Hughes et al.

A Comparison of Rolling vs. Non-rolling Cultures for the Early Detection of Viruses in Clinical Specimens

John H. Hughes, Michele A. Sturgill-Dible, and Vincent V. Hamparian

Department of Medical Microbiology and Immunology; and Department of Pediatrics
The Ohio State University 333 West Tenth Avenue, Columbus, OH 43210
and The Children's Hospital Viral Diagnostic Laboratory, Columbus, OH 43205

Correspondence should be addressed to: John H. Hughes Ph.D.
Email:
jhughes@magnus.acs.ohio-state.edu
Submitted for publication: July 1995


Key Words: Herpes simplex viruses, respiratory syncytial viruses, enteroviruses, adenoviruses, rapid viral diagnosis, viral isolations, rolling cultures, enhanced viral detection, motion and viruses.


Title Page Abstract Introduction Materials and Methods Results
Discussion Conclusions Acknowledgements References Table of Contents

Table I Table II Table III


ABSTRACT

A total of 30,303 specimens were submitted to the viral diagnostic laboratory at Columbus Children's Hospital over a 5-year period (1989-1993) for viral diagnosis. Specimens were inoculated into cell cultures that were either rolled at 2 to 3 rpm or not rolled (control). We found that more herpes simplex viruses were detected significantly sooner in cell cultures that had been continuously rolled, p < 0.001. In contrast, more enteroviruses were detected significantly sooner in 2 out of 3 cell lines that were not rolled, p < 0.001. Rolled monkey kidney cultures were better for detecting respiratory syncytial virus (RSV), while non-rolled HEp-2 cells detected RSV sooner. It is apparent that both rolled and non-rolled cell cultures should be used for viral isolations. Optimal rolling conditions remain to be established for each cell culture used for the isolation of viruses from clinical specimens.


INTRODUCTION

Diagnostic virology has been, when possible, based on the isolation of viruses that cause disease. For viral isolations, cell cultures are incubated either without motion or are rolled at approximately 0.2 revolutions per minute (rpm). The first report on rolling infected cell cultures was at 0.1 rpm for vaccinia virus (5). Recent studies with monkey kidney cell cultures infected with vaccinia virus at a low multiplicity of infection (< 0.1 MOI) and rolled at 96 rpm (1.9 Xg), showed that maximal cytopathic effects (CPE) occurred four days sooner with rolled than with non-rolled cultures (15). Also, cultures rolled at 96 rpm had a 75-fold geometric mean increase in viral yield when compared to cultures incubated without motion for the same time. In addition, cells infected with herpes simplex virus (HSV), coxsackievirus A21 or respiratory syncytial virus (RSV) and rolled at 96 rpm also produced significantly more viral CPE and virus (12,13,25,26,40). A revolution per minute response study with HSV-infected cultures rolled at 20, 96, and 383 rpm, revealed that 59-fold, 89-fold, and 53-fold more virus respectively was produced when compared to non-rolled cultures incubated for the same time (14).

Because rolling cultures may be important for the isolation and detection of certain viruses, we wanted to determine the effect of rolling at speeds 10-fold greater than routinely used on the isolation of viruses from clinical specimens. Traditionally, personnel in viral diagnostic laboratories roll inoculated cultures at 0.1 to 0.2 rpm. To our knowledge, studies using higher rpm's for isolating viruses in a diagnostic setting have not been reported. For this study, we present data involving over 5,000 viruses that were isolated from clinical specimens in either rolled (2 to 3 rpm) or non-rolled cell cultures.


MATERIALS AND METHODS

Cell cultures and specimens. Five different cell lines (primary African Green monkey kidney cells, SF foreskin and Flow 6000-fetal lung fibroblasts, HEp-2 and A549 epithelial cells) were used for viral isolations. Cell culture roller tubes (16 by 125 mm) were inoculated with fresh clinical specimens and were incubated either non-rolling (control) or rolling at 2 to 3 rpm. The culture tubes for rolling conditions were placed in commercial rotators purchased from Bellco (Bellco Biotechnology, Vineland, NJ). Viruses isolated from specimens were identified by CPE by one of five technologists in the Viral Diagnostic Laboratory at Children's Hospital and confirmed by a second technologist. When viruses could not be conclusively identified by CPE, identification was confirmed with a specific antiserum. Specimens for viral isolations came from such anatomical locations as the respiratory tract (bronchial lavage, bronchial brushings, nasopharyngeal and throat swabs, nasal washes and tracheal aspirates), central nervous system (cerebral spinal fluids), urinary tract (urine specimens), gastrointestinal tract (stools and rectal swabs), skin (lesions), blood (buffy coats), various tissues, body fluids (pericardial, peritoneal, amniotic and pleural), and the eye.

