U.S. patent application number 09/847156 was filed with the patent office on 2001-10-25 for mixed cell diagnostic systems.
This patent application is currently assigned to DIAGNOSTIC HYBRIDS, INC.. Invention is credited to Goodrum, Patricia Gail Ray, Huang, Yung T., Scholl, David R..
Application Number | 20010034022 09/847156 |
Document ID | / |
Family ID | 22067075 |
Filed Date | 2001-10-25 |
United States Patent
Application |
20010034022 |
Kind Code |
A1 |
Scholl, David R. ; et
al. |
October 25, 2001 |
Mixed cell diagnostic systems
Abstract
The present invention generally relates to the field of
diagnostic microbiology, and, more particularly, to compositions
and methods for detecting and differentiating one or more viruses
or other intracellular parasites present in a specimen. The present
invention also provides compositions and methods to evaluate the
susceptibility of a organisms to antimicrobial agents.
Inventors: |
Scholl, David R.; (Athens,
OH) ; Huang, Yung T.; (Richmond Heights, OH) ;
Goodrum, Patricia Gail Ray; (Athens, OH) |
Correspondence
Address: |
Peter G. Carroll
MEDLEN & CARROLL, LLP
Suite 2200
220 Montgomery Street
San Francisco
CA
94104
US
|
Assignee: |
DIAGNOSTIC HYBRIDS, INC.
One President Street
Athens
OH
45701
|
Family ID: |
22067075 |
Appl. No.: |
09/847156 |
Filed: |
May 1, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09847156 |
May 1, 2001 |
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09661849 |
Sep 14, 2000 |
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09661849 |
Sep 14, 2000 |
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09567295 |
May 8, 2000 |
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09567295 |
May 8, 2000 |
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09066072 |
Apr 24, 1998 |
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6168915 |
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Current U.S.
Class: |
435/5 ; 435/29;
435/347; 435/371 |
Current CPC
Class: |
C12N 2502/27 20130101;
C12N 2502/30 20130101; C12N 5/0697 20130101; C12N 2502/256
20130101; G01N 33/5091 20130101; C12Q 1/04 20130101; C12N 2503/04
20130101 |
Class at
Publication: |
435/5 ; 435/29;
435/347; 435/371 |
International
Class: |
C12Q 001/70; C12N
005/06 |
Claims
What is claimed is:
1. A method for the detection of enterovirus in a sample,
comprising: a) providing: i) a sample suspected of containing
enterovirus; and ii) a mixed cell culture comprising at least two
cell types, wherein said cell types comprise RD cells and human
mucoepidermoid cells; b) inoculating said mixed cell culture with
at least a portion of said sample to produce an inoculated culture;
and c) observing said inoculated culture for the presence of said
enterovirus.
2. The method of claim 1, wherein said human mucoepidermoid cell is
H292.
3. The method of claim 1, wherein said enterovirus is selected from
Coxsackie viruses, echoviruses, and polioviruses.
4. A method for the detection of enterovirus in a sample,
comprising: a) providing: i) a sample suspected of containing
enterovirus; and ii) a mixed cell culture comprising at least two
cell types, wherein one of said cell types comprises Caco-2 cells;
b) inoculating said mixed cell culture with at least a portion of
said sample to produce an inoculated culture; and c) observing said
inoculated culture for the presence of said enterovirus.
5. The method of claim 4, wherein said cell type other than said
Caco-2 cells is selected from RD cells, H292 cells, BGMK cells, and
A549 cells.
6. The method of claim 4, wherein said enterovirus is selected from
Coxsackie viruses, echoviruses, polioviruses, rotaviruses,
astroviruses, and adenoviruses.
7. A method for the detection and identification of parainfluenza
virus in a sample, comprising the steps of: a) providing: i) a
sample suspected of containing parainfluenza virus; and ii) a mixed
cell culture comprising at least two cell types, wherein one of
said cell types comprises mink lung cells; b) inoculating said
mixed cell culture with at least a portion of said sample to
produce an inoculated culture; and c) observing said inoculated
culture for the presence of said parainfluenza virus.
8. The method of claim 7, wherein said mink lung cells are Mv1Lu
cells.
9. The method of claim 7, wherein said parainfluenza virus is
selected from type 1, type 2 and type 3.
Description
[0001] This is a continuation-in-part of application Ser. No.
09/066,072, filed on Apr. 24, 1998.
FIELD OF THE INVENTION
[0002] The present invention generally relates to the field of
diagnostic microbiology, and more particularly, to compositions and
methods for detecting and differentiating one or more viruses or
other intracellular parasites present in a specimen. The present
invention also provides compositions and methods to evaluate the
susceptibility of a organisms to antimicrobial agents.
BACKGROUND OF THE INVENTION
[0003] Despite recent advances in methods for the detection of
viruses using molecular methods, the detection and identification
of these organisms in cell culture remains the "gold standard" by
which most viral diseases are definitively diagnosed and the method
against which newer methods are compared (See e.g., Wiedbrauk and
Johnston, Manual of Clinical Virology, Raven Press, Inc., New York,
N.Y. [1993], pp. 1-17). Cell cultures are also used for the
detection and identification of other intracellular parasites,
especially obligate intracellular parasites such as Chlamydia and
Rickettsia.
[0004] There are two general types of cell culture methods used for
virus identification. The first method uses identification of
virus-induced cytopathic effect (CPE) as an endpoint for virus
detection. The second method utilizes molecular methods to identify
the presence of virus before CPE is evident in the infected
cultures. Both methods utilize cell cultures, which may present
problems for small laboratories with limited expertise in cell
culturing methods, space, funding, equipment, and supplies.
Depending upon the cells used, cell cultures can be difficult to
maintain and often require the efforts of skilled laboratorians. In
addition, cell cultures require equipment such as cell culture
hoods, inverted microscopes (for observation of cells), incubators
with CO.sub.2 lines, and other equipment not readily available in
many laboratories.
CPE-Based Tests
[0005] CPE-based tests often require long incubation times, as
virus-induced CPE only becomes evident after multiple rounds of
viral replication and spread of virus to neighboring cells (i.e.,
the cells are "permissive" for viral infection). Virus spread
results in the destruction of the cells surrounding the cell
originally infected. CPE-based tests have been traditionally
conducted in tubes or flasks containing a single cell type that is
adhered or anchored to the sides and/or bottom of the tube or
flask. As the virus must infect a cell, replicate, and spread to
neighboring cells in which the process is repeated, results can be
delayed for at least 28 days. Indeed, results are often not
available for 7-28 days after inoculation of the cell culture with
the virus suspension (See e.g., Leland, Clinical Virology, W. B.
Saunders, Philadelphia [1996], pp. 60-65). The time necessary to
establish visible CPE is dependent upon the rate of viral
replication, which can vary among cell types and viruses. Thus, the
amount of time needed to detect virus in a sample can greatly
vary.
Pre-CPE Tests
[0006] In contrast to CPE-based tests, pre-CPE tests require only
entry of the virus into a susceptible host cell and detectable
expression of at least one early virus-specific antigen or nucleic
acid. Detection of the virus-specific analyte or other indicator is
accomplished by a number of methods (e.g., labeled antibodies, the
polymerase chain reaction [PCR], or the use of other reporters,
such as the ELVIS.TM. system). Expression of early viral genes has
been shown to be very rapid in many virus-host cell systems in
vitro. Thus, use of pre-CPE based virus tests significantly reduces
the time required to detect and identify viruses in clinical
specimens.
[0007] Pre-CPE detection of virus is often accomplished by using
monolayers of adherent cells grown on 12 mm round coverslips
contained in I dram shell vials (i.e., the "shell vial" method or
technique). The shell vial technique uses centrifugation of the
specimen to force viral introduction into cells and enhance viral
isolation. These vials are prepared by dispensing cells into
sterile shell vials containing coverslips. The vial are incubated
in an upright position until the ceils form a monolayer on the
coverslip. For shell vial inoculation, the culture medium is
decanted from the vial, processed sample (i.e., the clinical
specimen) is added to the cell monolayer, and the vial is
centrifuged at low speed, often for one hour. After centrifugation,
fresh culture medium is added to each vial. The vials are then
incubated for the desired period of time. At the end of the
incubation period, the coverslips are stained using an antigen
detection method (e.g., immunofluorescence) or the cells are
evaluated via molecular diagnostic techniques.
[0008] In addition to viruses, shell vials are also commonly used
for the detection and identification of Chlamydia, as other methods
available for the detection and identification of these organisms
are quite cumbersome, as well as time and reagent-consuming (See
e.g., Wiedbrauk and Johnston, supra, pp. 64-76).
[0009] The major advantage of these pre-CPE testing methods is that
rapid test results are often possible. One major disadvantage to
pre-CPE testing of shell vial cultures is that this type of test is
feasible and cost-effective only if one or a few viral agents are
sought for identification, and if a high proportion of specimens
are likely to be positive (See e.g., Schmidt and Emmons, "General
Principles of Laboratory Diagnostic Methods for Viral, Rickettsial,
and Chlamydial Infections," in Schmidt and Emmons (eds.),
Diagnostic Procedures for Viral, Rickettsial and Chlamydial
Infections, American Public Health Association, Washington, D.C.,
[1989], at p. 4).
Clinical Specimens
[0010] For example, the presence of skin vesicles in the genital
area of a patient is highly suspicious for infection by herpes
simplex virus (HSV). Typically, the physician will obtain a
specimen from the affected region (i.e., a vesicle) and order a CPE
or a pre-CPE virus test on a single, HSV-susceptible cell line.
These cell lines are often supplied either in tubes, shell vials,
or multi-well plates (e.g., microtiter plates). After inoculation
of the cell line and an appropriate incubation time, confirmation
of the presence of HSV in the sample can be accomplished using one
or more of the many analytical methods (e.g., immunofluorescence,
immunoperoxidase, nucleic acid probes, or substrates for
virus-induced reporter genes).
[0011] For detection of cytomegalovirus (CMV), shell vials
containing cells from a single cell line (e.g., human fibroblast
cell lines, such as lung [MRC-5 cells] or foreskin [HFF] cells) are
often used. The cells are grown to confluency on the coverslip
within the vial, the sample is added to the vial, the vial is
incubated for 24-48 hours or longer, and an immunofluorescent
method is used to detect expression of CMV early antigen.
