Evaluation of a Japanese quail fibrosarcoma cell line (QT-35) for use in the propagation and detection of metapneumovirus

A Japanese quail fibrosarcoma cell line (QT-35) was evaluated and compared to Vero cells for its utility in metapneumovirus propagation, titration and serological detection by indirect immunofluorescence staining. Cell characteristics such as growth kinetics at different passage levels and seeding density in 96-well plates using various media formulations were studied in order to determine suitable assay parameters. Specifically, QT-35 cells supported the replication of a subgroup A metapneumovirus, strain 14/1, when maintained in DMEM containing a high level of glucose (4500 mg/l) and 2% gamma-irradiated fetal bovine serum (gamma-FBS). There appeared to be a decreased ability of metapneumovirus produced in chicken embryo fibroblast (CEF) cells to replicate to high titers in QT-35 cells, however, this apparent restriction was overcome after the second passage resulting in high titered stock. Metapneumovirus produced in Vero cells and propagated in QT-35 cells produced high titered stock after the first passage. Viral titers determined in Vero and QT-35 cells were comparable, when the latter cell line was used at passage levels < or = 20 and seeded between 5.0 x 10(4) and 1.0 x 10(5) cells/0.33 cm(2) in hgDMEM containing 10% gamma-FBS, with a reduction to 2% gamma-FBS when the virus was applied to the cell monolayers 24 h post-seeding. After infection with metapneumovirus, QT-35 cells exhibited syncytia, similar to those in metapneumovirus-infected Vero cells, which were readily detected by indirect immunofluorescent (IF) staining.     

Plaque assay of variola virus in a cynomolgus monkey kidney cell line

Summary Variola virus produced plaques in a cynomolgus monkey kidney cell line, JINET, under agar overlay medium. The plaque morphology of variola virus in these cells was clearly distinct from that of other members of the variola-vaccinia subgroup of poxvirus and the plaque morphology marker of variola virus may be used for the identification of variola virus. The plaque number of variola virus was enhanced by the addition of DEAE-dextran and MgCl₂ to agar overlay medium at concentrations of 100 to 200 g/ml and 15 to 45 mm, respectively. The sensitivity of the plaque method in the presence of 100 g/ml of DEAE-dextran and 25 mm MgCl₂ in the overlay medium was equal to that of the usual CAM method.     

Comparison of avian cell substrates for propagating subtype C avian metapneumovirus

Avian metapneumovirus (AMPV) is a respiratory viral pathogen that causes turkey rhinotracheitis (TRT) or swollen head syndrome (SHS) in chickens. AMPV was first isolated in South Africa during the early 1970s and has subsequently spread worldwide during the 1980s to include Europe, Asia, and South America. In 1996, a genetically distinct AMPV subgroup C was isolated in the US following an outbreak of TRT. Vero cells are currently the best available substrate for AMPV propagation but are of non-avian origin. A number of different avian cell substrates have been compared to determine which is the most suitable for the propagation of AMPV to sufficiently high titers. Of the cell substrates tested, primary turkey turbinate and kidney and chicken kidney cells produced titers equal to or greater than Vero cells. Turkey turbinate and kidney epithelial cells that were life-span extended by the ectopic expression of human telomerase catalytic subunit (HTERT) initially displayed AMPV titers comparable to Vero cell controls, but declined in virus production with increased passage in culture. Interestingly, plaques emanating from Vero propagated virus were relatively small and dispersed, when analyzed by immunofluorescent assays (IFA), while both turkey turbinate and kidney cell propagated AMPV produced larger plaques. Even with these differences, there were no changes in the predicted amino acid sequences of the nucleocapsid (N) and phosphoprotein (P) genes of AMPV propagated in either turkey turbinate or Vero host cells. However, the fusion (F) gene showed 11 amino acid differences (98.7% identity) between the two host cell types. These results suggest that AMPV propagated in homologous avian cellular substrates may produce more infectious virus with possibly more effective fusion activity, compared to Vero cell propagation.     

Plaque assay for some strains of avian leukosis virus

Some strains of avian leukosis virus were found to produce plaques at 41 ° in cultures of chick embryo cells which had been fully infected with a temperature-sensitive mutant of Rous sarcoma virus (RSV) which fails to induce morphological transformation at the elevated temperature. The leukosis viruses caused no discernible lesion in normal chick embryo cultures. Macroscopically visible plaques appeared within 4 days after infection in the mutant-infected cultures, and by 7 to 10 days the plaques became nonstainable by neutral red. The titer of plaque-forming units of these virus preparations was comparable to the titer of virus-infectivity estimated by the interference assay. Avian leukosis viruses of subgroups B and D thus far tested produced plaques under these conditions, while viruses of subgroups A, C, and E did not. Some preparations of avian sarcoma virus, subgroups B and D, also induced plaques, but whether or not the sarcoma virus itself can induce plaques has not been clearly determined.     