Data analysis. Data were analyzed by the Wilcoxson signed-ranks test to determine which viruses were detected first in either rolled or non-rolled cultures. The student's t test was used to compare the mean detection times for viruses isolated in rolled vs non-rolled cultures. P- values less than 0.05 were considered to be statistically significant.


RESULTS

Over a 5-year interval (1989 to 1993), 30,303 specimens were received for viral isolations. From these specimens, 7,357 viruses (herpesviruses, adenoviruses, picornaviruses, myxoviruses and paramyxoviruses) were isolated for an isolation rate of 24.2%. A total of 197 (0.65%) of the specimens contained at least two viruses and at least one specimen had three viruses.

Herpes simplex viruses were detected significantly sooner in primary monkey kidney and A549 cells that were rolled at 2 to 3 rpm. See Table I. Although not significant, there was a trend for earlier detection of HSV in rolling diploid cell cultures (SF cells). No differences in earlier detection of cytomegaloviruses (CMV) or varicella-zoster viruses (VZV) were seen. However, the number of CMV and VZV isolates for each condition was small and a larger sample size would be required for a more definitive analysis.

For enteroviruses, significantly more viruses were detected sooner in non-rolled SF and A549 cells. In addition, non-rolled monkey kidney cells also appeared to be better for the rapid detection of enteroviruses. Although not significant, adenoviruses also had a tendency to be detected sooner in non-rolled SF and A549 cell cultures. The results with respiratory syncytial virus (RSV) varied by cell type. Monkey kidney cells that were rolled yielded more RSV isolates sooner than did non-rolled cultures (222 vs 82 respectively, p <0.005). See Table I. However, HEp-2 cultures that were not rolled yielded significantly more RSV isolates sooner than rolled cultures (363 vs 170 respectively, p <0.005). Rolling of diploid fibroblasts or A549 cells did not enhance the detection of RSV. However, more RSVs were always recovered in rolled fibroblasts and A549 cells.

A smaller and more controlled study was carried out with HSV specimens using primary monkey kidney cells and human diploid lung fibroblast cells (Flow-6000). For this study, cultures were also rolled between 2 and 3 rpm or were not rolled, and to reduce the variability of scoring for viral CPE by multiple technologists only a single technologist was used. The mean detection times for HSV in each cell and for each condition (rolled vs non-rolled) were determined. In addition, we determined at what time CPE was first detected in rolled or non-rolled cultures inoculated with the same clinical specimens. From Table II, it is clear that rolling either monkey kidney cells or human fibroblasts at 2 to 3 rpm significantly reduced the mean detection time of HSV by 0.5 to 1.0 day. Furthermore, fibroblasts were better than monkey kidney cells for detecting HSV earlier. In addition, significantly more herpes simplex viruses were also detected earlier in rolling monkey kidney cells or in rolling human fibroblasts cells when compared to non- rolled cultures. See Table III.


DISCUSSION

Increasing emphasis has been placed on the rapid diagnosis of viral infections. Motion or centrifugation can speed the diagnosis of some viral infections (12). For example, it has been demonstrated that rolling inoculated cultures at 0.1 to 0.3 rpm may enhance viral isolations and can enhance CPE and/or viral yields for enteroviruses (6,28,30,36), rhinoviruses (1,11,29,35,41), reoviruses (22) parainfluenza virus type 4 (2), rotaviruses (7,19,21,27,37,43), and herpesviruses (18). In a non-diagnostic setting, rolling at 2.0 rpm can enhance CPE and/or viral yields for coxsackievirus A21, respiratory syncytial virus, HSV and vaccinia virus (12,14,15,25,26,40). Since viral isolations are still important for diagnosis, we determined if cultures continuously rolled at 2.0 to 3.0 rpm would allow earlier detection of wild type viruses from clinical specimens

The results from this study indicate that conventional tube cultures continuously rolled at 2 to 3 rpm are better for the rapid detection of HSV from clinical specimens. However, not all cell lines displayed enhancement for early detection of viruses under rolling conditions. For RSV, more viruses were detected sooner in rolled monkey kidney cells, but this was not the case with HEp-2 cells. Previously, in a non-diagnostic setting and using young Hep-2 culture, we found that rolled cultures were better for the detection of RSV (40). In addition, cultures subjected to orbital motion also produce more RSV antigens. Generally, HEp-2 cells do not maintain well, and rolling may cause early overgrowth of these cells making it difficult to detect viral CPE in a diagnostic setting. Furthermore, the detection of RSV in HEp-2 cell cultures may be a problem in a busy diagnostic laboratory where older cultures may be used and time may not always be available to carefully scrutinize cultures for early or subtle CPE. Thus, the differences noted for RSV detection between this study and the previous study may be attributed to variations in cellular densities and CPE detection.

