Use of chicken cell line LSCC-H32 for titration of animal viruses and exogenous chicken interferon

The chicken embryo cell line LSCC-H32 was tested for the propagation and titration of several animal viruses of the families Toga-, Reo-, Rhabdo-, Herpeto-, Orthomyxo-, Paramyxo-, and Poxviridae and compared with secondary chicken embryo cells. The LSCC-H32 cells were demonstrated to be as susceptible for most of the tested viruses as were secondary chicken embryo cells. Both produced comparably sized virus plaques. The titers of Sindbis and Semliki Forest viruses in LSCC-H32 cells were 5- to 40-fold higher than in secondary chicken embryo cells or BHK-21 cells, respectively. Furthermore, exogenous chicken standard interferon was titrated in the LSCC-H32 cells, and a 50% plaque titer reduction of the challenging vesicular stomatitis virus was achieved by 0.12 IU of a standard chicken interferon preparation. Endogenous chicken interferon could not be induced by treatment of the cells with polyinosinic acid-polycytidylic acid. Due to its high plating efficiency and metabolic activities, the LSCC-H32 cell line provides a useful cell system for the titration and large-scale production of the tested animal viruses and for the titration of exogenous chicken interferon.     

Establishment and characterization of chicken embryo fibroblast clone LSCC-H32

Cell line CEC-32 and clone LSCC-H32 were established from primary chicken embryo cells spontaneously but not experimentally transformed at 32 degrees C. The lines consisted of fibroblastoid and polygonal cells and had a subtetraploid karyotype of 2N = 130 to 140. The cells showed increased plating efficiency and metabolic activities as demonstrated by hexose uptake and plasminogen activator assay. The established cells produced avian lymphoid leukosis viruses of subgroups A and B. The virus released from LSCC-H32 cells induced lymphoid leukosis in inoculated chickens 18 to 22 wk post infection (PI). The cells have been carried in continuous culture for 285 passages and they appeared to grow indefinitely. They were efficiently used to propagate several animal viruses and to titrate chicken interferon.     

A simple and efficient microassay method for titration of interferon.

A simple and efficient microassay method for the titration of interferon was developed by the use of microtest plates for handling a large number of samples. L929 cells pretreated with interferon were infected with vesicular stomatitis virus (VSV) and cultured in the presence of 3H-uridine. The activity was expressed by the reduction of extracellular radioactive RNA released after destruction of the infected cells, which was measured in terms of the radioactivity incorporated into cold TCA-insoluble materials in the culture fluid. The interferon titer determined by this method was in the same order as that by the plaque reduction method. The activity by this method was parallel to, but lower than that expressed by the yield reduction of infectious viruses. This method requires only 0.025 ml of each test sample with higher than 1 NIH ref. unit/ml to detect its interferon activity and takes 2 to 3 days for assaying hundreds of samples.     

Establishment of a novel ovine kidney cell line for isolation and propagation of viruses infecting domestic cloven-hoofed animal species

A sheep kidney-derived cell line, FLK-N3, was successfully established after serial (>100) passages. Persistent infection of this cell line with viruses and mycoplasma was not detected. The cells grew well and showed susceptibility to a wide variety of viruses derived from ovine, bovine, and porcine species, including orf virus, maedi visna virus, bovine herpesvirus 1, bovine parainfluenza virus 3, bovine viral diarrhea viruses 1 and 2, bovine coronavirus, bovine respiratory syncytial virus, bovine enterovirus, suid herpesvirus 1, and porcine enterovirus. These results suggest that the FLK-N3 cell line could be useful for isolation and propagation of viruses that affect cloven-hoofed animals.     

Replacement of primary chicken embryonic fibroblasts (CEF) by the DF-1 cell line for detection of avian leucosis viruses.

International regulations prescribe that the absence of avian leucosis viruses (ALV) in avian live virus vaccines has to be demonstrated. Primary chicken embryo fibroblasts (CEF) from special SPF chicken lines are normally used for detection of ALV. The suitability of the DF-1 cell line for ALV-detection, as alternative for primary CEF, was studied in three types of experiments: (1) in titration experiments without cell passage, (2) in experiments with passages in cell cultures according to European Pharmacopoeia requirements, and (3) in experiments with commercial live avian vaccines that had been spiked with known amounts of ALV. In all tests the sensitivity of ALV-A and ALV-J detections on DF-1 cells was at least as high as on primary CEF. The sensitivity of ALV-B detection was always superior when DF-1 cells were used. ALV were detected earlier in all comparative tests when DF-1 cells were used. ALV-A, ALV-B and ALV-J all induced CPE on DF-1 cells, whereas no clear CPE was seen on CEF-cells. For reasons of sensitivity, standardisation as well as reduction of animal use, the data support the use of DF-1 cells to monitor absence of ALV in vaccine virus seed lots or finished products.     

Genetic manipulation of an exogenous non-immunoglobulin protein by gene conversion machinery in a chicken B cell line

During culture, a chicken B cell line DT40 spontaneously mutates immunoglobulin (Ig) genes by gene conversion, which involves activation-induced cytidine deaminase (AID)-dependent homologous recombination of the variable (V) region gene with upstream pseudo-V genes. To explore whether this mutation mechanism can target exogenous non-Ig genes, we generated DT40 lines that bears a gene conversion substrate comprising the green fluorescent protein (GFP) gene as a donor and the blue fluorescent protein (BFP) gene as an acceptor. A few percent of the initially BFP-expressing cells converted their fluorescence from blue to green after culture for 2–3 weeks when the substrate construct was integrated in the Ig light chain locus, but not in the ovalbumin locus. This was the result of AID-dependent and the GFP gene-templated gene conversion of the BFP gene, thereby leading to the introduction of various sizes of GFP-derived gene segment into the BFP gene. Thus, G/B construct may be used to visualize gene conversion events. After switching off AID expression in DT40 cells, the mutant clones were isolated stably and maintained with their mutations being fixed. Thus, the gene conversion machinery in DT40 cells will be a useful means to engineer non-Ig proteins by a type of DNA shuffling.     