[0012] Accurate differential diagnosis is significantly more
difficult in virus diseases due to respiratory, gastrointestinal,
genital, or parenteral routes of transmission because many
pathogenic viruses are capable of eliciting similar symptoms or the
infection is sub-clinical (i.e., the signs and symptoms are not
readily apparent).
[0013] Of the respiratory viruses, rhinoviruses and corona viruses
are responsible for a large proportion of upper respiratory
infections. Once these viruses reach the upper respiratory mucosa,
they attach to and infect epithelial cells. Typically, these
infections last only a few days and self-resolve. Other respiratory
viruses, such as the influenzas, parainfluenzas, respiratory
syncytial virus (RSV), and various adenoviruses attach to and
infect ciliated, columnar epithelial cells. The virus-infected
cells lyse, resulting in the release of enzymes and activate
complement, resulting in a local mononuclear inflammatory response.
Normal airway clearance mechanisms fail because of the failure of
the epithelial cells to function normally. These cells may also
slough off. Cell debris from dead and dying cells often obstructs
airways, and the host becomes very susceptible to secondary
bacterial infection and/or superinfection. All of these viruses may
progress to lower respiratory involvement and pneumonia. After
replication in the respiratory epithelial cells, adenovirus may
travel via the blood to the lymphoid tissues in all areas of the
body, causing systemic infection or disease.
[0014] Standard clinical virology practice is to inoculate multiple
tubes of cell cultures with the specimen (e.g., throat swab,
nasopharyngeal swab, or sputum specimen) as the tropism of each
type of virus for specific cell types is often very narrow (i.e.,
only one type of virus may grow optimally on a single cell type).
This narrow tropism of virus for a limited number of cell types
creates at least two major practical problems for both CPE and
pre-CPE virus testing.
[0015] First, primary monkey kidney cells are currently the cell
line of choice for isolation of influenza viruses. The manufacture
of these cells requires the quarantine of source animals for long
periods prior to sacrifice and cell culture preparation. This
quarantine period is used to monitor the animals for good health
and allows time to test the animals for infection by endogenous
simian viruses such as foamy virus, SV5, and SV40. The quarantine
period also greatly reduces, but does not eliminate, the
possibility that the monkeys are infected with Monkey B Virus, a
herpesvirus that is highly fatal to humans. In addition, there are
other problems related to the use of monkeys for the production of
primary cell cultures, including the reduction in the stock of
suitable animals due to importation concerns and monkey population
considerations.
[0016] Second, additional continuous cell lines are required in
order to detect respiratory viruses other than influenza virus.
Thus, multiple cell lines are used in order to diagnose the viral
infection/disease of each patient. The need for multiple units of
individual cell lines is compounded in methods using pre-CPE tests
for detection and identification of respiratory viruses. Pre-CPE
testing for respiratory viruses requires the expenditure of
significant labor in handling coverslips, the added expense of
molecular reagents used with multiple cell lines for both positive
and negative specimens, and the significant labor associated with
microscopically reading each of the multiple cell lines inoculated
in the panel of cell lines.
[0017] However, despite these drawbacks, shell vial technology
using single cell types in multiple units (tubes, shell vials,
etc.), is still currently used to detect respiratory viruses, as it
is a proven method. For example, detection of RSV in 16 hours using
shell vials containing only HEp-2 cells yielded more positives than
antigen detection methods applied directly to the clinical
specimen, and as many positives as conventional cell cultures
(Smith et al., J. Clin. Microbiol., 29:463-465 [1991]). Isolation
of other respiratory viruses has also been possible with shell vial
cultures containing a monolayer of a single cell type. For example,
using vials of primary monkey kidney cells and A549 cells incubated
for 40 hours, 83% of adenoviruses, 94% of influenza B, and 80% of
parainfluenza virus types 1, 2, and 3 were identified (Rabalais et
al., J. Clin. Microbiol., 30:1505-1508 [1992]). In another report,
50% of adenoviruses, 94% of influenza A viruses, 100% of influenza
B viruses, and 100% of parainfluenza viruses, in shell vials of
primary rhesus monkey kidney cells, and 92% of RSV in shell vials
of HEp-2 cells incubated for 2-4 days (See e.g., Olsen et al., J.
Clin. Microbiol., 31:422-425 [1993]; and Leland, Clinical Virology,
W. B. Saunders Company, Philadelphia, Pa. [1996], at p. 85-86).
[0018] Although these methods provide relatively rapid results
(i.e., as opposed to the long incubation periods often necessary
for CPE tests), there remains a need in clinical and reference
virology laboratories for cell culture methods and compositions for
the reliable detection and identification of viruses in a single,
easy-to-manipulate unit that provides rapid detection and
identification in a cost-effective manner, while also providing the
sensitivity of a diagnostic assay system.
SUMMARY OF THE INVENTION
[0019] The present invention generally relates to the field of
diagnostic microbiology, and more particularly, to compositions and
methods for detecting and differentiating one or more viruses or
other intracellular parasites present in a specimen. The present
invention also provides compositions and methods to evaluate the
susceptibility of a organisms to antimicrobial agents.
[0020] In particular, the present invention provides methods and
compositions suitable for the detection of viruses using CPE-based
and pre-CPE methods. The preferred embodiments encompass mixed cell
cultures which contain at least two different cell types. In some
preferred embodiments, the mixed cell cultures contain two
different cell types, while in other embodiments, the mixed cell
cultures Contain three or more different cell types. Thus, it is
intended that the present invention encompass compositions in which
at least two cell types are mixed together in one container (e.g.,
flask, tube, shell vial, or any other container suitable for the
growth of cells). Importantly, each cell type within these mixed
cell cultures retains its susceptibility to viruses and other
intracellular parasites as if it was in a single cell culture
(i.e., a cell culture that contains only one cell type, as known in
the art). In addition, the mixed cell cultures of the present
invention remain viable for as long as required for their use in
diagnostic assays. In particularly preferred embodiments, the cell
types included within mixed cell cultures are present in
approximately the same ratio (i.e., for a two cell type mixed,
there is a 50:50 ratio of cell types). However, it is not intended
that the present invention be limited to any particular ratio of
cell types in mixed culture, as various detection systems may be
optimized using different ratios. For example, in some
circumstances, a two cell mixture of 45:55, 40:60, or even 35:75,
may be more suited than a 50:50 ratio.
[0021] The present invention also provides methods and compositions
suitable for the detection and identification of non-viral obligate
intracellular and intracellular parasites, such as members of the
Chlamydiales and Ricketsiales.
[0022] The present invention also contemplates compositions
comprising a cell culture suitable for the detection of
intracellular parasites, wherein the cell culture comprises at
least two cell types susceptible to infection by at least one
intracellular parasite. In some preferred embodiments of the
composition, the cell types comprise a first cell type and a second
cell type. In some embodiments, the first cell type consists of
buffalo green monkey kidney cells and the second cell type consists
of mink lung cells. In other embodiments, the first cell type
consists of mink lung cells and the second cell type consists of
human mucoepidermoid cells. In yet other embodiments, the first
cell type consists of human lung carcinoma cells and the second
cell type consists of human mucoepidermoid cells. In still other
embodiments, the first cell type consists of buffalo green monkey
kidney cells and the second cell type consists of human embryonic
lung cells. In further embodiments, the cell type consists of human
epidermoid laryngeal carcinoma cells and the second cell type
consists of McCoy cells. In additional embodiments, the first cell
type consists of mink lung cells and the second cell type consists
of human diploid lung cells.
[0023] In some preferred embodiments, the cell types of the
composition are susceptible to respiratory viruses, including but
not limited to influenza viruses of any type (e.g., Influenza A,
Influenza B, and Influenza C) and/or strain, RSV, cytomegalovirus,
parainfluenza viruses, respiratory syncytial virus, rhinoviruses,
coronoviruses, and adenoviruses. In yet other embodiments, the cell
types of the composition are susceptible to enteroviruses,
including but not limited to any type and/or strain of echovirus,
poliovirus, and Coxsackie virus (e.g., Coxsackie A and B viruses),
and numbered EV strains. In addition to enteroviruses, it is
contemplated that the present invention encompass cell types that
are susceptible to picornaviruses such as Hepatitis A.
[0024] The present invention also provides methods for the
detection and identification of intracellular parasites in a
sample, comprising the steps of providing a sample suspected of
containing one or more intracellular parasites; and a mixed cell
culture comprising at least two cell types; inoculating the mixed
cell culture with the sample to produce an inoculated culture; and
observing the inoculated culture for the presence of the one or
more intracellular parasites.
[0025] In some embodiments of the method, the intracellular
parasite is a virus. In some particularly preferred embodiments,
the virus is selected from the group consisting of cytomegalovirus,
influenza viruses, parainfluenza viruses, respiratory syncytial
virus, rhinoviruses, coronoviruses, and adenoviruses. In yet other
embodiments of the methods, the virus is an enterovirus. In other
particularly preferred embodiments, the enterovirus is selected
from the group consisting of poliovirus, Coxsackie viruses and
echoviruses (e.g., Coxsackie A and B viruses), and numbered EV
strains. In addition to enteroviruses, it is contemplated that the
present invention encompass cell types that are susceptible to
picornaviruses such as Hepatitis A. In still other preferred
embodiments, the virus is a herpes virus. In other particularly
preferred embodiments, the herpes virus is selected from the group
consisting of Herpes Simplex Type 1, Herpes Simplex Type 2,
Cytomegalovirus, Varicella-Zoster virus, Epstein-Barr virus, Human
Herpes Virus 6, Human Herpes Virus 7, and Human Herpes Virus 8. In
yet other preferred embodiments, the intracellular parasite is a
member of the genus Chlamydia. In still other particularly
preferred embodiments, the intracellular parasite is C.
trachomatis.