To our surprise, enteroviruses were detected significantly sooner in 2 out of 3 different cell lines that were not rolled. A similar trend for the earlier detection of adenoviruses in the same non-rolled cell lines existed. We speculate for certain viruses, such as enteroviruses, that rolling at 2 to 3 rpm may dislodge infected cells from a monolayer so that viral CPE cannot be detected sooner by a technologist. Thus, for certain enteroviruses, non-rolled cultures may be better for detecting early CPE induced by some of these viruses. Since the enteroviruses and adenoviruses in this study were not serotyped, we cannot conclude that non-rolled conditions would be best for the detection of all serotypes of these viruses.

The reason(s) for the enhanced effect of rolling on the earlier detection of some viruses in certain types of cells is not clear. Perhaps, such differences may be attributed to the density of the cells, the growth conditions used and the types of viruses isolated. The various conditions for detecting viruses by CPE need to be standardized. Since all technologists will not be equal for detecting viral CPE, alternative detection methods for rolled vs non-rolled cultures will be necessary to determine which conditions are best for viral replication.

The mechanism(s) involved with the enhancement of viral yields or CPE by rolling are not understood. Certainly, rolling would influence the physical transfer of virus and virus infected cells to non-infected cells. Data suggest that rolling cells prior to infection with HSV can enhance viral yields (12). Rolling may affect different cellular functions. Perhaps, rolling may activate cellular genes that can enhance the viral replication process. Kumei et al. (20) have shown that centrifugation can stimulate cellular proliferation, possibly via c-myc gene activation. Rolling may also induce cellular proteins needed for viral replication. Rolling cells also may enhance the expression of heat shock proteins which could stabilize and help maintain viral protein conformations for more efficient viral assembly. Previous reports have shown that heat shock proteins can be induced by viruses, are associated with certain viral proteins and these proteins may help to facilitate viral replication (3,4,8-10,16,17,23,24,31-34,38,39,42,44,45).


CONCLUSIONS

Data from this study indicate that rolling of primary monkey kidney cells, A-549 cells, and fibroblasts at 2 to 3 rpm resulted in significantly more herpes simplex viruses being detected sooner than in corresponding sister cultures that were not rolled. On the other hand, A-549 and fibroblast cultures not rolled allowed for the development of enterovirus CPE that permitted their earlier detection. Studies are warranted to determine the optimal rpm response for different cells and viruses. Our data indicate that personnel in viral diagnostic laboratories should use both rolled and non-rolled incubation conditions for the isolation of viruses.


ACKNOWLEDGMENTS

We thank the viral diagnostic employees (Annette Pagura, Jose Cuartas, Mary Connell, Kathy Mack and Janet Westlow-Schmidt) at The Children's Hospital Viral Diagnostic Laboratory for assistance with these studies and Laura Jo Hughes for editorial and typing assistance.


REFERENCES

  1. Andrewes, C.H., D.M. Chaproniere, A.E.H. Gompels, H.G. Pereira, and A.T. Roden. 1953. Propagation of common-cold virus in tissue cultures. Lancet i:546-547.

  2. Canchola, J., A.J. Vargosko, H.W. Kim, R.H. Parrott, E. Christmas, B. Jeffries, and R.M. Chanock. 1964. Antigenic variation among newly isolated strains of parainfluenza type 4 virus. Am. J. Hyg. 79:357-364.

  3. Collins, P.L. and L.E. Hightower. 1982. Newcastle disease virus stimulated the cellular accumulation of stress (heat shock) mRNAs and proteins. J. Virol. 44:703-707. MEDLINE

  4. Ellis, R.J. and S.M. van der Vies. 1991. Molecular chaperones. Annu. Rev. Biochem. 60:321-347. MEDLINE

  5. Feller, A.E., J.F. Enders, and T.H. Weller. 1940. The prolonged coexistence of vaccinia virus in high titre and living cells in roller tube cultures of chick embryonic tissues. J. Exp. Med. 72:367-388.