Evaluation of chicken-origin (DF-1) and quail-origin (QT-6) fibroblast cell lines for replication of avian influenza viruses

Avian influenza viruses (AIVs) are isolated routinely and propagated in specific pathogen free embryonated chicken eggs (ECE) and mammalian origin Madin-Darby Canine Kidney (MDCK) cell line. Continuous avian cell lines offer advantages for propagation of AIVs over MDCK cells because they maintain species specificity, and lower recurring costs compared to ECE. In this study, the characteristics of two avian fibroblast cell lines were evaluated, DF-1 (chicken-origin) and QT-6 (quail-origin), and their ability to support the growth of AIVs (n=19) belonging to nine different hemagglutinin subtypes from a variety of avian species. The replication efficiency of the AIVs in QT-6 and DF-1 cells was comparable to those in primary chicken embryo fibroblast (CEF) and MDCK cells. Receptor distribution analysis demonstrated high prevalence of SA alpha2,3-gal linked receptors in QT-6 and DF-1 cells which support a high growth of AIVs in these cell lines. Furthermore, the QT-6 and DF-1 cells supported high plaque-forming ability of representative highly pathogenic Eurasian H5N1 and H7N1 subtype AIVs. These two avian cell lines, especially QT-6 cells, also showed high transfection efficiency and could be useful for reverse genetics based rescue of AIVs. This study indicates that the DF-1 and QT-6 cell lines may be useful as a substitute for primary CEF and MDCK cells for AIV research in the areas of in vitro host range, molecular pathobiology and molecular genetics.     

Sequence comparison of avian interferon regulatory factors and identification of the avian CEC-32 cell as a quail cell line.

Interferon (IFN) regulatory factor-1 (IRF-1) is a well-characterized member of the IRF family. Previously, we have cloned cDNA of several members of the chicken IRF (ChIRF) family and studied the function of ChIRF-1 in the avian cell line CEC-32. The IRF-1 proteins from primary chicken embryo fibroblasts (CEF) and CEC-32 cells differed in their electrophoretic mobility. To characterize the different forms of IRF-1 in avian cells, we compared the sequences of IRF-1 cDNA from CEC-32 cells, primary CEF, and quail fibroblasts (QEF). The deduced amino acid sequences of IRF-1 cDNA from chicken and quail show high similarity. Comparison of genomic sequences of IRF-1 and IFN consensus sequence binding protein (ICSBP) also confirm the relatedness of the members of the IRF family in quail and chicken. Based on these data, it is concluded that the avian fibroblast cell line CEC-32 is derived from quail. This conclusion is further supported by deoxynucleotide sequence comparison of a DNA fragment in an avian MHC class II gene and by fluorescence in situ hybridization (FISH) using the vertebrate telomeric (TTAGGG) repeat. Chromosome morphology and the lack of interstitial hybridization signals in macrochromosomes suggest that the CEC-32 cell line has probably been derived from Japanese quail.     

Development of a nucleoprotein-based enzyme-linked immunosorbent assay using a synthetic peptide antigen for detection of avian metapneumovirus antibodies in Turkey sera

Avian metapneumoviruses (aMPV) cause an upper respiratory tract disease with low mortality but high morbidity, primarily in commercial turkeys, that can be exacerbated by secondary infections. There are three types of aMPV, of which type C is found only in the United States. The aMPV nucleoprotein (N) amino acid sequences of serotypes A, B, and C were aligned for comparative analysis. On the basis of the predicted antigenicity of consensus sequences, five aMPV-specific N peptides were synthesized for development of a peptide antigen enzyme-linked immunosorbent assay (aMPV N peptide-based ELISA) to detect aMPV-specific antibodies among turkeys. Sera from naturally and experimentally infected turkeys were used to demonstrate the presence of antibodies reactive to the chemically synthesized aMPV N peptides. Subsequently, aMPV N peptide 1, which had the sequence 10-DLSYKHAILKESQYTIKRDV-29, with variations at only three amino acids among aMPV serotypes, was evaluated as a universal aMPV ELISA antigen. Data obtained with the peptide-based ELISA correlated positively with total aMPV viral antigen-based ELISAs, and the peptide ELISA provided higher optical density readings. The results indicated that aMPV N peptide 1 can be used as a universal ELISA antigen to detect antibodies for all aMPV serotypes.     

Plaque assay and improved yield of human coronaviruses in a human rhabdomyosarcoma cell line.