[0026] In some preferred embodiments of the methods, the cell types
comprise a first cell type and a second cell type. In some
preferred embodiments, the first cell type is a mink lung cell, and
the second cell type is a human mucoepidermoid cell. In other
preferred embodiments, the first cell type is a buffalo green
monkey kidney cell and the second cell type is a human
mucoepidermoid cell. In yet another alternative embodiment, the
first cell type is a genetically engineered baby hamster kidney
cell and the second cell type is a mink lung cell. In still other
embodiments, the first cell type is a first genetically engineered
cell type and the second cell type is a second genetically
engineered cell type.
[0027] It is contemplated that the methods of the present invention
will be used in conjunction with controls of known positivity and
negativity for the virus(es) and/or other intracellular organism of
interest.
[0028] The present invention also provides methods for the
detection and identification of intracellular parasites in a
sample, comprising the steps of providing: a sample suspected of
containing one or more intracellular parasites; and a mixed cell
culture comprising a first cell type and a second cell type;
inoculating the mixed cell culture with the sample to produce an
inoculated culture; and observing the inoculated culture for the
presence of the one or more intracellular parasites.
[0029] In some particularly preferred embodiments, the
intracellular parasite is a virus. In some particularly preferred
embodiments, the virus is selected from the group consisting of
cytomegalovirus, influenza viruses, parainfluenza viruses,
respiratory syncytial virus, rhinoviruses, coronoviruses, and
adenoviruses. In yet other embodiments of the methods, the virus is
an enterovirus. In other particularly preferred embodiments, the
enterovirus is selected from the group consisting of poliovirus,
Coxsackie viruses and echoviruses (e.g., Coxsackie A and B
viruses), and numbered EV strains. In addition to enteroviruses, it
is contemplated that the present invention encompass cell types
that are susceptible to picornaviruses such as Hepatitis A. In
still other preferred embodiments, the virus is a herpes virus. In
other particularly preferred embodiments, the herpes virus is
selected from the group consisting of Herpes Simplex Type 1, Herpes
Simplex Type 2, Cytomegalovirus, Varicella-Zoster virus,
Epstein-Barr virus, Human Herpes Virus 6, Human Herpes Virus 7, and
Human Herpes Virus 8. In yet other preferred embodiments, the
intracellular parasite is a member of the genus Chlamydia. In still
other particularly preferred embodiments, the intracellular
parasite is C. trachomatis.
[0030] In some preferred embodiments of the methods, the cell types
comprise a first cell type and a second cell type. In some
preferred embodiments, the first cell type is a mink lung cell, and
the second cell type is a human mucoepidermoid cell. In other
preferred embodiments, the first cell type is a buffalo green
monkey kidney cell and the second cell type is a human
mucoepidermoid cell. In yet another alternative embodiment, the
first cell type is a genetically engineered baby hamster kidney
cell and the second cell type is a mink lung cell. In still other
embodiments, the first cell type is a first genetically engineered
cell type and the second cell type is a second genetically
engineered cell type.
[0031] It is contemplated that the methods of the present invention
will be used in conjunction with controls of known positivity and
negativity for the virus(es) and/or other intracellular organism of
interest.
[0032] The present invention further provides methods for the
detection of influenza virus, comprising the steps of providing a
sample suspected of containing influenza virus, and mink lung
cells; inoculating the mink lung cells with the sample; and
detecting the presence of the influenza within the mink lung cells.
In particularly preferred embodiments, the mink lung cells are
Mv1Lu cells. In alternative embodiments, the influenza virus is
selected from the group consisting of Influenza A, Influenza B, and
Influenza C.
[0033] It is contemplated that the methods of the present invention
will be used in conjunction with controls of known positivity and
negativity for the virus(es) and/or other intracellular organism of
interest.
[0034] In one embodiment, the present invention provides methods
for the detection of infectious virus in a specimen comprising the
steps of: a) providing a specimen suspected of containing a virus,
a cell line permissive for infection by the virus, and a
genetically engineered cell line containing an oligonucleotide
having a sequence comprising a promoter sequence derived from the
virus, wherein the promoter sequence is operably linked to a
reporter gene, and wherein the expression of the reporter gene is
dependent upon and quantitatively proportional to the presence of
the virus; b) mixing together the permissive cell line and the
genetically engineered cell line to create a mixed cell culture; c)
inoculating the mixed cell culture with the specimen under
conditions which permit the infection of the mixed cell culture by
the virus; and d) detecting the expression of the reporter gene and
thereby detecting the presence of virus in the specimen. In one
preferred embodiment, the mixed cell culture is a mixture
consisting of 80-99% of the permissive cell line and 1-20% of the
genetically engineered cell line. In other preferred embodiments,
the mixed cell culture is a mixture consisting of equal proportions
of the cell types used in the mixture.
[0035] In one embodiment of the method, the genetically engineered
cell line contains an oligonucleotide having a sequence comprising
a herpesvirus inducible promoter operably linked to a reporter gene
selected from the group comprising the Escherichia coli lacZ gene
and a luciferase gene. In one preferred embodiment of the method,
the genetically engineered cell line is BHKICP10LacZ. In an
alternative preferred embodiment, the genetically engineered cell
line is BHKICP6LacZ. However, it is not intended that the reporter
gene be limited to the lacZ and luciferase genes. Indeed, it is
contemplated that any suitable reporter gene known to those in the
art will be useful in the method of the present invention.
[0036] It is also contemplated that various permissive cell lines
will be useful in the method of the present invention. In one
embodiment, the permissive cell line is permissive for infection
with herpesvirus. In a particularly preferred embodiment, the
permissive cell line is MRC-5.
[0037] It is contemplated that the method of the present invention
will be used in conjunction with controls of known positivity and
negativity for the virus(es) of interest. Thus, for mixed cultures
in which genetically engineered cell lines are used, it is
contemplated that the pattern of reporter gene expression present
in a test sample (e.g., from a clinical specimen) will be compared
to the patterns of reporter gene expression in control samples
known to be positive and/or negative for the virus(es) of interest.
It is also contemplated that effects unrelated to the expression of
the reporter gene will be detectable, including but not limited to
CPE. These effects, alone and in combination with the reporter gene
expression may be used to detect the presence of viral
infection.
[0038] The present invention also provides methods for the typing
of infectious herpesvirus in specimens, comprising the steps of: a)
providing a specimen suspected of containing one or more members of
the herpesvirus family, a cell line permissive for infection by one
or more members of the herpesvirus family, a genetically engineered
cell line containing an oligonucleotide having a sequence
comprising a promoter sequence derived from a member of the
herpesvirus family wherein the promoter sequence is operably linked
to a reporter gene, and the expression of the reporter gene is
dependent upon and quantitatively proportional to the presence of
herpesvirus and wherein the expression of the reporter gene varies
in a distinguishable manner as a result of the presence of
different members of the herpesvirus family; b) mixing together the
permissive cell line and the genetically engineered cell line to
create a mixed cell culture; c) inoculating this mixed cell culture
with the specimen under conditions which permit the infection of
the mixed cell culture by members of the herpesvirus family,
wherein the infection results in a distinguishable pattern of
expression by the reporter gene; d) detecting the expression of the
reporter gene and thereby detecting the presence of one or more
members of the herpesvirus family in the specimen; and e)
identifying the presence of a specific member of the herpesvirus
family based upon the resulting distinguishable pattern. It is
contemplated that this pattern of expression will be observable by
various assisted and non-assisted methods, including visual
observation by eye, spectrophotometric observation, etc. It is not
intended that the detection of distinguishable pattern(s) be
limited to any particular method of detection.
[0039] In a preferred embodiment of the typing method of the
present invention, the mixed cell culture is a mixture consisting
of 80-99% of the permissive cell line and 1-20% of the genetically
engineered cell line. In yet other preferred embodiments, the cell
types are in approximate equal proportions in the mixed cell
cultures. As with the first method described, it is not intended
that the present invention be limited to any particular
herpesvirus. In one particular embodiment, the member of the
herpesvirus family detected and typed using the method of the
present invention is selected from the group comprising HSV-1,
HSV-2, CMV, VZV, EBV, and human herpes viruses such as HHV-6,
HHV-7, and HHV-8. It is intended that one or more herpesviruses may
be detected and typed in one specimen. In this manner, co-infection
with multiple herpesviruses may be diagnosed. For example, it is
contemplated that mixed infections with HSV-1 and HSV-2 may be
detectable and the infections distinguished using the methods of
the present invention.
[0040] In one embodiment of the typing method, the reporter gene
comprises E. coli lacZ gene. However, it is not intended that the
reporter gene be limited to lacZ. Indeed, it is contemplated that
any reporter gene may be used in this method. In one particularly
preferred embodiment, the detection of the reporter gene is
accomplished through the use of histochemical staining. It is
contemplated that one member of the herpesvirus family will produce
an histochemically pattern of expression that is distinguishable
from the histochemical patterns produced by other members of the
herpesvirus family. In this manner, it is possible to use the
methods of the present invention to distinguish infection with one
herpesvirus from infection with another herpesvirus.
[0041] It is contemplated that the method of the present invention
will be used in conjunction with controls of known positivity and
negativity for the virus(es) of interest. Thus, it is contemplated
that the pattern of expression present in a test sample (e.g., from
a clinical specimen) will be compared to the patterns of expression
in control samples known to be positive and/or negative for the
virus(es) of interest. It is also contemplated that effects
unrelated to the expression of the reporter gene will be
detectable, including but not limited to CPE. These effects, alone
and in combination with reporter gene expression may be used to
detect the presence of viral infection, as well as provide
information to distinguish between viruses.
[0042] In yet another embodiment, the present invention provides a
composition comprising a mixed cell culture, wherein the mixed cell
culture comprises the combination of a genetically engineered cell
line transformed with a promoter sequence from a virus, wherein the
promoter sequence is operably linked to a reporter gene, and
wherein expression of the reporter gene is dependent upon and
quantitatively proportional to the presence of virus, and a
non-engineered cell line which is permissive for virus
infection.
