An immunofluorescence test for detection of serum antibody to rodent coronaviruses

An indirect immunofluorescence test, using cultured cells infected with two strains of mouse hepatitis virus, was developed for detection of antibody to rodent coronaviruses. The immunofluorescence test detected serum antibody to mouse hepatitis virus and rat sialodacryoadenitis virus earlier than the neutralization test. In the case of mouse hepatitis virus, the results of the immunofluorescence test closely paralleled those obtained with a commercially available enzyme-linked immunosorbent assay.     

Immunofluorescence for detection of viral antigen in mice infected with Sendai virus

Sendai virus infection transmitted by contact from cagemates was followed by virus titration and immunofluorescence. The virus grew in the respiratory tract and caused macroscopic lesions in all contact mice. The virus grew to a higher titer in the lung than in the trachea. Tracheal smears, however, were found to be the most suitable for the diagnosis of Sendai virus infection by immunofluorescence, since they contained a large number of cells with intense fluorescence. Diagnosis of Sendai virus infection was made by immunofluorescence within a few hours after autopsy made at early stages of infection.     

Identification of a herpesvirus isolated from cytomegalovirus-transformed human cells.

Human cells transformed by cytomegalovirus and transplanted to athymic nude mice yielded a cytopathic virus, Hershey Medical Center virus, following prolonged in vitro passage of the tumor cells. The virus is a double-enveloped herpesvirus, is sensitive to ether, and is inhibited by iododeoxyuridine. No significant antigenic relationship to herpes simplex virus was detected using herpes simplex virus-immune sera in neutralization and immunofluorescence tests, but indirect immunofluorescence tests revealed cytomegalovirus-related antigenicity. Further immunological tests revealed that Hershey Medical Center virus is antigenically indistinguishable from infectious bovine rhinotracheitis virus. Thus, it appears that Hershey Medical Center virus is infectious bovine rhinotracheitis virus, which presumably appeared in the cell culture as a contaminant from fetal calf serum.     

Immunofluorescence on avian sarcoma virus-transformed cells: localization of the src gene product.

The localization of the avian sarcoma virus src gene product (termed p60src) was examined by indirect immunofluorescence in cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup D (SR-RSV-D). Antiserum to p60src was obtained from rabbits bearing SR-RSV-D-induced tumors, and immunofluorescence was performed on chicken embryo fibroblasts (CEF) transformed with SR-RSV-D, as well as normal rat kidney (NRK) cells transformed by the same virus (termed SR-RK cells). Both acetone and formaldehyde fixation were used for the immunofluorescence tests. The specificity of the anti-tumor serum was first demonstrated in both cell systems by gel electrophoresis of immunoprecipitates prepared from 35S--methionine-labeled cells. Anti-tumor serum precipitated p60src from SR-RSV-D-transformed CEF but not from CEF infected with a transformation-defective mutant of SR-RSV-D. All viral structural proteins and precursors contained in these immunoprecipitates could be eliminated by competition with unlabeled virus. Similar experiments on SR-RK cells indicated that no viral proteins other than p60src were expressed in these cells, and this observation was supported by immunofluorescence tests using antiserum to whole virus. For immunofluorescence localization of p60src, reactions with viral structural proteins were blocked with unlabeled virus. This presaturation step, obligatory for p60src detection in the SR-RSV-D-transformed CEF, was unnecessary when antitumor serum was tested on SR-RK cells, since p60src was the only viral protein detectable in these cells. With acetone-fixed cells, p60src-specific immunofluorescence revealed a characteristic fluorescence pattern which was similar in both cell systems. The principal pattern was diffuse and situated in the cytoplasm. A clear nuclear fluorescence was never observed. Immunofluorescence on formaldehyde-fixed cells also indicated the cytoplasmic location of p60src and revealed a specific subcytoplasmic concentration of the fluorescence. With both fixation methods, an additional fluorescence pattern was seen between cells in contact, and was also found in both SR-RK cells and SR-RSV-D-transformed CEF. Immunofluorescence on viable cells suggested that p60src was not on the surface of these transformed cells. The fluorescence patterns were specific for avian sarcoma virus-transformed cells and were not found in uninfected cells, cells infected with a transformation-defective mutant of SR-RSV-D or cells transformed by an antigenically unrelated murine sarcoma virus. Furthermore, anti-tumor serum did not contain antibodies to proteins of the microtubules or intermediate filaments.     

Sea bream Sparus aurata, an asymptomatic contagious fish host for nodavirus.

