Skip to main content
PMC home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1977 Dec;6(6):610–617. doi: 10.1128/jcm.6.6.610-617.1977

Rotavirus isolation and cultivation in the presence of trypsin.

L A Babiuk, K Mohammed, L Spence, M Fauvel, R Petro
PMCID: PMC274833  PMID: 201663

Abstract

Rotaviruses are generally difficult to isolate and culture in vitro; therefore, virus isolation has not been used as a method of diagnosing this group of agents. The present report describes a simple procedure for isolating bovine rotaviruses directly from feces after pretreatment of fecal samples with trypsin. This procedure resulted in virus isolation from five of five samples that contained virus particles, as demonstrated by electron microscopy, and four of seven samples where virus particles could not be observed but were considered positive by the presence of immunofluorescent-staining cells in feces. Virus could not be isolated from "normal" feces. If the virus was not passaged in the presence of trypsin, the infectivity was gradually lost, but infectivity could be restored again if trypsin was added, resulting in increased virus spread and concomitant increase in virus yield. The application of this technique as a diagnostic tool for bovine and other rotaviruses is briefly discussed.

Full text

PDF
610

Images in this article

612Image
on p.612
613Image
on p.613

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Albrey M. B., Murphy A. M. Letter: Rotavirus growth in bovine monolayers. Lancet. 1976 Apr 3;1(7962):753–753. doi: 10.1016/s0140-6736(76)93136-6. [DOI] [PubMed] [Google Scholar]
  2. Appleyard G., Maber H. B. Plaque formation by influenza viruses in the presence of trypsin. J Gen Virol. 1974 Dec;25(3):351–357. doi: 10.1099/0022-1317-25-3-351. [DOI] [PubMed] [Google Scholar]
  3. Babiuk L. A., Acres S. D., Rouse B. T. Solid-phase radioimmunoassay for detecting bovine (neonatal calf diarrhea) rotavirus antibody. J Clin Microbiol. 1977 Jul;6(1):10–15. doi: 10.1128/jcm.6.1.10-15.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Banatvala J. E., Totterdell B., Chrystie I. L., Woode G. N. In-vitro detection of human rotaviruses. Lancet. 1975 Oct 25;2(7939):821–821. doi: 10.1016/s0140-6736(75)80057-2. [DOI] [PubMed] [Google Scholar]
  5. Banfield W. G., Kasnic G., Blackwell J. H. Further observations on the virus of epizootic diarrhea of infant mice. An electron microscopic study. Virology. 1968 Nov;36(3):411–421. doi: 10.1016/0042-6822(68)90166-9. [DOI] [PubMed] [Google Scholar]
  6. Bishop R. F., Davidson G. P., Holmes I. H., Ruck B. J. Virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis. Lancet. 1973 Dec 8;2(7841):1281–1283. doi: 10.1016/s0140-6736(73)92867-5. [DOI] [PubMed] [Google Scholar]
  7. Bridger J. C., Woode G. N. Characterization of two particle types of calf rotavirus. J Gen Virol. 1976 May;31(2):245–250. doi: 10.1099/0022-1317-31-2-245. [DOI] [PubMed] [Google Scholar]
  8. Bryden A. S., Davies H. A., Thouless M. E., Flewitt T. H. Diagnosis of rotavirus infection by cell culture. J Med Microbiol. 1977 Feb;10(1):121–125. doi: 10.1099/00222615-10-1-121. [DOI] [PubMed] [Google Scholar]
  9. Fernelius A. L., Ritchie A. E., Classick L. G., Norman J. O., Mebus C. A. Cell culture adaptation and propagation of a reovirus-like agent of calf diarrhea from a field outbreak in Nebraska. Arch Gesamte Virusforsch. 1972;37(1):114–130. doi: 10.1007/BF01241157. [DOI] [PubMed] [Google Scholar]
  10. Flewett T. H., Bryden A. S., Davies H., Woode G. N., Bridger J. C., Derrick J. M. Relation between viruses from acute gastroenteritis of children and newborn calves. Lancet. 1974 Jul 13;2(7872):61–63. doi: 10.1016/s0140-6736(74)91631-6. [DOI] [PubMed] [Google Scholar]
  11. Homma M. Trypsin action on the growth of Sendai virus in tissue culture cells. I. Restoration of the infectivity for L cells by direct action of tyrpsin on L cell-borne Sendai virus. J Virol. 1971 Nov;8(5):619–629. doi: 10.1128/jvi.8.5.619-629.1971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kapikian A. Z., Cline W. L., Kim H. W., Kalica A. R., Wyatt R. G., Vankirk D. H., Chanock R. M., James H. D., Jr, Vaughn A. L. Antigenic relationships among five reovirus-like (RVL) agents by complement fixation (CF) and development of new substitute CF antigens for the human RVL agent of infantile gastroenteritis. Proc Soc Exp Biol Med. 1976 Sep;152(4):535–539. doi: 10.3181/00379727-152-39434. [DOI] [PubMed] [Google Scholar]
