the cytotoxicity reactions were studied in the rabies system. The antibodydependent complement cytotoxicity (ADC), the cellular cytotoxicity (CC), and the antibody-dependent cellular cytotoxicity (ADCC) are shown, being the cytotoxic effect as evidenced by the ⁵¹Cr released from the cells infected with the Pasteur strain of rabies virus. Some parameters such as time of cellular infection, the amount of infected cells, the concentration of complement, and the incubation time of the ADC reaction, which help to increase the performance of this reaction, are discussed. The detection and the level of the cellular response against the Pasteur strain of rabies virus in immunized mice is shown. Evidence is presented that in the ADCC test, specific human antibodies and non-immune human lymphoid cells are able to mediate in vitro lysis of cells infected with rabies virus. A comparison of the ADCC test with serum neutralization and immunoenzymatic tests is shown.     

Quantitation of flaviviruses by fluorescent focus assay

An indirect immunofluorescence assay for quantitation of flaviviruses was developed as an alternative to the standard plaque assay. The assay was validated with West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and Dengue virus (DENV) types 1-4. Vero cells were plated in 8-well chamber slides, and infected with 10-fold serial dilutions of virus. About 1-3 days after infection, cells were fixed, incubated with specific monoclonal antibody, and stained with a secondary antibody labeled with a fluorescent tag. Fluorescent foci of infection were observed and counted using a fluorescence microscope, and viral titers were calculated as fluorescent focus units (FFU) per ml. The optimal time for performing the fluorescent focus assay (FFA) on Vero cells was 24 h for WNV, and 48 h for SLEV and the four DENV serotypes. In contrast, the time required to complete a standard Vero cell plaque assay for these viruses range from 3 days for WNV to 11 days for DENV-1. Thus, the FFA method of virus titration is useful for viruses whose plaques develop slowly. In addition, these viruses can be quantitated by FFA on a mosquito cell line (C6/36), which does not support plaque formation. The FFA for flaviviruses was validated for accuracy, precision, specificity, and robustness of the assay.     

Role of beta-chemokines in HIV-1 infection of dendritic cells maturing from CD34+ stem cells.

OBJECTIVES: To study the susceptibility to infection by different strains of HIV-1 viruses and the roles of chemokines (macrophage inflammatory protein-1alpha [MIP-1alpha], MIP-1beta, and regulated-on-activation-T-expressed-and-secreted [RANTES]) in CD34+ stem cells maturing into dendritic cells (DC). DESIGN: It has been controversial whether CD34+ stem cells are susceptible to HIV-1 infection and whether high levels of beta-chemokines are beneficial for suppressing HIV-1 infection during DC maturation. These questions were addressed using different strains of HIV-1 and CD34+ stem cells taken from cord blood and cultured with granulocyte-macrophage colony stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha) to generate mature DC. METHODS: CD34+ stem cells were exposed with M-tropic virus Ba-L or T-tropic viruses IIIB or Rut at day 1. Beta-chemokines were added to some cells before the virus and kept throughout the culture. Virus replication was measured throughout the maturation of these cells into CD1a+ DC and CD1a- CD14+ cells using enzyme-linked immunosorbent assay (ELISA) for p24, nested polymerase chain reaction (PCR) for env and intracellular p24 detection by flow cytometry. RESULTS: First, CD34+ stem cells acquired or were infected by live virus because maturing cells showed infection by both M- and T-tropic viruses. Second, the viruses replicated actively during the maturation of CD34+ stem cells toward CD1a+ DC and CD1a- CD14+ cells. Third, beta-chemokines suppressed infection by M-tropic virus Ba-L. And finally, beta-chemokines enhanced infection by T-tropic viruses IIIB and Rut. CONCLUSIONS: In addition to the initial anti-M-tropic virus effect by beta-chemokines, selective pressure on viruses may also result because of an increase in susceptibility to T-tropic virus. Caution should be taken when evaluating the effect of beta-chemokine receptor agonists in AIDS therapy.     

Development of a flow cytometry based method for rapid and sensitive detection of a novel marine fish iridovirus in cell culture

A sensitive and accurate flow cytometry (FCM) based method has been developed to detect and quantitate a novel marine fish iridovirus (Singapore grouper iridovirus, SGIV) after amplification in cell cultures. Confluent grouper cell (GP) monolayers were infected with SGIV. When advanced cytopathic effect (CPE) appeared, the cell cultures were fixed and permeabilized, and then reacted with monoclonal antibodies specific against SGIV, followed by a second antibody conjugated with FITC (anti-mouse IgG-FITC). A Coulter EPICS Elite ESP flow cytometer was used to directly detect and analyze the percentage of virus-infected cells. Three fixation and permeabilization methods were evaluated. The kinetics of the virus infection process was determined. The FCM procedure enables large amounts of cells to be screened rapidly for infectivity, and it can also detect low levels of virus infection. As early as 8 h after inoculation with the virus, 0.34% of infected cells were detected in cell culture. The maximum level of infection was obtained at 72 h. The efficiency and reliability of the FCM procedure were compared with those of the standard methods of immunofluorescence microscopy and PCR.     

Isolation of a phylogenetically distinct rabies virus from a tufted capuchin monkey (Cebus apella) in Brazil

A rabies virus isolate (BRmk1358 strain) was discovered from a rabid tufted capuchin monkey in Brazil. The present study determined the nucleotide sequence of the BRmk1358 strain and compared with the rabies viruses isolated from marmosets and other animals in the Americas. Phylogenetic analyses showed that the BRmk1358 strain formed a lineage distant from that of marmoset rabies virus within the Chiroptera-related rabies virus cluster. This result suggests that the source of rabies infection in the tufted capuchin monkey may have been bat, and that they have a risk to act as rabies reservoir in Brazil.     

Rapid cytopathic effect with rabies virus in fused hamster embryo cells

Summary A line of hamster embryo cells fused with inactivated Sendai virus was infected with a strain of fixed rabies virus. The growth of rabies virus in such a system produced a characteristic cytopathic effect.Work done while the senior author was on leave from the Panamerican Zoonosis Center, Casilla de Correos 23, Ramos Mejía, Buenos Aires, Argentina.