Feline Embryo Cells Research Articles

Molecular cloning and phylogenetic analysis of a cDNA encoding the cat ( Felis domesticus) Ig epsilon constant region

A feline splenic cDNA library was screened with a 32P-labelled cDNA probe encoding the canine IgE epsilon heavy chain subunit. A cDNA sequence of 1614 nucleotides encoding the complete feline IgE heavy chain, as well as a portion of a variable region, was identified. A search of the GenBank database revealed an identity of 82% at the nucleotide level and 76% at the amino acid level between the feline epsilon heavy chain sequence and the canine homologue. In a separate study, feline genomic DNA, isolated from whole feline embryo cells, was subjected to PCR amplification using primers based on known partial genomic DNA sequences for the feline Cε gene. Following removal of an intron from the 683 bp PCR product, the coding sequence yielded an ORF of 506 bp. The DNA sequence of this PCR clone differed by a single nucleotide from the cDNA clone. This difference is silent, and therefore the proteins encoded by the two sequences are identical over the regions cloned and sequenced. Phylogenetic analysis of the constant regions of nine immunoglobulin epsilon genes revealed that the feline cDNA is most similar to the canine homologue.

Restriction endonuclease analysis of DNA from isolates of feline herpesvirus type 1.

DNAs prepared from whole feline embryo cells infected with a standard laboratory strain of FHV-1, B927, and 50 wild type isolates were digested with a variety of restriction endonucleases. The resulting fragments were separated on agarose gels and Southern blotting performed. To visualize the fragments, B927 DNA was purified by pulsed field gel electrophoresis, labelled with alpha 32P and used as a hybridization probe. With most enzymes a large number of the isolates displayed altered mobilities of several fragments with some fuzzy band heterogeneity. A few isolates gave distinct cleavage patterns, in particular using the enzymes Bam HI, Cla I and Sac I. It is suggested that different strains of FHV-1 exist but that changes in the viral genome occur only sporadically and thus may not be readily detected.

Variable sensitivity of a feline embryo cell line and of three kitten kidney cell cultures to feline herpes- and caliciviruses

The number of plaques produced in a feline embryo (FEmb) cell line and in three independently derived kitten kidney (KK) cell cultures varied in a consistent and reproducible manner when each was inoculated with the same number of feline herpesvirus 1 (FHV1) plaque forming units (PFU); the three KK cells produced 2–9 times more plaques than FEmb cells. One of the three KK cells produced FHV1 plaques that were smaller in diameter than those FEmb cells. Each of the three KK cell cultures inoculated with the same number of FEmb cell PFU of a strain of feline calicivirus (FCV) produced different numbers of plaques; two of the three KK cell cultures produced 2–3 times more plaques than FEmb cells. The plaque diameter of FCV in the three KK cells was 30–50% smaller than the plaque diameter in FEmb cells.

Parvovirus (feline panleucopaenia virus) plaque formation.

A plaque assay was developed for feline parvovirus (FPV; feline panleucopaenia virus) in a feline embryo (FEmb) cell line. Higher numbers and larger diameter plaques were obtained with a) seeding rates of 0.7 X 10(5) and 1.5 X 10(5) cells cf. 3 X 10(5) and 6 X 10(5) cells/well of 35 mm diameter, b) synchronised cells infected at the G1-S interface cf. nonsynchronised cells and c) 5 to 6 days incubation post inoculation. The plaque assay was standardised by using serum deprivation for 24 hours to synchronize cells, a seeding rate of 1.5 X 10(5) cells/35 mm diameter well, inoculation of virus 16 hours post seeding followed by 5 days incubation. The standardised assay gave consistent, reproducible results. A dose-response curve using the assay showed a linear, 45 degrees slope, relationship between plaque forming units and virus dilution which further verified the sensitivity and reliability of the assay. Plaques produced by "wild" type and plaque purified virus were invariably non uniform in diameter; diameter of plaques in fact followed a normal frequency distribution under standard assay conditions.