Biotechnology and the chicken B cell line DT40

Protein optimization is a major focus of the biotech and pharmaceutical industry. Various in vitro technologies have been developed to accelerate protein evolution and to achieve protein optimization of functional characteristics such as substrate specificity, enzymatic activity and thermostability. The chicken B cell line DT40 diversifies its immunoglobulin (Ig) gene by gene conversion and somatic hypermutation. This machinery can be directed to almost any gene inserted into the Ig locus. Enormously diverse protein libraries of any gene of interest can be quickly generated in DT40 by utilizing random shuffling of complex genetic domains (gene conversion) and by the introduction of novel non-templated genetic information (random mutagenesis). The unique characteristics of the chicken cell line DT40 make it a powerful in-cell diversification system to improve proteins of interest within living cells. One essential advantage of the DT40 protein optimization approach is the fact that variants are generated within an in-cell system thus allowing the direct screening for desired features in the context of intracellular networks. Utilizing specially designed selection strategies, such as the powerful fluorescent protein technology, enables the reliable identification of protein variants exhibiting the most desirable traits. Thus, DT40 is well positioned as a biotechnological tool to generate optimized proteins by applying a powerful combination of gene specific hypermutation, gene conversion and mutant selection.     

RNA recombination in animal and plant viruses.

An increasing number of animal and plant viruses have been shown to undergo RNA-RNA recombination, which is defined as the exchange of genetic information between nonsegmented RNAs. Only some of these viruses have been shown to undergo recombination in experimental infection of tissue culture, animals, and plants. However, a survey of viral RNA structure and sequences suggests that many RNA viruses were derived form homologous or nonhomologous recombination between viruses or between viruses and cellular genes during natural viral evolution. The high frequency and widespread nature of RNA recombination indicate that this phenomenon plays a more significant role in the biology of RNA viruses than was previously recognized. Three types of RNA recombination are defined: homologous recombination; aberrant homologous recombination, which results in sequence duplication, insertion, or deletion during recombination; and nonhomologous (illegitimate) recombination, which does not involve sequence homology. RNA recombination has been shown to occur by a copy choice mechanism in some viruses. A model for this recombination mechanism is presented.     

Antiviral and cell multiplication inhibitory activities of mouse interferon poreparations tested on an interferon sensitive murine sarcoma cell line

The cell multiplication inhibitory effect of SDS-treated mouse interferon separated into antiviral (AV) and cell multiplication inhibitory (CMI) fractions was compared to that of untreated similar interferon on a line of murine osteosarcoma cells. The untreated interferon poreparatin and the CMI fractions dose-dependently inhibited the multiplication of the cells as measured by cell count and incorporation of 3H-thymidine into the cultures. The AV fractions, containing comparable antiviral activites as the untreated interferon preparations, had only a minor effect on cell multiplication. The biochemical properties of the fractions studied remain unknown.     

Enhanced wound healing in animal models by interferon and an interferon inducer.

Healing of wounds is a significant biological process that involves the interactions of different cell types, growth factors, cytokines, and extracellular matrix molecules. Mice and rats were treated in vivo with interferon (IFN) alpha/beta or polyinosinic-polycytidylic acid (Poly I:C), a ds-RNA, a potent inducer of IFN. We observed faster and enhanced closure of wounds as compared to untreated controls on day 7 (wound area measured on Macintosh II CX using NIH image 1.30u program), increased migration of dermal fibroblasts in the wound bed, complete re-epithelialization evidenced by routine histology and scanning electron microscopic procedures, and increased collagen synthesis, which correlates to greater tensile strength. In addition, classical immunofluorescence procedures using frozen sections showed that dermal fibroblasts synthesized much more laminin following Poly I:C treatment, whereas no effect was observed on fibronectin synthesis. These results suggest that Poly I:C and IFN treatment result in a faster restoration of tissue integrity in both full skin punch biopsy and skin incision models.     

Optimization of the BGM cell line culture and viral assay procedures for monitoring viruses in the environment.

An in-depth study of the continuous cell line designated BGM is described herein, and recommendations are made for standardizing cell culture and viral assay procedures. Based on data gathered from a survey of 58 laboratories using this cell line, a research plan was developed that included the study of growth media, sera, NaHCO3 levels, culture bottles, cell concentration, overlay media, agar, virus infection conditions, and cell-dissociating agents. Additionally, a comparative virus isolation study with BGM cells and nine other cell types was conducted with 37 sewage samples collected from nine different geographic areas. The results of the study indicated that the BGM cell line is superior for virus isolation when compared with the other cell types and that certain media and additives tend to increase BGM cell sensitivity to a specific group of viruses. A standardized procedure for cultivation of BGM cells is described which provides a more effective enterovirus assay system.     

Antiproliferative effect of interferon on a Burkitt''s lymphoma cell line

The effect of interferon (IF) on the growth of a Burkitt's lymphoma cell line was analysed. The degree of depression of cell doublings was the same if the cells were in a steady state mode of exponential growth or in a resting state (G0) when IF was added. As IF had a lag time of 24 h before decreased growth could be observed, cells in G0 did not seem to be more sensitive when growth was estimated by cell counts expressed as cell doublings. IF inhibited cells to proceed into the cell cycle and the possibility that IF may increase the escape into a G0 loop is discussed.