During an epidemiological survey of viral encephalopathy and retinopathy (VER) in diseased sea bass Dicentrarchus labrax, a nodavirus isolate was recovered from net pen-reared sea bream Sparus aurata harboured in the same farming premises. After the virus was isolated and identified by immunofluorescence on SSN-1 cells, sequence analysis with a PCR product from the T4 region of the capsid protein gene indicated that the virus shared 100% identity with a pathogenic virus strain isolated from sea bass. Infection trials demonstrated the pathogenicity of the sea bream virus isolate for juvenile sea bass whereas sea bream infected with the same virus isolate remained asymptomatic even following intramuscular injection of virus. Nevertheless, the sea bream appeared to be a potential carrier of nodavirus, as juvenile sea bass became infected when maintained in a tank containing experimentally contaminated sea bream.     

Production and characterisation of monoclonal antibodies to salmon pancreas disease virus.

Six mouse monoclonal antibodies (mAbs) specific to salmon pancreas disease virus (SPDV) were produced following immunisation with purified virus preparations. These mAbs and 2 mAbs resulting from an earlier investigation were characterised. None of the mAbs possessed virus neutralising activity but all reacted with 4 geographically different SPDV isolates as determined by indirect immunofluorescence. Three mAbs produced positive immunostaining with Western blots of SPDV proteins. The 4H1 mAb reacted with the 53 kDa structural E1 glycoprotein present in virus-infected cells and in gradient-purified virus. Two mAbs, 5A5 and 7B2, which exhibited unusual immunofluorescence staining of the nuclear margin, reacted with a 35 kDa protein, which is present in gradient-purified virus and which is considered to be the capsid protein. A sandwich ELISA, based on the use of mAb 2D9 for capture and a biotinylated conjugate of mAb 7A2 for detection, detected SPDV antigen in virus-infected Chinook salmon embryo-214 cells and gradient-purified virus. These mAbs may be of use in pathogenesis studies and in diagnostic test development.     

Murine retroviruses use at least six different receptors for entry into Mus dunni cells.

Murine retroviruses have been divided into six interference groups that use different receptors for cell entry: the ecotropic, xenotropic, polytropic, amphotropic, 10A1, and Mus dunni endogenous virus groups. Some interference is observed between xenotropic and polytropic viruses and between amphotropic and 10A1 viruses, indicating some overlap in receptor specificity between these groups, but otherwise these interference groups appear completely independent. In contrast, one study found interference among many of these groups when Mus dunni wild mouse cells were examined with an immunofluorescence assay to detect infection by the challenge virus. Here we have used a more direct assay for cell entry by using pseudotyped retroviral vectors to measure interference in M. dunni cells, and we find no evidence for extensive interference between members of different murine retrovirus groups. Indeed, our results in M. dunni cells are consistent with interference results observed in other cell types and indicate that the anomalous interference results previously observed in M. dunni cells with the immunofluorescence assay were most likely due to factors other than those that affect receptor-mediated virus entry. In summary, our results show that murine retroviruses use at least six different receptors for entry into M. dunni cells.     

Human lymphoblastoid cells as hosts for parvoviruses H-1 and rat virus.

A human T-cell line (Molt-4) was shown by viral hemagglutination and infectivity assays to support the replication of rat virus (RV) and H-1 virus. In addition, H-1 virus, but not RV, multiplied in two human B-cell lines, AV-1 and NC-37. The ability to bind radioactively labeled RV was demonstrated for each of the cell lines, but viral adsorption occurred to a greater degree with Molt-4 cells than with either AV-1 or NC-37 cells. After challenge with RV, virus-specific antigens were detected in cells of the B-cell lines by the indirect immunofluorescence technique. Infection of AV-1 or NC-37 cells by RV apparently results in an abortive cycle of virus replication. Differences among the three cell lines that might influence with H-1 virus or RV are discussed.     

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.     

Monoclonal antibodies specific for African swine fever virus proteins.

We have obtained 60 stable hybridomas which produced immunoglobulins that recognized 12 proteins from African swine fever virus particles and African swine fever virus-infected cells. Most of the monoclonal antibodies were specific for the three major structural proteins p150, p72, and p12. The specificity of some monoclonal antibodies for the structural proteins p150 and p37 and the nonstructural proteins p220 and p60 indicated that proteins p150 and p220 are antigenically related to proteins p37 and p60. The association of some viral antigens to specific subcellular components was determined by immunofluorescence and analysis of the binding of monoclonal antibodies to infected cells. A host protein (p24) seemed to be associated with the virus particles.     