Feline panleukopenia virus replicates in cells in which cellular DNA synthesis is blocked

The components of the cell cycle for a feline embryo cell line were defined. Thymidine (6mM)-supplemented medium reversibly arrested cells 1 h into the S phase of the cell cycle and was used in a double blocking procedure to synchronize cells to the early S phase. The kinetics of feline panleukopenia virus replication in synchronized cells was studied by using (i) inclusion body formation, (ii) a plaque assay for cell-associated and cell-free virus under one-step growth conditions, (iii) an enzyme immunoassay for viral protein, (iv) electron microscopy of infected cells, and (v) the detection and identification of viral replicative form DNA by restriction endonuclease analysis. Parallel studies by each of these procedures of the replication of feline panleukopenia virus in cells in which a 6 mM thymidine block was maintained indicated that parvovirus replicated with essentially similar kinetics in both unblocked, synchronized cells and in cells in which the block was maintained. Accordingly, a 6 mM thymidine-supplemented medium, although it effectively blocks cellular DNA synthesis, does not block the replication of parvovirus.

Characterization of clones of tumorigenic feline cells transformed by a chemical carcinogen.

Tumorigenic clonal lines derived from soft agar colonies induced by DMBA-transformed feline embryo cells were isolated and characterized. The morphologically altered clonal cells formed large aggregates, growing in this aggregate form when suspended in liquid growth medium above an agar base and forming colonies in soft agar with high efficiency. When inoculated into athymic nude mice, chemically altered clonal cells produced progressively growing sarcomas. Cells established from the tumors morphologically resembled the DMBA-transformed feline embryo cells and were characterized as cat cells by karyological analysis. The tumorigenic lines were negative for feline oncornavirus-associated cell membrane antigen (FOCMA), and for "gag-X" the transformation-related polyprotein which is encoded by the replication defective feline sarcoma virus.

Two populations of virus-specific particles released from feline calicivirus-infected cells

In harvests of a feline calicivirus (FCV) grown in feline embryo cells two populations of viral components were observed after centrifugation in sucrose and CsCl gradients. Particles in the first (PI) were mature virions since they had a buoyant density of 1.39 g · cm −3, a sedimentation coefficient of 170 S, were infectious and showed characteristic calicivirus morphology by electron microscopy. The second particle (P2), which in terms of protein was 10 times as abundant as P1, had a buoyant density of 1.31 g · cm −3, a sedimentation coefficient of 15 S, and contained little or no infectivity. Evidence that P2 was virus specific was that P2 as well as P1 contained a polypeptide with a molecular weight of 65,000 which is the weight of the viral capsid protein, and P1 and P2 shared an antigenic determinant which was responsible for inducing neutralizing antibodies. The 15 S component was not generated from the virion by conditions encountered during viral growth and purification. It is considered likely that the 15 S particle is a stable product of FCV infection and may be a subunit in the assembly of the viral capsid.

EQUINE HERPESVIRUSES: ON THE DIFFERENTIATION OF RESPIRATORY FROM FOETAL STRAINS OF TYPE 1

SUMMARY Confirmation of the occurrence of equine herpesvirus type 1 (EHV1) abortion in both epizootic and sporadic form epizootic in Australia for the first time in 1977 against a background in which a reasonably diligent search for such viruses during the preceeding 11 years failed to associate EHV1 with abortion, provided a special opportunity to compare the properties of the newly isolated foetal (F) strains with a collection of endemic respiratory (R) strains that had been recovered at fairly regular intervals since 1967. Using plaque size and host cell range all of 7 R strains tested were clearly distinguishable from 7 of 11 F isolates. The remaining 4 F strains had plaque diameters of R strains but 3 of the 4 viruses conformed in their host cell range with F strains. Only one F isolate (from Tasmania) had both plaque morphology and host cell range of R strain viruses.The mean diameter of plaques produced by R strains in equine foetal kidney (EFK) cells after 4 days under a methyl cellulose overlay was 1.52 mm (range 1.30–1.84 mm) while the mean diameter of small plaques produced by F strains was 0.82 mm (range 0.68–0.91 mm).In addition to EFK cells all R and F strains grew in an equine dermal (EDerm) cell line and all but two of 19 isolates grew in a pig kidney (PK) cell line. None of the low passage R strains grew in bovine embryo tracheal (EBTr) or feline embryo (FEmb) cells whereas all but one of 11 F isolates grew in EBTr cells. 8/11F isolates also grew in FEmb cell line.Growth of viruses at 33° and 40.5°cf. a usual growth temperature of 37° was of no detectable value in differentiating R and F strains of EHV1.In a limited geographic and time frame the criteria of plaque size in EFK cells and growth in EBTr cells unambiguously distinguished between R and F isolates and represent simple markers worthy of additional study.

Transformation of feline embryo cells in culture by a chemical carcinogen.