  13. Kapikian A. Z., Kalica A. R., Shih J. W., Cline W. L., Thornhill T. S., Wyatt R. G., Chanock R. M., Kim H. W., Gerin J. L. Buoyant density in cesium chloride of the human reoviruslike agent of infantile gastroenteritis by ultracentrifugation, electron microscopy, and complement fixation. Virology. 1976 Apr;70(2):564–569. doi: 10.1016/0042-6822(76)90301-9. [DOI] [PubMed] [Google Scholar]
  14. Kapikian A. Z., Kim H. W., Wyatt R. G., Rodriguez W. J., Ross S., Cline W. L., Parrott R. H., Chanock R. M. Reoviruslike agent in stools: association with infantile diarrhea and development of serologic tests. Science. 1974 Sep 20;185(4156):1049–1053. doi: 10.1126/science.185.4156.1049. [DOI] [PubMed] [Google Scholar]
  15. Lecce J. G., King M. W., Mock R. Reovirus-like agent associated with fatal diarrhea in neonatal pigs. Infect Immun. 1976 Sep;14(3):816–825. doi: 10.1128/iai.14.3.816-825.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Matsuno S., Inouye S., Kono R. Plaque assay of neonatal calf diarrhea virus and the neutralizing antibody in human sera. J Clin Microbiol. 1977 Jan;5(1):1–4. doi: 10.1128/jcm.5.1.1-4.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McNulty M. S., Allan G. M., Pearson G. R., McFerran J. B., Curran W. L., McCracken R. M. Reovirus-like agent (rotavirus) from lambs. Infect Immun. 1976 Dec;14(6):1332–1338. doi: 10.1128/iai.14.6.1332-1338.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mebus C. A., Kono M., Underdahl N. R., Twiehaus M. J. Cell culture propagation of neonatal calf diarrhea (scours) virus. Can Vet J. 1971 Mar;12(3):69–72. [PMC free article] [PubMed] [Google Scholar]
  19. Petric M., Szymanski M. T., Middleton P. J. Purification and preliminary characterization of infantile gastroenteritis virus (orbivirus group). Intervirology. 1975;5(3-4):233–238. doi: 10.1159/000149919. [DOI] [PubMed] [Google Scholar]
  20. SPENDLOVE R. S., SCHAFFER F. L. ENZYMATIC ENHANCEMENT OF INFECTIVITY OF REOVIRUS. J Bacteriol. 1965 Mar;89:597–602. doi: 10.1128/jb.89.3.597-602.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Schoub B. D., Lecatsas G., Prozesky O. W. Antigenic relationship between human and simian rotaviruses. J Med Microbiol. 1977 Feb;10(1):1–6. doi: 10.1099/00222615-10-1-1. [DOI] [PubMed] [Google Scholar]
  22. Snodgrass D. R., Wells P. W. Rotavirus infection in lambs: studies on passive protection. Arch Virol. 1976;52(3):201–205. doi: 10.1007/BF01348017. [DOI] [PubMed] [Google Scholar]
  23. Spence L., Fauvel M., Petro R., Bloch S. Comparison of Counterimmunoelectrophoresis and electron microscopy for laboratory diagnosis of human reovirus-like agent-associated infantile gastroenteritis. J Clin Microbiol. 1977 Feb;5(2):248–249. doi: 10.1128/jcm.5.2.248-249.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Spendlove R. S., McClain M. E., Lennette E. H. Enhancement of reovirus infectivity by extracellular removal or alteration of the virus capsid by proteolytic enzymes. J Gen Virol. 1970 Aug;8(2):83–94. doi: 10.1099/0022-1317-8-2-83. [DOI] [PubMed] [Google Scholar]
  25. Welch A. B., Twiehaus M. J. Cell culture studies of a neonatal calf diarrhea virus. Can J Comp Med. 1973 Jul;37(3):287–294. [PMC free article] [PubMed] [Google Scholar]
  26. Woode G. N., Bridger J. C., Jones J. M., Flewett T. H., Davies H. A., Davis H. A., White G. B. Morphological and antigenic relationships between viruses (rotaviruses) from acute gastroenteritis of children, calves, piglets, mice, and foals. Infect Immun. 1976 Sep;14(3):804–810. doi: 10.1128/iai.14.3.804-810.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Woode G. N., Bridger J. C. Viral enteritis of calves. Vet Rec. 1975 Jan 25;96(4):85–88. doi: 10.1136/vr.96.4.85. [DOI] [PubMed] [Google Scholar]
  28. Wyatt R. G., Kapikian A. Z., Thornhill T. S., Sereno M. M., Kim H. W., Chanock R. M. In vitro cultivation in human fetal intestinal organ culture of a reovirus-like agent associated with nonbacterial gastroenteritis in infants and children. J Infect Dis. 1974 Nov;130(5):523–528. doi: 10.1093/infdis/130.5.523. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES