key: cord-0004957-it0dr0xd authors: Otsuki, K.; Tsubokura, M. title: Plaque formation by avian infectious bronchitis virus in primary chick embryo fibroblast cells in the presence of trypsin date: 1981 journal: Arch Virol DOI: 10.1007/bf01320246 sha: e89b89ec98a931a6733e876ecc80bae870357e7d doc_id: 4957 cord_uid: it0dr0xd Ten strains of avian infectious bronchitis virus (IBV) were titrated as plaqueforming units in primary chick embryo fibroblast cells. In the absence of trypsin, plaques were only formed by Beaudette-42 and Iowa-609 strains. When trypsin was incorporated in the overlay medium of cell monolayers, all the IBV strains tested produced plaques within 4 days after inoculation. Incorporation of 20–40 µg of trypsin per ml of the overlay medium seemed to be suitable for plaque formation of IBV. A preliminary investigation was made of the mode of action of trypsin. Avian infectious bronchitis virus (IBV) replicates in various cultivated cells, but distinct cytopathic effect (CPE) or plaque formation occurs only in chick embryo kidney ceils and chick kidney {CK) cells. A few strains of IBV grow in VEI~O cells (4, 5) , BHK-21 cells (10) and chick embryo fibroblast (CE) cells (9, 10) . CE cells are easier to prepare than CK cells and it would be advantageous if, by incorporating some agent into the medium, plaque formation could be made to occur. Trypsin markedly enhances the in vitro infectivity of various viruses, i.e. rotavirus (11, 12) , influenza viruses (1, 2, 8, 15) , Sendal virus (7) , reovirus (13) and vaeeinia virus (6) . Quite recently, S:ro~z et al. (14) reported that trypsin enhanced plaque formation of an enteropathogenic bovine coronavirus in bovine fetal thyroid and bovine fetal brain cells. In the present report, we describe plaque formation by IBV in CE cells when trypsin is incorporated into the overlay medium. The ten strains of IBV studied were: Beaudette-42 (Be-42), Massachusetts 41 (IB-41), Connecticut A-5968 (A-5968), Connaught, Holte, Iowa-609, KH, Nerima, Ishida and Shiga. None of these strains would produce CPE or plaques in CE cells (10) , but after I0 further passages in CK cells Be-42 and Iowa-609 strains both produced CPE in CE cells. We examined all I0 strains after passage in CK cells for their ability to form plaques. Monolayers of primary CK and CE cells were prepared as described previously (10) in Eagle's MEM containing 2 per cent heat inactivated calf serum with penicillin and streptomycin at a final concentration of 200 unit, s and 200 y.g per ml respectively. The overlay was prepared using an 0.95 per cent Bacto-agar (DIFCO) and 0.1 per cent tryptose phosphate broth (DIFCO) in Eagle's MEM; the medium was sterilized by autoclaving prior to use. After removal of the growth medium, cell monolayers were washed twice with Eagle's MEM. Glass culture bottles (30 × 30 × 60 ram) containing cell monolayers were inoculated with 0.2-ml of virus and incubated at 37 ° C for 60 minutes. Excess inoculum was then removed and 3-ml portions of the overlay was added to each bottle. Cultivation was continued for 2 days at 37 ° C. Then, 2-ml of overlay containing 0.004 per cent neutral red was added, and cultivation was recontinued for a further 5 days. The number of plaques on each monolayers were then counted. Ten strains of IBV, all passaged in CK cells, were tested for their ability to produce plaques in CE cells with and without trypsin (20 ,ag per ml) in the overlay. Be-42 and Iowa-609 strains both produced plaques in the absence of trypsin, b u t their diameter was small when compared with those produced in the presence of trypsin. None of the other strains produced plaques after 7 days i n c u b a t i o n in the absence of trypsin (Table 1) . However, the incorporation of trypsin into the m e d i u m in the remaining 8 strains of IBV produced plaques with diameters ranging from 1 to 4 m m after 4 days incubation. The n u m b e r of plaques in CE cells approximated those produced in CK cells. To stmiy the effect of t r y p s i n concentration on the n u m b e r a n d size of plaques, I B V A-5968 strain was grown in CE cells in tile presence of 10 to 60 y.g of trypsin IBV A-5968 strain which forms small plaques not only in CK cells but also in CE cells was used in the following studies: To exclude the possibility that plaque enhancement was caused by an impurity in the trypsin preparation, a solution of trypsin containing 500 Fg per ml was mixed with 1,000 ~zg per ml of soybean