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.
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.
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.
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.
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.
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).
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.
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.