[0043] In one embodiment of the composition, the mixed cell culture
is a mixture consisting of 1-20% of the genetically engineered cell
line and 80-99% of the permissive cell line. In yet other preferred
embodiments, the cell types are in approximate equal proportions in
the mixed cell cultures. In one preferred embodiment of the
composition, the genetically engineered cell line component may
comprise a promoter for a gene that encodes ribonucleotide
reductase. In an alternative preferred embodiment, the promoter may
comprise genes that encode one or more subunits of ribonucleotide
reductase. In one particularly preferred embodiment, the
genetically engineered cell line is BHKICP10LacZ, while in another
particularly preferred embodiment, the genetically engineered cell
line is BHKICP6LacZ. In an alternative embodiment of the
composition, the genetically engineered cell line comprises an E.
coli lacZ gene positioned 3' to a virus inducible promoter. It is
contemplated that this lacZ gene be positioned immediately 3' to
this virus-inducible promoter. However, it is not intended that
these sequences will be contiguous. Indeed, it is contemplated only
that the reporter and promoter genes are operably linked.
Furthermore, it is contemplated that the composition will comprise
promoter sequences from any virus, including but not limited to
members of the herpesvirus family. It is also contemplated that the
non-engineered cell line be permissive for infection by any number
of viruses, including but not limited to members of the herpesvirus
family.
[0044] In one preferred embodiment, the composition includes a
genetically engineered cell line which includes a promoter for a
gene that encodes a ribonucleotide reductase large subunit and the
virus is a member of the herpesvirus family selected from the group
consisting of HSV-1, HSV-2, CMV, VZV, EBV, HHV-6, HHV-7, and HHV-8.
However, it is not intended that the present invention be limited
to any particular herpesvirus. In one preferred embodiment, the
genetically engineered cell line component contains an ICP10
promoter and the herpesvirus family member is HSV-2, while in
another preferred embodiment, the genetically engineered cell line
comprises an ICP6 promoter and the herpesvirus family member is
HSV-1.
[0045] It is contemplated that the detection of reporter gene
expression be accomplished through the use of various methods,
including, but not limited to colorimetric, fluorimetric or
luminometric assays or assay systems. In one preferred embodiment,
the reporter gene encodes .beta.-galactosidase.
[0046] In one embodiment, the composition includes a genetically
engineered cell line that is a mammalian cell line susceptible to
infection by virus. In one preferred embodiment, the genetically
engineered cell line comprises baby hamster kidney cells (e.g.,
cell lines derived from BHK cells). In one embodiment, the
composition includes a permissive cell line that is permissive to
infection by herpesviruses, including but not limited to HSV-1 and
HSV-2. In a particularly preferred embodiment, the permissive cell
line is MRC-5. It is not intended that the composition of the
present invention be limited to detection of viral infection based
on the expression of the reporter gene, as effects such as CPE may
also be detectable.
[0047] The present invention also provides a kit for assaying for
the presence of infectious herpesvirus in a specimen. The kit
includes: a) a supply of a mixed cell line comprised of a cell line
of genetically engineered mammalian cells susceptible to infection
by herpesvirus, wherein the cell line contains an oligonucleotide
having a sequence comprising a virus promoter sequence operably
linked to a reporter gene, and where the expression of the reporter
gene is dependent upon and quantitatively proportional to the
presence of virus in the specimen; and a cell line permissive for
virus; and b) a supply of reagents to detect the expression of the
reporter gene. It is not intended that the promoter sequences
present within the genetically engineered cell line be limited to
any particular virus or virus family. It is contemplated that any
virus promoter will be useful in the kit of the present invention.
However, in one preferred embodiment, herpesvirus promoter
sequences are present in the genetically engineered cell line.
[0048] It is contemplated that various promoter sequences will be
useful in the kit of the present invention. However, in a preferred
embodiment, the promoter encodes either a complete ribonucleotide
reductase enzyme, or in the alternative, subunits of ribonucleotide
reductase. In one particularly preferred embodiment, the promoter
sequence contains a promoter for a gene that encodes a
ribonucleotide reductase large subunit and the herpesvirus is a
herpesvirus family member selected from the group consisting of
HSV-1, HSV-2, CMV, VZV, EBV, HHV-6, HHV-7, and HHV-8. However, it
is not intended that the kit will be limited to this list of
herpesviruses. Indeed, it is contemplated that any herpesvirus may
be detected using the present kit. In one particularly preferred
embodiment of the kit, the promoter sequence contains an ICP10
promoter and the herpesvirus family member is HSV-2, while in an
alternative preferred embodiment, the promoter sequence contains an
ICP6 promoter and the herpesvirus family member is HSV-1.
[0049] In one preferred embodiment of the kit, the promoter
sequence present in the genetically engineered cell line comprises
an E. coli lacZ gene that is operably linked to a herpesvirus
inducible promoter. In one particularly preferred embodiment, the
genetically engineered mammalian cells are BHKICP10LacZ cells,
while in an alternative embodiment, the cells are BHKICP6LacZ
cells.
[0050] In one preferred embodiment, the reporter gene encodes
.beta.-galactosidase. However, it is not intended that the present
invention be limited to any particular reporter gene. It is also
contemplated that the reporter gene will encode any number of
enzymes that are amenable to detection by various methods,
including but not limited to such methods as colorimetric,
fluorimetric or luminometric assay systems. In one preferred
embodiment of the kit, the reagents provided for the detection of
reporter gene expression may include, but are not limited to,
solutions of 5-bromo-4-chloro-3-indolyl-.beta.-D-gala-
ctopyranoside, o-nitrophenyl-galactopyranoside solution, and
fluorescein di-.beta.-D-galactopyranoside. However, it is not
intended to limit the kit to these assay systems, as other systems
(e.g., radiometric assay systems) may be useful.
[0051] It is contemplated that the kit of the present invention may
also include additional components, such as materials suitable for
positive and negative controls and instructions for use. It is not
intended that the kit of the present invention be limited to the
mixed cell line and reagents for the detection of reporter gene
expression. It is also intended that the kit will be useful for
detection of viral effects on cells other than and unrelated to
reporter gene expression. For example, it is contemplated that the
kit may be useful for detection of CPE.
[0052] The invention further provides methods for the detection and
identification of enterovirus in a sample by using mixed cell type
cultures which contain RD cells and human mucoepidermoid cells. In
particular, the invention provides a method for the detection and
identification of enterovirus in a sample, comprising: a)
providing: i) a sample suspected of containing enterovirus; and ii)
a mixed cell culture comprising at least two cell types, wherein
the cell types comprise RD cells and human mucoepidermoid cells; b)
inoculating the mixed cell culture with at least a portion of the
sample to produce an inoculated culture; and c) observing the
inoculated culture for the presence of the enterovirus. Without
intending to limit the invention to any particular mucoepidermoid
cell, in one preferred embodiment, the human mucoepidermoid cell is
H292. Also while not intending to limit the invention to the type
of enterovirus, in an alternative preferred embodiment, the
enterovirus is selected from Coxsackie viruses, echoviruses, and
polioviruses.
[0053] Also provided herein are methods for the detection and
identification of enterovirus in a sample by using mixed cell type
cultures containing Caco-2 cells in combination with other cell
types. In particular, the invention provides a method for the
detection and identification of enterovirus in a sample,
comprising: a) providing: i) a sample suspected of containing
enterovirus; and ii) a mixed cell culture comprising at least two
cell types, wherein one of the cell types comprises Caco-2 cells;
b) inoculating the mixed cell culture with at least a portion of
the sample to produce an inoculated culture; and c) observing the
inoculated culture for the presence of the enterovirus. It is not
intended that the invention be limited to the type or source of the
cell types which are preset in mixed cell culture with Caco-2
cells. Nonetheless, in one embodiment, the cell type other than the
Caco-2 cells is selected from RD cells, H292 cells, BGMK cells, and
A549 cells. The methods provided herein are not intended to be
limited to the type of enterovirus. However, in one embodiment, the
enterovirus is selected from Coxsackie viruses, echoviruses,
polioviruses, rotaviruses, astroviruses, and adenoviruses.
[0054] Also provided by the instant invention are methods for the
detection and identification of parainfluenza virus using mixed
cell type cultures which contain mink lung cells. Specifically
provided is a method for the detection and identification of
parainfluenza virus in a sample, comprising the steps of: a)
providing: i) a sample suspected of containing parainfluenza virus;
and ii) a mixed cell culture comprising at least two cell types,
wherein one of the cell types comprises mink lung cells; b)
inoculating the mixed cell culture with at least a portion of the
sample to produce an inoculated culture; and c) observing the
inoculated culture for the presence of the parainfluenza virus.
Without intending to limit the invention to any particular type of
mink lung cell, in one preferred embodiment, the mink lung cells
are Mv1Lu cells. Also without intending to limit the invention to a
particular parainfluenza virus, in a preferred embodiment of the
methods, the parainfluenza virus is selected from type 1, type 2
and type 3.
[0055] The invention further provides a method for the detection
and identification of cytomegalovirus in a sample, comprising the
steps of: a) providing: i) a sample suspected of containing
cytomegalovirus; and ii) a mixed cell culture comprising MRC-5
cells and at least one cell type selected from McCoy cell,
genetically engineered baby hamster kidney cell, A549 cell, HEp-2
cell, Mv1Lu cell, and H292 cell; b) inoculating the mixed cell
culture with the sample to produce an inoculated culture; and c)
observing the inoculated culture for the presence of the
cytomegalovirus.
[0056] Also provided herein is a method for the detection and
identification of enterovirus in a sample, comprising the steps of:
a) providing: i) a sample suspected of containing enterovirus; and
ii) a mixed cell culture comprising CV-1 cells and at least one
cell type which comprises a first cell type selected from MRC-5
cell, McCoy cell, genetically engineered baby hamster kidney cell,
A549 cell, HEp-2 cell, Mv1Lu cell, and H292 cell; b) inoculating
the mixed cell culture with the sample to produce an inoculated
culture; and c) observing the inoculated culture for the presence
of the enterovirus.
[0057] The invention additionally discloses a method for the
detection and identification of enterovirus in a sample, comprising
the steps of: a) providing: i) a sample suspected of containing
enterovirus; and ii) a mixed cell culture comprising buffalo green
monkey cells and at least one cell type which comprises a first
cell type selected from MRC-5 cell, CV-1 cell, McCoy cell,
genetically engineered baby hamster kidney cell, A-549 cell, HEp-2
cell, Mv1Lu cell, and H292 cell; b) inoculating the mixed cell
culture with the sample to produce an inoculated culture; and c)
observing the inoculated culture for the presence of the
enterovirus.