trypsin inhibitor and then used in the plaque assay at a concentration of 20 Ixg of trypsin per ml. The enhancing action of trypsin was abolished by trypsin inhibitor. Hence, trypsin itself was responsible for the effect on plaque formation. To test whether or not trypsin might act by destroying an inhibitor in the medium, agar overlay medium was incubated with 50 Fg per ml of trsTsin for 24 hours at 37 ° C. The medium was then heated at 100 ° C and used as overlay. This medium did not enhance plaque formation. When 20 Fg per mI of trypsin was incorporated into this medium it enhanced plaque formation. This result suggested that trypsin did not act by removing a pre-existing virus inhibitor from the medium. To test whether or not trypsin might act by modifying the surface of CE cells, 5-ml volumes of maintenance medium containing trypsin (20 Fg per ml) were poured into bottles containing cell monolayers and these were incubated for 5 hours at 37 ° C. IBV A-5938 strain was then inoculated onto the cells, and after 60 minutes adsorption, the inoculum was replaced with overlay without trypsin. No plaques were formed. Therefore, trypsin did not act by modifying CE cells at the time of virus entry. CtIOM[AK et al. (3) reported that the Beaudette strain of IBV produced CPE in CE cells after many passages in cell culture. In the present investigation, we showed that the Be-42 and Iowa-609 strains both produced plaques in CE cells in the absence of trypsin. We further showed that the addition of trypsin to the overlay medium enables other strains of IBV to produce plaques in CE cells. In many other investigators (1, 6, 7, 8, 11, 12, 13, 14, 15) , trypsin has been incorporated into the overlay medium at a concentration of 10 9g per ml. The results of the present investigation, however, show that the incorporation of 20--40 y.g of trypsin per ml of overlay is optimal for plaque formation by IBV. The mechanism by which trypsin enhances plaque formation is uncertain, but it is possible that it enhances the replication or release of IBV. STORZ et al. (14) reported that eoronaviral particles produced in trypsin-treated bovine cells had uniformly shorter surface projection than did the viral forms generated by trypsinfree cell cultures. The results reported here show that trypsin was required during the whole period of plaque development and we therefore assume that it acts on successive cycles of virus replication. CA~]~ et al. (2) suggested that pancreatin produced its effect by an action on the host cells. In the present investigation, however, IBV A-5968 strain did not form plaques in CE cells which had been pre-incubated with trypsin. Possibly trypsin facilitates the development of IBV plaques by acting on the released virus particles, as has been described for reovirus (13), influenza virus (8) and rotavirus (12) . Further studies are necessary to establish the mechanism(s) by which trypsin enhances plaque formation of IBV. Plaque formation by influenza viruses in the presence of trypsin Effect of panereatin of plaque formation by influenza viruses The propagation and eytopathogenic effect of an-egg-adapted strain of infectious bronchitis virus in tissue culture Serial passage of 3 strains of avian infectious bronchitis virus in African green monkey kidney eetls (VER0) Replication of avian infectious bronchitis virus in African green monkey kidney cell line VERO Enhancement of vaeeinia virus plaque formation Trypsin action on the growth of Sendai virus in tissue culture cells. I. t~esoration of the infectivity for L cells by direct action of trypsin on L celt-born Sendal virus Activation of influenza A viruses by trypsin treatment Immunofluoreseenee of avian infectious bronchitis virus in primary chicken embryo kidney, liver, lung, and fibroblast cell cultures. Arch. gas. Virusforsch Studies on avian infectious bronchitis virus (IBV). II. Propagation of IBV in several cultured ceils Simian rotavirus SA-11 plaque formation in the presence of trypsin The role of trypsin in plaque formation by simian rotavirus SA-11 Enzymatic enhancement of infectivity of reovirus Enhancement of plaque formation and cell fusion of an enteropathogenie coronavirus by trypsin treatment Genetic recombination for antigenic markers of antigenically different strains of influenza B virus Authors' address : Dr. K. 0wSlJl~i, Department of Veterinary Microbiology, Faculty of Agriculture, Tottori University, Tottori City, Japan.l~eeeived June 2, t981