A feline embryo cell line was treated in vitro with various levels of 7,12-dimethylbenz[a]anthracene (DMBA) or dimethyl sulfoxide (control). Repeat treatment of DMBA only induced in vitro transformation of feline embryo cells following clonal growth and selection. The morphologically altered cells formed large cell aggregates and grew in this aggregate form when suspended in liquid growth medium above an agar base, formed colonies in soft agar, and grew to high saturation densities. However, no progressively growing tumors were produced when cells were inoculated into nude athymic mice. The transformed lines were negative for feline oncornavirus-associated cell membrane antigen (FOCMA).

Pneumoviruses: the cell surface of lytically and persistently infected cells.

Human embryonic lung (MRC-5), feline embryo (FEA), mink lung (Mv1Lu) and monkey kidney (BSC-1) cells infected by respiratory syncytial virus showed characteristic morphological changes when viewed by scanning electron microscopy. The surfaces of respiratory syncytial virus-infected cells developed a profusion of slender filaments after 48 h incubation at 31 degrees C. Similar changes in surface morphology were observed in BSC-1 cells infected by murine pneumonia virus. Filament production therefore appears to be a common property of pneumo-viruses. Filaments were not observed in cells infected with either syncytial and non-syncytial herpes simplex virus, the cytocidal vesicular stomatitis and Batai (Bunyaviridae) viruses, or the focus-inducing rabbit fibroma virus. Filament production was not observed in cells infected with ts mutants of respiratory syncytial (RS) virus during incubation at the restrictive temperature, or in a persistently infected culture of BSC-1 cells at 37 degrees C. The persistently infected cells (the RS ts 1/BSC-1 line) had some of the characteristics of cells transformed by oncogenic viruses, namely ability to overlap adjacent cells and agglutination by a low concentration of concanavalin A. The pseudo-transformed phenotype was temperature-dependent, however, and suppressed by raising the temperature of incubation to 39 degrees C. The presence of virus antigen at the cell surface was similarly temperature-dependent in these cells, diminished at high temperature (39 degrees C) and enhanced at low temperature (31 degrees C), suggesting that the changes in the host cell were the result of insertion of virus protein into the cell membrane. Evidently, persistent infection by a cytoplasmic virus can produce alterations in the host cell usually associated with transformation by nuclear viruses.

Feline Syncytium-forming Virus: DNA Provirus Size and Structure

An infectious DNA assay has been used to investigate the size and structure of the genome of feline syncytium-forming virus (FSFV). The dose response between DNA extracted from FSFV-infected cells and plaque number on feline embryo cells followed two-hit kinetics and the mol. wt. of the proviral DNA was estimated as approx. 6 x 10(6).

Feline syncytium-forming virus: identification of a virion associated reverse transcriptase and electron microscopical observations of infected cells.

The maturation of feline syncytium-forming virus (FSFV), a member of the foamy virus sub-family (Spumavirinae), has been studied by electron microscopy of thin sections of infected feline embryo (FEA) cells. The initial event observed was formation of crescent-shaped nucleoids at the plasma membrane. As budding progressed, the nucleoid became circular in outline with an electron-lucent centre in fully mature extracellular particles. These observations suggested that the maturation of FSFV in fully permissive FEA cells resembled that of C-type RNA tumour viruses, rather thant the B-type mouse mammary tumour virus. In this respect FSFV may be distinct from other foamy viruses. However, like other foamy viruses FSFV possessed reverse transcriptase activity. Polymerase activity co-sedimented with infectivity in an equilibrium density gradient and exhibited a preference for poly(rA).oligo(dT)10 over poly(dA).oligo(dT)10 as exogenous template.

Neutralisation of respiratory syncytial virus by cat serum.

RESPIRATORY SYNCYTIAL (RS) virus has been identified as a major cause of lower respiratory infection in infants, and the epidemiology of RS virus infection is characterised uniquely by annual winter outbreaks in the infant population and by antigenie stability of the virus1. The initial isolation of RS virus was from a captive chimpanzee2 and more recently an antigenically similar virus has been associated with respiratory disease in cattle3–5. The bovine and human RS viruses, although antigenically similar, can be differentiated by their different host range in vitro6. The bovine RS virus had a wider host range, multiplying in bovine, hamster, swine and primate cells, whereas the human RS virus multiplied only in bovine and primate cells. We have observed recently, however, that human RS virus (but not bovine RS virus) can multiply efficiently in secondary cultures of feline embryo cells. The cytopathology induced by RS virus infection of feline embryo cells was similar to that described previously for African green monkey (BS-C-1) cells7: the surfaces of infected cells were seen to be covered by long slender filaments when viewed by scanning electron microscopy or by immunofluorescent staining (C.R.P. and J. E. Parry, unpublished data). The susceptibility of feline embryo cells to RS virus, and an observation that the sera of some laboratory cats in the USA inhibited RS virus (R.M. Chanock, personal communication), suggested that cats might be susceptible to RS virus infection. We therefore examined a random sample of cat sera, and we show that antibody to RS virus is widespread in domestic cats in Britain.