A touchdown nucleic acid amplification protocol as an alternative to culture backup for immunofluorescence in the routine diagnosis of acute viral respiratory tract infections

Background

Immunofluorescence and virus culture are the main methods used to diagnose acute respiratory virus infections. Diagnosing these infections using nucleic acid amplification presents technical challenges, one of which is facilitating the different optimal annealing temperatures needed for each virus. To overcome this problem we developed a diagnostic molecular strip which combined a generic nested touchdown protocol with in-house primer master-mixes that could recognise 12 common respiratory viruses.

Results

Over an 18 month period a total of 222 specimens were tested by both immunofluorescence and the molecular strip. The specimens came from 103 males (median age 3.5 y), 80 females (median age 9 y) and 5 quality assurance scheme specimens. Viruses were recovered from a number of specimen types including broncho-alveolar lavage, nasopharyngeal secretions, sputa, post-mortem lung tissue and combined throat and nasal swabs. Viral detection by IF was poor in sputa and respiratory swabs. A total of 99 viruses were detected in the study from 79 patients and 4 quality control specimens: 31 by immunofluorescence and 99 using the molecular strip. The strip consistently out-performed immunofluorescence with no loss of diagnostic specificity.

Conclusions

The touchdown protocol with pre-dispensed primer master-mixes was suitable for replacing virus culture for the diagnosis of respiratory viruses which were negative by immunofluorescence. Results by immunofluorescence were available after an average of 4–12 hours while molecular strip results were available within 24 hours, considerably faster than viral culture. The combined strip and touchdown protocol proved to be a convenient and reliable method of testing for multiple viruses in a routine setting.     

Cultivation of Tuleniy virus (strain Murman) in vitro.

In vitro culturability of Murman strain of Tuleniy flavivirus isolated recently in the northern regions of the USSR was studied. Stable PS pig kidney line was found suitable as a primary sensitive cell substrate for the isolation, proliferation and serial propagation of the virus. The pronounced pathogenicity of the virus to PS cells permits the testing of its infective activity comparable with i.c. titrations on mice, VNT in vitro and the plaquing technique. PS line is suitable for the demonstration and identification of the virus antigen and/or for the study of reproduction of the virus on cellular level using the technique of immunofluorescence.     

Respiratory syncytial virus ts mutants and nuclear immunofluorescence.

A replicated sector-plating procedure was used to isolate 35 induced temperature-sensitive (ts) mutants and one spontaneous ts mutant from a wild-type stock of respiratory syncytial (RS) virus cloned from recent clinical material. Seven of these mutants were ts for plaque formation at 37 degrees C as well as at the restrictive temperature of 39 degrees C. The wild-type strain did not differ markedly from standard laboratory strains of RS virus. It was dependent on exogenous arginine (84 mug/ml) for optimal growth, and was not significantly inhibited by mitomycin C (10 mug/ml). It was sensitive to actinomycin D (2.5 mug/ml) during the early part of the growth phase. A characteristic focal cytopathic effect was obtained in BS-C-1 cells. Staining of infected monolayers by an indirect immunofluorescence procedure revealed a profusion of filamentous processes extending from the plasma membrane, and a similar modification of the surface of infected cells could be visualized by scanning electron microscopy. Filament production was inhibited when certain ts mutants were incubated at 39 degrees C, confirming the virus-specific nature of the phenomenon. Thirty-four of the mutants were classified into three groups by immunofluorescence. Complementation was observed in mixed infection with a single mutant from each group. Nuclear, as well as cytoplasmic, immunofluorescence was detected in RS virus-infected cells using a high-titer bovine anti-bovine RS virus serum. Visualization of nuclear antigen was dependent on the inhibition of cytoplasmic fluorescence obtained when ts mutants in groups I and III were incubated at restrictive temperature.     

Ribavirin cures cells of a persistent infection with foot-and-mouth disease virus in vitro.

Ribavirin (1-beta-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide) eliminates foot-and-mouth disease virus from persistently infected cell cultures. The latter are 10-fold more sensitive to ribavirin than lytically infected cells. In treated cells no viral RNA or proteins could be detected by dot-blot hybridization to cDNA probes, virus and RNA infectivity assays, or immunofluorescence. A potential application of the drug for the treatment of animals carrying the virus is suggested.     

Localization of the simian virus 40 small t antigen in the nucleus and cytoplasm of monkey and mouse cells.