Propagation and plaque assay of human coronavirus prototypes were studied in two human cell lines: a diploid fetal tonsil (FT) and a heteroploid rhabdomyosarcoma (RD) cell lines. Plaques, observed within 2 to 3 days on FT cell monolayers with both 229E and OC43 viruses, appeared as colorless areas after staining with neutral red or crystal violet, whereas neutral red staining was required for visualization of plaques on RD cells. The plating efficiencies were approximately equal between the two cell lines, but virus assay by plaque formation was 15- to 30-fold more efficient than tube dilution assay with 50% endpoints. The discrepancy between 50% endpoint and plaque-forming unit values was striking and appeared to result from the fact that killing of cells (particularly RD cells) by coronaviruses was not accompanied by visible changes in the cells but killing was detected by the failure of infected cells to stain with a vital dye. The latent phase in one-step growth curves was 5 to 6 h for both viruses in either cell line, but the maximum yield of intracellular virus was reached in 18 to 20 h for FT cells and 24 to 28 h for RD cells. Virus release also differed between the two cell lines: in FT cells, the maximum yield of extracellular virus was reached 2 to 3 h later than that of intracellular virus, whereas in RD cells, the difference was 5 h for 229E virus and 10 h for OC43 virus. Although both cell lines appear equally useful for plaque assay, RD cells would be preferred for mass virus propagation because yields (5 X 10(8) plaque-forming units per ml) were 10-fold higher than in FT cells, a finding true for both virus prototypes.     

Permissibility of different cell types for the growth of avian metapneumovirus

Vero cells are commonly used for the growth of avian metapneumovirus subtype C (aMPV-C). This study was conducted to evaluate 17 different cell types for the growth of a Minnesota strain of aMPV-C. The virus was inoculated into these cell types and virus growth was monitored by the development of cytopathic effects (cpe) and immunofluorescence. Virus growth was obtained in 6 of 17 cell types tested with the highest virus titers observed in BGM and DF-1 cells. The flow cytometric analysis of cells at 72 h post inoculation found the highest number of infected cells in BGM cells followed by QT-35 cells. At 48 h post inoculation, DF-1 and BGM cells showed the highest number of infected cells. These results suggest that BGM, QT-35, and DF-1 cells can be used for high titer propagation of aMPV-C.     

Development and evaluation of a blocking enzyme-linked immunosorbent assay for detection of avian metapneumovirus type C-specific antibodies in multiple domestic avian species

The first cases of infection caused by avian metapneumoviruses (aMPVs) were described in turkeys with respiratory disease in South Africa during 1978. The causative agent was isolated and identified as a pneumovirus in 1986. aMPVs have been detected in domestic nonpoultry species in Europe, but tests for the detection of these viruses are not available in the United States. To begin to understand the potential role of domestic ducks and geese and wild waterfowl in the epidemiology of aMPV, we have developed and evaluated a blocking enzyme-linked immunosorbent assay (bELISA) for the detection of aMPV type C (aMPV-C)-specific antibodies. This assay method overcomes the species-specific platform of indirect ELISAs to allow detection of aMPV-C-specific antibodies from potentially any avian species. The bELISA was initially tested with experimental turkey serum samples, and the results were found to correlate with those of virus neutralization assays and indirect enzyme-linked immunosorbent assay (iELISA). One thousand serum samples from turkey flocks in Minnesota were evaluated by our bELISA, and the level of agreement of the results of the bELISA and those of the iELISA was 94.9%. In addition, we were able to show that the bELISA could detect aMPV-C-specific antibodies from experimentally infected ducks, indicating its usefulness for the screening of serum samples from multiple avian species. This is the first diagnostic assay for the detection of aMPV-C-specific antibodies from multiple avian species in the United States.     

Properties of rabies virus (MNIIVP-74 strain) adapted to Japanese quail embryo cell culture

Summary The Pasteur strain of fixed rabies virus was adapted to primary cell cultures of Japanese quail embryos and designated as MNIIVP-74. In the course of adaptation the virus pathogenicity for rabbits by the intracerebral route decreased considerably and the pathogenicity for rabbits and adult white mice by extraneural routes was completely lost. After inoculation of Japanese quail embryo cell cultures, a titer of the virus in the culture fluid at 4 days was 6.25–7.0 lg LD₅₀/ml (by the intracerebral inoculation of adult white mice). Viral antigen could be detected by immunofluorescence in the cytoplasm of approximately 60 per cent of the cells. Virus multiplication was accompanied by intensive interferon production. In cultures of BHK-21/13S cells the titer of the virus reached was 5.75 lg LD₅₀/ml at 24 hours and about 30 per cent of the cells were affected. The MNIIVP-74 virus showed a high immunogenic activity in rabbits, guinea pigs and mice.With 3 Figures