Initiation and maintenance of persistent infection by respiratory syncytial virus.

Propagation of cells infected with temperature-sensitive (ts) mutants of respiratory syncytial (RS) virus at nonpermissive temperature (39 degrees C) resulted in cytolytic, abortive, or persistent infection, depending on the mutant used to initiate infection. Five mutants from complementation group B produced cytolytic or abortive infections, whereas a single mutant (ts1) from group D and a noncomplbmenting mutant produced persistent infections. The persistently infected culture initiated by mutant ts1 (RS ts1/BS-C-1) has been maintained in serial culture for greater than 100 transfers, and infectious-center assays and immunofluorescent staining indicated that all cells harbored the RS virus genome. RS ts1/BS-C-1 cultures were resistant to superinfection by homologous and some heterologous viruses, and interferon-like activity against some heterologous viruses was present in the culture medium. Small amounts (0.002 to 0.2 PFU/cell) of infectious virus were present in the culture fluid, but autointerfering defective particles were not detected. This released virus formed small plaques and produced persistent infection of BS-C-1 cells at 37 degrees C. The RS ts1/BS-C-1 cells contained abundant RS virus antigen internally, but little at the surface, although the cells showed enhanced agglutinability by concanavalin A. Nucleocapsids and the 41,000-molecular-weight nucleoprotein were present in extracts of both nucleated and enucleated cells. No infectious RS virus was obtained by transfection of DNA from RS tsl/BS-C-1 cells to susceptible BS-C-1 or feline embryo cells under conditions allowing efficient transfection of a foamy virus proviral DNA. It was concluded that persistent infection was maintained in part by a non-ts variant of RS virus partially defective in maturation. The karyotype of the RS ts1/BS-C-1 culture differed from that of unifected cells.

Growth of an autonomously replicating parvovirus (feline panleukopenia): Kinetics and morphogenesis

Feline embryo (FEmb) cell cultures, in which 90 percent of cells were dividing (cycling), were synchronized, by serum deprivation, to the degree that 88 per cent of the cells divided within a 12 hour period. When such cultures were infected with feline panleukopenia virus (FPV) at a multiplicity of infection of 5.7, a maximum level of cell associated virus was attained 28 hours post-infection (p.i.). There was a tendency for virus to remain cell associated in that cell lysis did not begin until 40 hours p.i. The genesis of FPV inclusion bodies was studied by light microscopy. Inclusions were intranuclear, weakly basophilic and Feulgen positive; they were first observed 8 hours p.i., and increased to be present in 90 percent of cells by 40 hours. Mitosis was markedly inhibited in FPV infected monolayers. The earliest changes observed by electron microscopy of infected cells were the presence of virus particles within nuclei, progressive chromatin margination, and nucleolar changes involving apparent segregation of the fibrillar and granular components. Virus particles measured ≃20 nm in diameter, and appeared either uniformly electron dense or possessed a dense margin and a pale center; many of the latter contained a single, central, dark spot. Virions ultimately became closely packed in all areas unoccupied by other nuclear components. In some nuclei a linear arrangement of virions was noted, but paracrystalline arrays were not seen. Other changes observed in infected nuclei included the presence of nucleolar remnants sometimes in the form of solid or hollow bodies comprised of nucleolar granules or filaments; distension of the space between the two membranes of the nuclear envelope; and the presence of aggregates of abnormal, electron dense material within the nucleus. Discontinuities of the plasma membrane and swelling of cytoplasmic organelles were commonly seen in cells showing advanced nuclear changes, but at least the inner membrane of the nuclear envelope generally remained intact. The characteristic, well defined inclusions of light microscopy were not observed by electron microscopy, and thus probably represented a preparation (shrinkage) artifact.