[0058] Further provided by this disclosure is a method for the
detection and identification of herpes simplex virus in a sample,
comprising the steps of: a) providing: i) a sample suspected of
containing herpes simplex virus; and ii) a mixed cell culture
comprising genetically engineered baby hamster kidney cells and at
least one cell type which comprises a first cell type selected from
MRC-5 cell, CV-1 cell, buffalo green monkey kidney cell, McCoy
cell, A549 cell, HEp-2 cell, Mv1Lu cell, and H292 cell; b)
inoculating the mixed cell culture with the sample to produce an
inoculated culture; and c) observing the inoculated culture for the
presence of the herpes simplex virus.
[0059] Yet another method is provided for the detection and
identification of adenovirus in a sample, comprising the steps of:
a) providing: i) a sample suspected of containing adenovirus; and
ii) a mixed cell culture comprising A549 cells and at least one
cell type which comprises a first cell type selected from MRC-5
cell, CV-1 cell, buffalo green monkey kidney cell, McCoy cell,
genetically engineered baby hamster kidney cell, HEp-2 cell, Mv1Lu
cell, and H292 cell; b) inoculating the mixed cell culture with the
sample to produce an inoculated culture; and c) observing the
inoculated culture for the presence of the adenovirus.
[0060] Also provided herein is a method for the detection and
identification of respiratory syncytial virus in a sample,
comprising the steps of: a) providing: i) a sample suspected of
containing respiratory syncytial virus; and ii) a mixed cell
culture comprising HEp-2 cells and at least one cell type which
comprises a first cell type selected from MRC-5 cell, CV-1 cell,
buffalo green monkey kidney cell, McCoy cell, genetically
engineered baby hamster kidney cell, A549 cell, Mv1Lu cell, and
H292 cell; b) inoculating the mixed cell culture with the sample to
produce an inoculated culture; and c) observing the inoculated
culture for the presence of the respiratory syncytial virus.
[0061] Additionally provided is a method for the detection and
identification of a virus selected from influenza virus and
parainfluenza virus in a sample, comprising the steps of: a)
providing: i) a sample suspected of containing a virus selected
from influenza virus and parainfluenza virus; and ii) a mixed cell
culture comprising Mv1Lu cells and at least one cell type which
comprises a first cell type selected from MRC-5 cell, CV-1 cell,
buffalo green monkey kidney cell, McCoy cell, genetically
engineered baby hamster kidney cell, A549 cell, HEp-2 cell, and
H292 cell; b) inoculating the mixed cell culture with the sample to
produce an inoculated culture; and c) observing the inoculated
culture for the presence of the virus selected from influenza virus
and parainfluenza virus.
[0062] The invention also provides a method for the detection and
identification of a virus selected from respiratory syncytial
virus, adenovirus, cytomegalovirus, and parainfluenza virus in a
sample, comprising the steps of: a) providing: i) a sample
suspected of containing a virus selected from respiratory syncytial
virus, adenovirus, cytomegalovirus, and parainfluenza virus; and
ii) a mixed cell culture comprising H292 cells and at least one
cell type which comprises a first cell type selected from MRC-5
cell, CV-1 cell, buffalo green monkey kidney cell, McCoy cell,
genetically engineered baby hamster kidney cell, A549 cell, HEp-2
cell, and Mv1Lu cell, and a second cell type; b) inoculating the
mixed cell culture with the sample to produce an inoculated
culture; and c) observing the inoculated culture for the presence
of the virus selected from respiratory syncytial virus, adenovirus,
cytomegalovirus, and parainfluenza virus. Without intending to
limit the second cell type to any particular cell, in one
embodiment, the second cell type is selected from MRC-5 cell, CV-1
cell, buffalo green monkey kidney cell, genetically engineered baby
hamster kidney cell, A549 cell, HEp-2 cell, Mv1Lu cell, and H292
cell.
DESCRIPTION OF THE INVENTION
[0063] The present invention generally relates to the field of
diagnostic microbiology, and more particularly, to compositions and
methods for detecting and differentiating one or more viruses or
other intracellular parasites present in a specimen. The present
invention also provides compositions and methods to evaluate the
susceptibility of a organisms to antimicrobial agents.
[0064] The present invention provides methods and compositions for
the detection of several different viruses, as well as other
intracellular organisms present in clinical and other specimens in
a single cell culture unit comprised of a mixture of cells grown in
a manner to co-exist as a monolayer of relatively equivalent ratio
and demonstrating complementary susceptibilities to a wider range
of viruses and/or other organisms than could be detected by each
individual cell line. For example, the viral assays involve
inoculating a cell mixture with a specimen suspected of containing
a virus, allowing a sufficient period of time for the virus
infectious cycle to proceed, followed by the detection and/or
quantification of the number of virus-infected cells to determine
the number of infectious virions in the specimen. This detection
step may be accomplished using any number of available confirmation
methods, including specific viral antigen detection using
antigen-specific antibodies, nucleic acid probes, and reporter gene
detection. The assay also provides reliable methods and
compositions for the quantification of the number of infectious
virions present in a sample. In addition, the methods and
compositions of the present invention are sufficiently sensitive
that the presence of a single virion in a specimen may be
detected.
[0065] The present invention also provides compositions comprising
novel mixtures of various cell types traditionally used in single
cell assays. In preferred embodiments, the cells are mixed to
produce mixed monolayer cell cultures. One such mixed cell culture
includes mink lung (e.g., Mv1Lu) cells co-cultivated with human
mucoepidermoid cells (e.g., NCI-H292; also referred to as "H292"
cells). This cell mixture is susceptible to viruses such as
influenza A, influenza B, RSV, parainfluenza types 1, 2, and 3,
adenovirus, and CMV (i.e., the group of viruses most commonly
associated with respiratory virus disease). In other mixed
cultures, buffalo green monkey kidney cells (BGMK) are
co-cultivated with NCI-H292 cells for the detection and
identification of enteroviruses, such as poliovirus, echoviruses
and Coxsackie B virus (e.g., Coxsackie A and B viruses), and
numbered EV strains. In addition to enteroviruses, it is
contemplated that the present invention encompass cell types that
are susceptible to picornaviruses such as Hepatitis A.
[0066] The present invention also provides compositions comprising
novel mixtures of different cell types traditionally used in single
cell assays that are co-cultivated with genetically engineered
cells. In particularly preferred embodiments, the genetically
engineered cell line is a DNA-transfected cell line that is
susceptible to infection by a virus, the cell line having been
stably transformed with a chimeric gene comprising a
virus-inducible promoter and a gene coding for an enzyme, the
expression of the enzyme being dependent upon the presence of the
virus. Such genetically engineered cells are, for example,
described in U.S. Pat. No. 5,418,132, herein incorporated by
reference. In one preferred embodiment, a cell mixture includes
human lung fibroblasts (e.g., MRC-5 cells) co-cultivated with a
stable baby hamster kidney (BHK) cell line, the genome of which has
been engineered to contain the E. coli lacZ gene behind (i.e., 3'
to) an inducible HSV promoter, HSV-1 ICP6 promoter (BHK/ICP6LacZ-5
cells are available from the ATCC as CRL-12072). This cell mixture
is susceptible to infection by CMV and HSV types 1 and 2.
[0067] In yet another embodiment, the present invention provides
compositions comprising novel mixtures of different types of
genetically engineered cells. In particularly preferred
embodiments, the genetically engineered cell line is a
DNA-transfected cell line that is susceptible to infection by a
virus, the cell line having been stably transformed with a chimeric
gene comprising a virus-inducible promoter and a gene coding for an
enzyme, the expression of the enzyme being dependent upon the
presence of the virus. The second genetically engineered cell line
is a DNA-transfected cell line susceptible to viral infection,
which is stably transformed with a chimeric gene comprising a
virus-inducible promoter, and a gene encoding a second enzyme
(i.e., an enzyme that is different from that associated with the
first cell line), in which the expression of the second enzyme is
dependent upon the presence of a second virus. In one preferred
embodiment, a cell mixture is prepared in which engineered BHK
cells (e.g., BHK/ICP6/LacZ-5 cells) are co-cultivated with a stable
mink lung cell line (Mv1Lu), the genome of which has been
engineered to contain the an inducible CMV promoter (the CMV UL45
promoter); these cells are referred to as "MLID5" cells, and are
disclosed in U.S. patent appln. Ser. No. 08/846,026, herein
incorporated by reference. This cell mixture is susceptible to
infection by CMV and HSV virus types 1 and 2 (HSV-1 and HSV-2),
with CMV infecting the genetically engineered BHK cells, and HSV-1
and HSV-2 preferentially infecting the mink lung cells. In another
embodiment, the present invention contemplates the use of
genetically engineered cells (e.g., mink lung cells) in which the
cell genome is engineered to contain the firefly luciferase gene
behind (i.e., 3' to) an inducible CMV promoter; these cells are
also described in U.S. patent appln. Ser. No. 08/846,026. However,
it is not intended that the present invention be limited to any
particular cell types or cell lines, nor is it intended that the
present invention be limited to any particular combinations of
cells. It is also not intended that the present invention be
limited in terms of the genetically engineered cells.
[0068] The following Table provides a matrix which indicates the
ability of various cells to form single, confluent monolayers, as
well as co-cultivated, confluent, mixed cell monolayers.
1TABLE 1 Cell Cultures NCI- MRC-5 CV-1 BGMK McCoy BHK* A549 HEp-2
Mv1Lu H292 1 2 3 4 5 6 7 8 9 MRC-5 A ++ + No + + + + + + CV-1 B ++
No + + + + + + BGMK C ++ + + + + + + McCoy D ++ + + Yes + + BHK* E
++ + + + + A549 F ++ + + + HEp-2 G ++ + + Mv1Lu H ++ + NCI- I ++
H292 ++ Denotes single cell types producing confluent monolayers +
Denotes some degree of dimorphic, mixed monolayer Yes Denotes cell
mixtures that appear very uniform, with an even distribution No
Denotes cell mixtures that did not appear to work *Denotes
genetically engineered ELVIS BHK cells.
[0069] In yet another embodiment, the present invention provides
kits for assaying samples for the presence of infectious viruses.