  6. Frothingham, T.E. 1959. Effect of aging and rotation on human amnion cell response to polio and sindbis viruses. Proc. Soc. Exp. Biol. Med. 100:505-510.

  7. Fukusho, A.,Y. Shimizu, and Y. Ito. 1981. Isolation of cytopathic porcine rotavirus in cell roller culture in the presence of trypsin. Arch. Virol. 69:49-60. MEDLINE

  8. Furlini, G., M.C. Re, M. Musiani, M.L. Zerbini, and M. LaPlaca. 1990. Enhancement of HIV-1 marker detection in cell cultures treated with mild heat-shock. Microbiologica 13:21-26. MEDLINE

  9. Garry, R.F., E.T. Ulug, and H.R. Bose, Jr. 1983. Induction of stress proteins in Sindbis virus- and vesicular stomatitis virus-infected cells. Virology 129:319-332. MEDLINE

  10. Hightower, L.E. 1991. Heat shock, stress proteins, chaperones, and proteotoxicity. Cell 66:191-197. MEDLINE

  11. Holper, J.C., L.F Miller, Y. Crawford, J.C. Sylvester, and G.S. Marquis, Jr. 1960. Further studies on multiplication, serology and antigenicity of 2060 and JH viruses. J. Infect. Dis. 107:395-401.

  12. Hughes, J.H. 1993. Physical and chemical methods for enhancing rapid detection of viruses and other agents. Clin. Micro. Rev. 6:150-175. MEDLINE

  13. Hughes, J.H., V.V. Hamparian, and C.T. Mavromoustakis. 1989. Continuous high- speed rolling versus centrifugation for detection of herpes simplex virus. J. Clin. Microbiol. 27:2884-2886. MEDLINE

  14. Hughes, J.H., C.T. Mavromoustakis, R. Wamsley, R. Vieth, and V.V. Hamparian. 1994. The effect of rolling and orbital motion on herpesvirus replication in tube cultures and shell vials. Clin. and Diag. Virol. 2:53-62.

  15. Hughes, J.H. and J.F. Sheridan. 1988. Enhanced production of poxvirus vectors by high speed rolling. J. Virol. Methods 22 MEDLINE

  16. Jindal, S. and R.A. Young. 1992. Vaccinia virus infection induces a stress response that leads to association of HSP70 with viral proteins. J. Virol. 66:5357-5362. MEDLINE

  17. Khandjian, E.W. and H. Turler. 1983. Simian virus 40 and polyoma virus induce synthesis of heat shock proteins in permissive cells. Mol. Cell. Biol. 3:1-8. MEDLINE

  18. King, P. and S.M. Goyal. 1987. Comparison of stationary and roller cultures for the isolation of herpesviruses affecting livestock. Microbiologica 10:241-245. MEDLINE

  19. Koves, B. 1979. Isolation of cytopathogenic rotavirus from neonatal calves. Acta Microbiol. Acad. Sci. Hung. 26:225-231. MEDLINE

  20. Kumei, Y., T. Nakajima, A. Sato, N. Kamata, and S. Enomoto. 1989. Reduction of G1 phase duration and enhancement of c-myc gene expression in HeLa cells at hypergravity. J. Cell Sci. 93:221-226. MEDLINE

  21. Kurogi, H., Y. Inaba, E. Takahashi, K. Sato, Y. Goto, and T. Omori. 1976. Cytopathic effects of Nebraska calf diarrhoea virus (Lincoln strain) on secondary bovine kidney cell monolayer. Natl. Inst. Anim. Health Q. 16:133-134. MEDLINE

  22. Lerner, A.M., J.D. Cherry, and M. Finland. 1962. Enhancement of cytopathic effects of reoviruses in rolled cultures of rhesus kidney. Proc. Soc. Exp. Biol. Med. 110:727-729.

  23. Macejak, D.G. and R.B. Luftig. 1991. Association of HSP70 with the adenovirus type 5 fiber protein in infected Hep-2 cells. Virology 180:120-125. MEDLINE

  24. Macejak, D.G. and P. Sarnow. 1992. Association of heat shock protein 70 with enterovirus capsid precursor P1 in infected human cells. J. Virol. 66:1520-1527. MEDLINE

  25. Mavromoustakis, C.T., D.T. Witiak, and J.H. Hughes. 1988. Effect of rolling on foci development and viral replication of herpes simplex virus (HSV). J. Virol. Methods 20:95- 100.