An Improved Assay for Feline Leukaemia Virus Pseudotypes of Murine Sarcoma Virus

An assay is described for feline leukaemia virus pseudotypes of murine sarcoma virus which increased the virus titre by about 100-fold over conventional assays. The titre is independent of dilution and no secondary focus formation occurs. The assay may be used to study virus neutralization and to detect and type feline leukaemia virus in feline embryo cells by interference.

Ultrastructural studies of the development of feline calicivirus in a feline embryo cell line

The ultrastructural changes in a feline embryo continuous cell line infected with feline calicivirus at a multiplicity of infection of approximately 1 were studied. Virus was found only in the cytoplasm and was observed as single particles, as extensive, non-regular accumulations, as paracrystalline arrays, and as single or multiple linear arrays associated with microfbrils. Mature virus particles were readily distinguished from ribosomes in that they were larger (35nm diameter) and consisted of a central, electron-dense core 20 nm diameter surrounded by a less electron-dense coat. Other changes ovserved in infected cells included rounding of the cell and nucleus and loss of pseudopodia. There was extensive production of smooth-membrane bound vesicles in the cytoplasm. Virus accumulations of each type, but especially paracrystalline arrays, were frequently closely associated with collections of these vesicles. The cisternae of the endoplasmic reticulum and the space between the two layers of the nuclear membrane was distended. By Feulgen staining and light microscopy, as well as electron microscopy, it was established that nuclear chromatin undergoes profound changes consisting of condensation usually into a single, rounded, central mass.

Determination of subgroup-specific feline oncornavirus-neutralizing antibody

A microneutralization assay was developed for antibody-to-subgroup-specific feline oncornaviruses. This study combines the economic advantage of a microtiter system and the quantitative focus reduction method which permits contruction of multiplicity curves for determination of virus-neutralizing titers. A twofold increase in Synder-Theilen feline sarcoma virus (ST-FeSV) on feline embryo cells decreased by approximately twofold the titer of reference goat serum prepared against Kawakami-Theilen feline leukemia virus. Similar dose effects with FeLV serotype virus preparations were not observed. An assay system utilizing FeLV serotypes on sarcoma-positive leukemia-negative cells demonstrated slightly greater sensitivity than one employing ST-FeSV on FE cells. Differential antibody responses to the three subgroup-specific feline oncornaviruses (A, B ,and C) were observed in reference goat sera. This test demonstrated good reproducibility as well as sensitivity and constitutes a significant improvement over end point dilution assay systems.

Characterization of non‐producer human cells induced by kirsten sarcoma virus

Non-producer (NP) human cells induced by the Kirsten sarcoma virus were characterized. These morphologically altered NP cells produced neither infectious virus nor complement-fixing antigens of the murine sarcoma-leukemia virus complex. The NP cells did not release RNA-dependent DNA polymerase and type-C virus particles with a density of approximately 1.15 g/ml in sucrose gradients by 3H-uridine labelling. The NP cells produced tumors when transplanted subcutaneously into athymic nude mice. The tumor cells re-established in culture resembled the orginal NP cells, were confirmed as human cells by karyological analysis and were also found to be "non-producer". The sarcoma virus genome in NP cells could be rescued not only by co-cultivation with "helper virus"-releasing cells but also by superinfection with helper type-C viruses. Murine (Rauscher, Ki-MuLV, AT-124 and two other xenotropic viruses), feline, RD-114 and Simian (woolly monkey and baboon) type-C viruses possessed the ability to rescue the sarcoma genome from NP cells but not AKR leukemia virus. In addition, the feline leukemia virus titer obtained by the rescuing technique in NP cells was the same as those obtained in feline embryo and NP cells by CF induction assay.

<italic>Brief Communication:</italic> Aberrant Viruses in Cells Infected With Murine Sarcoma Virus-Feline Leukemia Virus<xref ref-type="fn" rid="fn2">2</xref>

This study describes an unusual type of virus seen when a feline leukemia virus (FeLV) pseudotype of murine sarcoma virus (MuSV) obtained by cocentrifugation procedures infected feline embryo cells (FEF) and two Crandell cat cell lines (CrFK1, CrFK2). When all three cell cultures were infected with MuSV-FeLV, only FEF and CrFK2 were transformed and only these showed normal and aberrant virus. The CrFK1 infected with MuSV-FeLV did not transform but did replicate normal type-C virus with a 50-A intermediate coat. The virus replicated in the two transformed lines showed three particles; a normal particle with a 50-A intermediate coat, a normal particle with a 100-A intermediate coat, and an aberrant particle with a 100-A intermediate coat.