Monkey and mouse cells producing simian virus 40 small t antigen in the absence of clearly detectable intact or truncated large T antigens were subjected to indirect immunofluorescence and biochemical cell compartment analyses. Results revealed specific immunofluorescence and small t polypeptide in both the nucleus and cytoplasm of these cells.     

Expression of cloned herpesvirus genes. I. Detection of nuclear antigens from herpes simplex virus type 2 inverted repeat regions in transfected mouse cells.

Three different recombinant plasmids containing the entire 15-kilobase L and S inverted repeat sequence of herpes simplex virus type 2 DNA have been introduced into cultured Ltk- or BSC cells by both the calcium and DEAE-dextran transfection procedures. In each case, after 24 h approximately 1% of the cells gave strongly positive nuclear staining when assayed by immunofluorescence with hyperimmune antisera made against early and immediate-early infected-cell polypeptides. The nuclear fluorescence pattern and intensity mimicked that observed within 2 to 3 h after infection of Ltk- cells with either herpes simplex virus type 1 or type 2 wild-type virus. Herpes simplex virus type 1 (KOStsB2)-infected Ltk- cells under nonpermissive conditions did not express these antigens in the nucleus. Therefore, we conclude that either one or both of the 185,000- and 110,000-molecular-weight immediate early proteins, or some other as yet unknown gene product encoded entirely within the inverted repeats, can be transiently expressed in large amounts in transfected cells in the absence of other viral genes or accompanying virion components. Permanent mouse cell lines derived from transfection with these plasmids by using the thymidine kinase coselection procedure did not express sufficient nuclear antigen to be detectable by immunofluorescence.     

Establishment of a persistent measles virus infection in HEp-2 cells.

A productive measles virus persistent infection has been established in HEp-2 cells. Greater than 90% of the persistently infected HEp-2 cells (H2MV) exhibited measles specific immunofluorescence and haemadsorption. Although most of the H2MV cells contained measles specific antigens, only a small percentage (less than 1%) actually produced infectious measles virus as determined by infectious centre assays. The measles virus produced by H2MV cells exhibited properties different from the initiating parent Edmonston strain virus, being reduced in virulence and also temperature sensitive for replication at 39 degrees C. The role of these altered virus properties in the establishment of persistence is considered.     

Propagation of human parvovirus B19 in primary culture of erythroid lineage cells derived from fetal liver.

Erythroid lineage cells derived from fetal liver were demonstrated to be target cells for human parvovirus B19 infection. B19 virus antigen-positive serum was inoculated into primary cultures containing erythroid lineage cells enriched from fetal liver. The B19 virus antigen was detected on about 5% of cells in the culture by immunofluorescence staining, and the stained cells were identified as erythroid lineage cells by double staining with anti-B19 virus-positive serum and anti-erythroid lineage monoclonal antibody. The immunofluorescence staining study also revealed that the B19 virus antigen localized in the nucleus and the periphery of cytoplasm. We also detected B19 virus DNA, which was generated by replication in the infected cells, not only in the cells but also in the culture supernatants, in which the amount of B19 DNA increased depending on the period of culture, indicating that the cells infected with B19 virus produced B19 virus and released it into the medium. The ability of B19 virus released into the medium to infect fetal erythroid lineage cells was demonstrated quantitatively. Because of the absence of any cytopathic effect of B19 virus during culture periods of at least 15 days, this culture system should be useful in the study of B19 virus replication and in vitro generation of B19 virus. In addition, the present study may contribute to a better understanding of the pathogenesis of hydrops fetalis, which is probably associated with B19 virus infection during pregnancy.     

Role of extracellular virus on the maintenance of the persistent infection induced in Aedes albopictus (mosquito) cells by Sindbis virus.

Sindbis virus infection of cultured mosquito cells was found to have no effect on the growth of these cells; instead, a persistent infection of the culture followed an initial acute phase of rapid virus synthesis. Nearly all of the cells in the acute stage of infection were found to actively release virus in an infectious-center assay and to contain significant amounts of virus antigen as determined by immunofluorescence. Cells in the persistent phase of infection released few virions into the media, and only a small percentage of the cultured cells could be demonstrated to contain detectable amounts of virus antigen by immunofluorescence assay. In spite of the fact that nearly 100% of the cells in the persistent phase of infection were found to be virus negative by the two assays described above, the culture as a whole totally excluded the expression of superinfecting virus, as did cells in the acute phase, suggesting that most of the persistently infected cells did, indeed, contain virus information. Prevention of reinfection of the cells in the persistent phase by eliminating extracellular virus resulted in a curing of the culture such that it responded to infection by added virus much as would an uninfected culture.