In these kits, mixed cell cultures are provided which facilitate
the detection and identification of particular virus groups
(e.g.,viruses associated with respiratory infections/diseases). In
the kits, co-cultivated cells are supplied either frozen or
dispensed (i.e., ready for use) in shell vials, tubes, or multiwell
plates. These cells are susceptible to infection by the virus group
of interest as indicated by the sample type. In preferred
embodiments, the kits also include reagents necessary to detect
expression of viral antigens or virus-induced reporter gene
expression.
[0070] One of the several advantages of the present invention is
that it provides rapid and sensitive assay systems for the
detection and identification of a single virus type from a
multiplicity of possibilities, in a single mixed cell unit that is
suitable for diagnostic assay. Thus, the present invention
eliminates the need for multiple cell lines cultured in individual
containers, provides reliable results in 1-3 days following
inoculation of the cell cultures, rather than 1-28 days, eliminates
the necessity of working with primary cell cultures, provides an
efficient screening method for grouping and preliminary
identification of viruses, and provides assay systems that are
highly specific for viruses capable of inducing reporter gene
expression. Thus, the present invention clearly fulfills a need
that has been heretofore unmet in the field of diagnostic
virology.
Definitions
[0071] The terms "sample" and "specimen" in the present
specification and claims are used in their broadest sense. On the
one hand, they are meant to include a specimen or culture. On the
other hand, they are meant to include both biological and
environmental samples. These terms encompasses all types of samples
obtained from humans and other animals, including but not limited
to, body fluids such as urine, blood, fecal matter, cerebrospinal
fluid (CSF), semen, sputum, and saliva, as well as solid tissue.
These terms also refers to swabs and other sampling devices which
are commonly used to obtain samples for culture of
microorganisms.
[0072] Biological samples may be animal, including human, fluid or
tissue, food products and ingredients such as dairy items,
vegetables, meat and meat by-products, and waste. Environmental
samples include environmental material such as surface matter,
soil, water, and industrial samples, as well as samples obtained
from food and dairy processing instruments, apparatus, equipment,
disposable, and non-disposable items. These examples are not to be
construed as limiting the sample types applicable to the present
invention.
[0073] Whether biological or environmental, a sample suspected of
containing microorganisms may (or may not) first be subjected to an
enrichment means to create a "pure culture" of microorganisms. By
"enrichment means" or "enrichment treatment," the present invention
contemplates (i) conventional techniques for isolating a particular
microorganism of interest away from other microorganisms by means
of any culture medium and/or technique, and (ii) novel techniques
for isolating particular microorganisms away from other
microorganisms. It is not intended that the present invention be
limited only to one enrichment step or type of enrichment means.
For example, it is within the scope of the present invention,
following subjecting a sample to a conventional enrichment means,
to subject the resultant preparation to further purification such
that a pure culture of a strain of a species of interest is
produced. This pure culture may then be analyzed by the medium and
method of the present invention.
[0074] As used herein, the term "organism" and "microorganism," are
used to refer to any species or type of microorganism, including
but not limited to viruses and bacteria, including rickettsia and
chlamydia. Thus, the term encompasses, but is not limited to DNA
and RNA viruses, as well as organisms within the orders
Rickettsiales and Chlamydiales.
[0075] As used herein, the term "culture," refers to any sample or
specimen which is suspected of containing one or more
microorganisms. "Pure cultures" are cultures in which the organisms
present are only of one strain of a particular genus and
species.
[0076] This is in contrast to "mixed cultures," which are cultures
in which more than one genus and/or species of microorganism are
present.
[0077] As used herein, the term "cell type," refers to any cell,
regardless of its source or characteristics.
[0078] As used herein, the term "cell line," refers to cells that
are cultured in vitro, including primary cell lines, finite cell
lines, continuous cell lines, and transformed cell lines.
[0079] As used herein, the terms "primary cell culture," and
"primary culture," refer to cell cultures that have been directly
obtained from animal or insect tissue. These cultures may be
derived from adults as well as fetal tissue.
[0080] As used herein, the term "finite cell lines," refer to cell
cultures that are capable of a limited number of population
doublings prior to senescence.
[0081] As used herein, the term "continuous cell lines," refer to
cell cultures that have undergone a "crisis" phase during which a
population of cells in a primary or finite cell line apparently
ceases to grow, but yet a population of cells emerges with the
general characteristics of a reduced cell size, higher growth rate,
higher cloning efficiency, increased tumorigenicity, and a variable
chromosomal complement. These cells often result from spontaneous
transformation in vitro. These cells have an indefinite
lifespan.
[0082] As used herein, the term "transformed cell lines," refers to
cell cultures that have been transformed into continuous cell lines
with the characteristics as described above. Transformed cell lines
can be derived directly from tumor tissue and also by in vitro
transformation of cells with whole virus (e.g., SV40 or EBV), or
DNA fragments derived from a transforming virus using vector
systems.
[0083] As used herein, the term "hybridomas," refers to cells
produced by fusing two cell types together. Commonly used
hybridomas include those created by the fusion of
antibody-secreting B cells from an immunized animal, with a
malignant myeloma cell line capable of indefinite growth in vitro.
These cells are cloned and used to prepare monoclonal
antibodies.
[0084] As used herein, the term "mixed cell culture," refers to a
mixture of two types of cells. In some preferred embodiments, the
cells are cell lines that are not genetically engineered, while in
other preferred embodiments the cells are genetically engineered
cell lines. In some embodiments, the one or more of the cell types
is re "permissive" (i.e., virus is capable of replication and
spread from cell to cell within the culture). The present invention
encompasses any combination of cell types suitable for the
detection, identification, and/or quantitation of viruses in
samples, including mixed cell cultures in which all of the cell
types used are not genetically engineered, mixtures in which one or
more of the cell types are genetically engineered and the remaining
cell types are not genetically engineered, and mixtures in which
all of the cell types are genetically engineered.
[0085] As used herein, the term "suitable for the detection of
intracellular parasites," refers to cell cultures that can be
successfully used to detect the presence of an intracellular
parasite in a sample. In preferred embodiments, the cell cultures
are capable of maintaining their susceptibility to infection and/or
support replication of the intracellular parasite. It is not
intended that the present invention be limited to a particular cell
type or intracellular parasite.
[0086] As used herein, the term "susceptible to infection" refers
to the ability of a cell to become infected with virus or another
intracellular organism. Although it encompasses "permissive"
infections, it is not intended that the term be so limited, as it
is intended that the term encompass circumstances in which a cell
is infected, but the organism does not necessarily replicate and/or
spread from the infected cell to other cells. The phrase "viral
proliferation," as used herein describes the spread or passage of
infectious virus from a permissive cell type to additional cells of
either a permissive or susceptible character.
[0087] As used herein, the terms "monolayer," "monolayer culture,"
and "monolayer cell culture," refer to cells that have adhered to a
substrate and grow in as a layer that is one cell in thickness.
Monolayers may be grown in any format, including but not limited to
flasks, tubes, coverslips (e.g., shell vials), roller bottles, etc.
Cells may also be grown attached to microcarriers, including but
not limited to beads.
[0088] As used herein, the term "suspension," and "suspension
culture," refers to cells that survive and proliferate without
being attached to a substrate. Suspension cultures are typically
produced using hematopoietic cells, transformed cell lines, and
cells from malignant tumors.
[0089] As used herein, the terms "culture media," and "cell culture
media," refers to media that are suitable to support the growth of
cells in vitro (i.e., cell cultures). It is not intended that the
term be limited to any particular culture medium. For example, it
is intended that the definition encompass outgrowth as well as
maintenance media. Indeed, it is intended that the term encompass
any culture medium suitable for the growth of the cell cultures of
interest.
[0090] As used herein, the term "obligate intracellular parasite,"
(or obligate intracellular organism) refers to any organism which
requires an intracellular environment for its survival and/or
replication. Obligate intracellular parasites include viruses, as
well as many other organisms, including certain bacteria (e.g.,
most members of the orders Rickettsiales [e.g., Coxiella,
Rickettsia and Ehrlichia] and Chlamydiales [e.g., C. trachomatis,
C. psittaci], etc). The term "intracellular parasite," refers to
any organism that may be found within the cells of a host animal,
including but not limited to obligate intracellular parasites
briefly described above. For example, intracellular parasites
include organisms such as Brucella, Listeria, Mycobacterium (e.g.,
M. tuberculosis and M. leprae), and Plasmodium, as well as
Rochalimea.
[0091] As used herein, the term "antimicrobial," is used in
reference to any compound which inhibits the growth of, or kills
microorganisms. It is intended that the term be used in its
broadest sense, and includes, but is not limited to compounds such
as antibiotics which are produced naturally or synthetically. It is
also intended that the term includes compounds and elements that
are useful for inhibiting the growth of, or killing
microorganisms.
[0092] As used herein, the terms "chromogenic compound," and
"chromogenic substrate," refer to any compound useful in detection
systems by their light absorption or emission characteristics. The
term is intended to encompass any enzymatic cleavage products,
soluble, as well as insoluble, which are detectable either visually
or with optical machinery. Included within the designation
"chromogenic" are all enzymatic substrates which produce an end
product which is detectable as a color change. This includes, but
is not limited to any color, as used in the traditional sense of
"colors," such as indigo, blue, red, yellow, green, orange, brown,
etc., as well as fluorochromic or fluorogenic compounds, which
produce colors detectable with fluorescence (e.g., the yellow-green
of fluorescein, the red of rhodamine, etc.). It is intended that
such other indicators as dyes (e.g., pH) and luminogenic compounds
be encompassed within this definition.
[0093] As used herein, the commonly used meaning of the terms "pH
indicator," "redox indicator," and "oxidation-reduction indicator,"
are intended. Thus, "pH indicator," encompasses all compounds
commonly used for detection of pH changes, including, but not
limited to phenol red, neutral red, bromthymol blue, bromcresol
purple, bromcresol green, bromchlorophenol blue, m-cresol purple,
thymol blue, bromcresol purple, xylenol blue, methyl red, methyl
orange, and cresol red. The terms "redox indicator," and
"oxidation-reduction indicator," encompasses all compounds commonly
used for detection of oxidation/reduction potentials (i.e., "eH")
including, but not limited to various types or forms of
tetrazolium, resazurin, and methylene blue.