    MEDLINE

  26. Mavromoustakis, C.T., D.T. Witiak, and J.H. Hughes. 1988. Effect of high-speed rolling on herpes simplex virus detection and replication. J. Clin. Microbiol. 26:2328-2331. MEDLINE

  27. McNulty, M.S., G.M. Allan, and J.B. McFerran. 1977. Cell culture studies with a cytopathic bovine rotavirus. Arch. Virol. 54:201-209. MEDLINE

  28. Melnick, J.L. and J.T. Riordan. 1952. Poliomyelitis viruses in tissue cultures. IV. Protein-free nutrient media in stationary and roller tube cultures. Proc. Soc. Exp. Biol. Med. 81:208-213.

  29. Mogabgab, W.J. and B. Homes. 1961. 2060 and JH viruses in secondary monkey kidney cultures. J. Infect. Dis.108:59-62.

  30. Mufson, M.A., K.M. Johnson, H.H. Bloom, and R.M. Chanock. 1962. Multiplication and cytopathology of coxsackie A-21 virus in rotated and stationary tissue culture. Proc. Soc. Exp. Biol. Med. 110:198-203.

  31. Mulvey, M. and D.T. Brown. 1995. Involvement of the molecular chaperone BiP in maturation of sindbis virus envelope glycoproteins. J. Virol. 69:1621-1627. MEDLINE

  32. Nevins, J.R. 1982. Induction of the synthesis of a 70,000 Dalton mammalian heat shock protein by the adenovirus E1A gene product. Cell 29:913-919. MEDLINE

  33. Notarianni, E.L. and C.M. Preston. 1982. Activation of cellular stress protein genes by herpes simplex virus temperature-sensitive mutants which overproduce immediate early polypeptides. Virology 123:113-122. MEDLINE

  34. Oglesbee, M., S. Ringler, and S. Krakowka. 1990. Interaction of canine distemper virus nucleocapsid variants with 70K heat-shock proteins. J. Gen. Virol. 71:1585-1590. MEDLINE

  35. Price, W.H., H. Emerson, I. Ibler, R. Lachaine, and A. Terrell. 1959. Studies of the JH and 2060 viruses and their replationship to mild upper respiratory disease in humans. Am. J. Hyg. 69:224-249.

  36. Robbins, F.C., T.H. Weller, and J.F. Enders. 1952. Studies on the cultivation of poliomyelitis viruses in tissue culture. J. Immunol. 69:673-694.

  37. Saif, L.J., L.A. Terrett, K.L. Miller, and R.F. Cross. 1988. Serial propagation of porcine group C rotavirus (pararotavirus) in a continuous cell line and characterization of the passaged virus. J. Clin. Microbiol. 26:1277-1282. MEDLINE

  38. Sagara, J. And A. Kawai. 1992. Identification of heat shock protein 70 in the rabies virion. Virology 190:845-848. MEDLINE

  39. Sedger, L. and J. Ruby. 1994. Heat shock response to vaccinia virus infection. J. Virol. 68:4685-4689. MEDLINE

  40. Sturgill, M.A. and J.H. Hughes. 1989. Use of high-speed rolling to detect respiratory syncytial virus in cell culture. J. Clin. Microbiol. 27:577-579. MEDLINE

  41. Tyrrell, D.A.J. and R. Parsons. 1960. Some virus isolations from common colds. III. Cytopathic effects in tissue cultures. Lancet i:239-242.

  42. Walter, G., A. Carbone, and W.J. Welch. 1987. Medium tumor antigen of polyomavirus transformation-defective mutant N659 is associated with 73-kilodalton heat shcok protein. J. Virol. 61:405-410. MEDLINE

  43. Ward, R.L., D.R. Knowlton, and M.J. Pierce. 1984. Efficiency of human rotavirus propagation in cell culture. J. Clin. Microbiol. 19:748-753. MEDLINE

  44. Zerbini, M., M. Musiani, and M. LaPlaca. 1985. Effect of shock on Epstein-Barr virus and cytomegalovirus expression. J. Gen. Virol. 66:633-636. MEDLINE

  45. Zerbini, M., M. Musiani, and M. LaPlaca. 1986. Stimulating effect of heat shock on the early stage of human cytomegalovirus replication cycle. Virus Res. 6:211-216. MEDLINE


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