[0094] As used herein, the term "inoculating suspension," or
"inoculant," is used in reference to a suspension which may be
inoculated with organisms to be tested. It is not intended that the
term "inoculating suspension," be limited to a particular fluid or
liquid substance. For example, inoculating suspensions may be
comprised of water, saline, or an aqueous solution. It is also
contemplated that an inoculating suspension may include a component
to which water, saline or any aqueous material is added. It is
contemplated in one embodiment, that the component comprises at
least one component useful for the intended microorganism. It is
not intended that the present invention be limited to a particular
component.
[0095] As used herein, the term "kit," is used in reference to a
combination of reagents and other materials.
[0096] As used herein, the term "primary isolation," refers to the
process of culturing organisms directly from a sample. As used
herein, the term "isolation," refers to any cultivation of
organisms, whether it be primary isolation or any subsequent
cultivation, including "passage," or "transfer," of stock cultures
of organisms for maintenance and/or use.
[0097] As used herein, the term "presumptive diagnosis," refers to
a preliminary diagnosis which gives some guidance to the treating
physician as to the etiologic organism involved in the patient's
disease. Presumptive diagnoses are often based on "presumptive
identifications," which as used herein refer to the preliminary
identification of a microorganism.
[0098] As used herein, the term "definitive diagnosis," is used to
refer to a final diagnosis in which the etiologic agent of the
patient's disease has been identified. The term "definitive
identification" is used in reference to the final identification of
an organism to the genus and/or species level.
[0099] The term "recombinant DNA molecule," as used herein refers
to a DNA molecule which is comprised of segments of DNA joined
together by means of molecular biological techniques.
[0100] DNA molecules are said to have "5' ends" and "3' ends"
because mononucleotides are reacted to make oligonucleotides in a
manner such that the 5' phosphate of one mononucleotide pentose
ring is attached to the 3' oxygen of its neighbor in one direction
via a phosphodiester linkage. Therefore, an end of an
oligonucleotides is referred to as the "5' end" if its 5' phosphate
is not linked to the 3' oxygen of a mononucleotide pentose ring and
as the "3' end" if its 3' oxygen is not linked to a 5' phosphate of
a subsequent mononucleotide pentose ring. As used herein, a nucleic
acid sequence, even if internal to a larger oligonucleotide, also
may be said to have 5' and 3' ends. In either a linear or circular
DNA molecule, discrete elements are referred to as being "upstream"
or 5' of the "downstream" or 3' elements. This terminology reflects
the fact that transcription proceeds in a 5' to 3' fashion along
the DNA strand. The promoter and enhancer elements which direct
transcription of a linked gene are generally located 5' or upstream
of the coding region (enhancer elements can exert their effect even
when located 3' of the promoter element and the coding region).
Transcription termination and polyadenylation signals are located
3' or downstream of the coding region.
[0101] The term "an oligonucleotide having a nucleotide sequence
encoding a gene," refers to a DNA sequence comprising the coding
region of a gene or, in other words, the DNA sequence which encodes
a gene product. The coding region may be present in either a cDNA
or genomic DNA form. Suitable control elements such as enhancers,
promoters, splice junctions, polyadenylation signals, etc. may be
placed in close proximity to the coding region of the gene if
needed to permit proper initiation of transcription and/or correct
processing of the primary RNA transcript. Alternatively, the coding
region utilized in the vectors of the present invention may contain
endogenous enhancers and/or promoters, splice junctions,
intervening sequences, polyadenylation signals, etc. or a
combination of both endogenous and exogenous control elements.
[0102] The term "transcription unit," as used herein refers to the
segment of DNA between the sites of initiation and termination of
transcription and the regulatory elements necessary for the
efficient initiation and termination. For example, a segment of DNA
comprising an enhancer/promoter, a coding region, and a termination
and polyadenylation sequence comprises a transcription unit.
[0103] The term "regulatory element," as used herein refers to a
genetic element which controls some aspect of the expression of
nucleic acid sequences. For example, a promoter is a regulatory
element which facilitates the initiation of transcription of an
operably linked coding region. Other regulatory elements are
splicing signals, polyadenylation signals, termination signals,
etc. (defined infra).
[0104] The terms "reporter gene construct," or "reporter gene
vector," as used herein refers to a recombinant DNA molecule
containing a sequence encoding the product of a reporter gene and
appropriate nucleic acid sequences necessary for the expression of
the operably linked coding sequence in a particular host organism.
Eukaryotic cells are known to utilize promoters, enhancers, and
termination and polyadenylation signals.
[0105] The term "reporter gene," refers to an oligonucleotide
having a sequence encoding a gene product (typically an enzyme)
which is easily and quantifiably assayed when a construct
comprising the reporter gene sequence operably linked to a
heterologous promoter and/or enhancer element is introduced into
cells containing (or which can be made to contain) the factors
necessary for the activation of the promoter and/or enhancer
elements. Examples of reporter genes include but are not limited to
bacterial genes encoding .beta.-galactosidase (lacZ), the bacterial
chloramphenicol acetyltransferase (cat) genes, firefly luciferase
genes and genes encoding .beta.-glucuronidase (GUS).
[0106] Transcriptional control signals in eukaryotes comprise
"promoter" and "enhancer" elements. Promoters and enhancers consist
of short arrays of DNA sequences that interact specifically with
cellular proteins involved in transcription (Maniatis, et al.,
Science 236:1237 [1987]). Promoter and enhancer elements have been
isolated from a variety of eukaryotic sources including genes in
yeast, insect and mammalian cells and viruses (analogous control
elements [i.e., promoters, are also found in prokaryotes]). The
selection of a particular promoter and enhancer depends on what
cell type is to be used to express the protein of interest. Some
eukaryotic promoters and enhancers have a broad host range while
others are functional in a limited subset of cell types (for review
see Voss, et al., Trends Biochem. Sci., 11:287 [1986], and
Maniatis, et al., supra [1987]). For example, the SV40 early gene
enhancer is very active in a wide variety of cell types from many
mammalian species and has been widely used for the expression of
proteins in mammalian cells (Dijkema, et al., EMBO J. 4:761
[1985]). Two other examples of promoter/enhancer elements active in
a broad range of mammalian cell types are those from the human
elongation factor 1.alpha. gene (Uetsuki et al., J. Biol. Chem.,
264:5791 [1989]; Kim et al., Gene 91:217 [1990]; and Mizushima and
Nagata, Nuc. Acids. Res., 18:5322 [1990]) and the long terminal
repeats of the Rous sarcoma virus (Gorman et al., Proc. Natl. Acad.
Sci. USA 79:6777 [1982]), and the human cytomegalovirus (Boshart et
al., Cell 41:521 [1985]).
[0107] The term "promoter/enhancer," denotes a segment of DNA which
contains sequences capable of providing both promoter and enhancer
functions (for example, the long terminal repeats of retroviruses
contain both promoter and enhancer functions). The
enhancer/promoter may be "endogenous," or "exogenous," or
"heterologous." An endogenous enhancer/promoter is one which is
naturally linked with a given gene in the genome. An exogenous
(heterologous) enhancer/promoter is one which is placed in
juxtaposition to a gene by means of genetic manipulation (i.e.,
molecular biological techniques).
[0108] The presence of "splicing signals," on an expression vector
often results in higher levels of expression of the recombinant
transcript. Splicing signals mediate the removal of introns from
the primary RNA transcript and consist of a splice donor and
acceptor site (Sambrook et al., Molecular Cloning: A Laboratory
Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York
[1989], pp. 16.7-16.8). A commonly used splice donor and acceptor
site is the splice junction from the 16S RNA of SV40.
[0109] Efficient expression of recombinant DNA sequences in
eukaryotic cells requires signals directing the efficient
termination and polyadenylation of the resulting transcript.
Transcription termination signals are generally found downstream of
the polyadenylation signal and are a few hundred nucleotides in
length. The term "poly A site," or "poly A sequence," as used
herein denotes a DNA sequence which directs both the termination
and polyadenylation of the nascent RNA transcript. Efficient
polyadenylation of the recombinant transcript is desirable as
transcripts lacking a poly A tail are unstable and are rapidly
degraded. The poly A signal utilized in an expression vector may be
"heterologous" or "endogenous." An endogenous poly A signal is one
that is found naturally at the 3' end of the coding region of a
given gene in the genome. A heterologous poly A signal is one which
is isolated from one gene and placed 3' of another gene. A commonly
used heterologous poly A signal is the SV40 poly A signal. The SV40
poly A signal is contained on a 237 bp BamHI/BclI restriction
fragment and directs both termination and polyadenylation
(Sambrook, supra, at 16.6-16.7). This 237 bp fragment is contained
within a 671 bp BamHI/PstI restriction fragment.
[0110] The term "genetically engineered cell line," refers to a
cell line that contains heterologous DNA introduced into the cell
line by means of molecular biological techniques (i.e., recombinant
DNA technology).
[0111] The term "stable transfection," or "stably transfected,"
refers to the introduction and integration of foreign DNA into the
genome of the transfected cell. The term "stable transfectant,"
refers to a cell which has stably integrated foreign DNA into the
genomic DNA.
[0112] The term "stable transfection" (or "stably transfected"),
refers to the introduction and integration of foreign DNA into the
genome of the transfected cell. The term "stable transfectant,"
refers to a cell which has stably integrated foreign DNA into the
genomic DNA.
[0113] The term "selectable marker," as used herein refers to the
use of a gene which encodes an enzymatic activity that confers
resistance to an antibiotic or drug upon the cell in which the
selectable marker is expressed. Selectable markers may be
"dominant"; a dominant selectable marker encodes an enzymatic
activity which can be detected in any mammalian cell line. Examples
of dominant selectable markers include the bacterial aminoglycoside
3' phosphotransferase gene (also referred to as the neo gene) which
confers resistance to the drug G418 in mammalian cells, the
bacterial hygromycin G phosphotransferase (hyg) gene which confers
resistance to the antibiotic hygromycin and the bacterial
xanthine-guanine phosphoribosyl transferase gene (also referred to
as the gpt gene) which confers the ability to grow in the presence
of mycophenolic acid. Other selectable markers are not dominant in
that their use must be in conjunction with a cell line that lacks
the relevant enzyme activity. Examples of non-dominant selectable
markers include the thymidine kinase (tk) gene which is used in
conjunction with tk.sup.- cell lines, the CAD gene which is used in
conjunction with CAD-deficient cells and the mammalian
hypoxanthine-guanine phosphoribosyl transferase (hprt) gene which
is used in conjunction with hprt.sup.- cell lines. A review of the
use of selectable markers in mammalian cell lines is provided in
Sambrook et al., supra at pp.16.9-16.15.
[0114] The terms "nucleic acid molecule encoding," "DNA sequence
encoding," and "DNA encoding," refer to the order or sequence of
deoxyribonucleotides along a strand of deoxyribonucleic acid. The
order of these deoxyribonucleotides determines the order of amino
acids along the polypeptide (protein) chain. The DNA sequence thus
codes for the amino acid sequence.
[0115] The terms "confluent" or "confluency" as used herein in
reference to an adherent cell line define a condition wherein cells
throughout a culture are in contact with each other creating what
appears to be a continuous sheet or "monolayer" of cells.
[0116] The terms "cytopathic effect" or "CPE" as used herein
describe changes in cellular structure (i.e., a pathologic effect)
resulting from external agents such viruses. Common cytopathic
effects include cell destruction, syncytia (i.e., fused giant
cells) formation, cell rounding vacuole formation, and formation of
inclusion bodies. CPE results from actions of a virus on permissive
cells that negatively affect the ability of the permissive cellular
host to preform its required functions to remain viable. In in
vitro cell culture systems, CPE is evident when cells, as part of a
confluent monolayer, show regions of non-confluence after contact
with a specimen that contains a virus. The observed microscopic
effect is generally focal in nature and the foci is initiated by a
single virion. However, depending upon viral load in the sample,
CPE may be observed throughout the monolayer after a sufficient
period of incubation. Cells demonstrating viral induced CPE usually
change morphology to a rounded shape, and over a prolonged period
of time can die and be released form their anchorage points in the
monolayer. When many cells reach the point of focal destruction,
the area is called a viral plaque, which appears as a hole in the
monolayer. Cytopathic effects are readily discernable and
distinguishable by those skilled in the art.
[0117] The abbreviation "ONPG," represents
o-Nitrophenyl-.beta.-D-Galactop- yranoside. ONPG is a substrate for
the enzyme .beta.-galactosidase (.beta.-gal). The reaction between
ONPG and .beta.-gal produces a yellow product which can be
quantified spectrophotometrically at 405 nm.
[0118] The abbreviation "X-gal," represents the chemical compound
5-bromo-4-chloro-3-indolyl-.beta.-D-galactopyranoside, a substrate
for the enzyme .beta.-galactosidase. The reaction between X-gal and
.beta.-galactosidase results in the formation of a blue precipitate
which is visually discernable.
[0119] The term "hybriwix," represents a product of Diagnostic
Hybrids, Inc., Athens, Ohio which allows for quantification of
certain viral DNA in an infected monolayer of cells by DNA
hybridization. "DNA hybridization" is the annealing of two
complementary DNA molecules whose base sequences match according to
the rules of base pairing. DNA hybridization is used to identify or
quantify an unknown or "target" DNA by hybridization to a known DNA
or "probe." The probe is typically labeled with a reporter molecule
such as .sup.125I, a radioisotope which can be detected and
quantified with a gamma counter.
[0120] The phrase "plaque reduction assay," or "PRA," as used
herein describes a standard method used to determine efficacy of
anti-viral drugs by enumerating a decrease in plaque formation in a
cell monolayer exposed to a drug. A "plaque" is a defined area of
"CPE." It is usually the result of infection of the cell monolayer
with a single infectious virus which then replicates and spreads to
adjacent cells of the monolayer. A plaque may also be referred to
as a "focus of viral infection."
[0121] The term "permissive" as used herein describes the sequence
of interactive events between a virus and its putative host cell.
The process begins with viral adsorption to the host cell surface
and ends with release of infectious virions. A cell is "permissive"
if it readily permits the spread of virus to other cells. Many
methods are available for the determination of the permissiveness
of a given cell line, including, but not limited to plaque
reduction assays, comparisons of the production and/or quantitation
of viral proteins based on results obtained from gel
electrophoresis, relative comparisons using hybridization analysis
to analyze DNA or RNA content, etc.
[0122] The term "susceptible," as used herein describes the extent
that a permissive or non-permissive host cell can adsorb and be
penetrated by a virus. A cell line may be susceptible without being
permissive in that it can be penetrated but not release virions. A
permissive cell line however must be susceptible.
[0123] The phrase "seed on," as used herein describes the act of
transferring an aqueous solution of suspended cells into a vessel
containing cells adhered to a surface, after which the vessel is
stored for a sufficient period of time to allow the suspended cells
or "seeds" to settle out by gravity and attach in a relatively
uniform manner to the adhered cells and become integrated into the
final cell monolayer as a mixture. A "mixed cell monolayer,"
results from the "seed on" process.
[0124] The phrase "seed in," as used herein describes the mixing of
two or more aqueous solutions of suspended tissue culture cells,
each cell suspension having different cellular properties, and
transfer of such mixture of cells into a vessel which is stored for
a sufficient period of time to allow the suspended cells to settle
out by gravity and attach in a relatively uniform manner such that
the distribution of any single cell type is indicative of the
relative ratio of the cells in the original mixture.
[0125] The term "starts," as used herein refers to the reporter
cells which represent a primary infection of virus. The virus
infects a reporter cell (a genetically engineered cell) and induces
the expression of the reporter gene. A reporter cell can be
non-permissive (i.e. permissiveness of the reporter cells is not
required) and still produce starts.
EXPERIMENTAL
[0126] The following examples are provided in order to demonstrate
and further illustrate certain preferred embodiments and aspects of
the present invention and are not to be construed as limiting the
scope thereof.
[0127] In the experimental disclosure which follows, the following
abbreviations apply: eq (equivalents); M (Molar); .mu.M
(micromolar); N (Normal); mol (moles); mmol (millimoles); .mu.mol
(micromoles); nmol (nanomoles); g (grams); mg (milligrams); .mu.g
(micrograms); ng (nanograms); 1 or L (liters); ml (milliliters);
.mu.l (microliters); cm (centimeters); mm (millimeters); .mu.m
(micrometers); nm (nanometers); xg (times gravity); .degree. C.
(degrees Centigrade); FBS (fetal bovine serum); PBS (phosphate
buffered saline; HEPES (N-[2-hydroxyethyl]piperazi-
ne-N'-[2-ethanesulfonic acid]); HBSS (Hank's Balanced Salt
Solution); MEM (Minimal Essential Medium); EMEM (Eagle's Minimal
Essential Medium); BBL (Becton Dickinson Microbiology Systems,
Cockeysville, Md.); DIFCO (Difco Laboratories, Detroit, Mich.);
U.S. Biochemical (U.S. Biochemical Corp., Cleveland, Ohio);
Chemicon (Chemicon, Inc., Temecula, Calif.); Dako (Dako
Corporation, Carpinteria, Calif.); Fisher (Fisher Scientific,
Pittsburgh, Pa.); Sigma (Sigma Chemical Co., St. Louis, Mo.); ATCC
(American Type Culture Collection, Rockville, Md.); Bartel's
(Bartels, Issaquah, Wash.); and BioWhittaker (BioWhittaker,
Walkersville, Md.).
[0128] The cells used during the development of the present
invention and described in the following Examples, were obtained
from the ATCC, with the exception being that the BGMK and PRMK
cells were obtained from BioWhittaker, and the MRC-5 cells were
obtained from both ATCC and BioWhittaker. The ATCC numbers of the
cells are indicated in the following Table.
2TABLE 2 ATCC Cell Lines Cell Line ATCC Number BHK/ICP6LacZ-5
CCL-12072 A549 CCL-185 CV-1 CCL-70 HEp-2 CCL-23 hs27 HFF; CRL-1634
Mv1Lu CCL-64 McCoy CCL-1696 NCI-H292 CCL-1848 MRC-5 CCL-171 WI-38
CCL-75 Vero CCL-81 MDCK (NBL-2) CCL-34 BHK21 CCL-10 HEL299 CCL-137
HeLa CCL-2
EXAMPLE 1
Co-Cultivation of Cells
[0129] In this Example, mixed cell cultures were established in
which single, dimorphic cell sheets were produced at
confluency.
[0130] In these experiments, all of the cell lines were cultured to
confluency in sterile polystyrene flasks in EMEM (Eagle's Minimal
Essential Medium) with 25 mM HEPES, 7% fetal bovine serum (FBS), 2
mM L-glutamine, and penicillin/streptomycin (100 Units/100 .mu.g
per ml of medium each).
[0131] Cells to be cultured were harvested by first rinsing source
cell monolayers with Hank's Balanced Salt Solution (HBSS) without
magnesium or calcium. Depending upon the cell line, the cells were
dissociated by adding trypsin (0.125% in HBSS, without calcium or
magnesium) or trypsin-EDTA (0.25% in 1 mM EDTA in HBSS, without
calcium or magnesium), or directly to the cell monolayer, and
incubating for approximately 5 minutes at ambient temperature. Ten
volumes of cell culture medium was added to each trypsinized cell
suspension and the cells were repeatedly pipetted in order to
produce near-single cell suspensions (i.e., without cell
aggregates). Each trypsinized cell suspension was diluted in an
adequate volume of culture medium to produce an optical density of
cell suspension suitable to produce a confluent monolayer of cells
within 2-3 days of incubation in a 96-well microtiter plate. For
single cell monolayers (i.e., one cell type per well), 0.2 ml of
suspension was used to inoculate each well. For example, the final
cell preparations ranged from a final optical density at 500 nm of
0.012 OD units/ml for CV-1 cells to 0.03 OD units/ml for HEp-2
cells.
[0132] Cell mixture monolayers were produced by co-planting two
distinct cell types at an equal volume of each diluted cell
suspension (i.e., 0.1 ml of each cell type was used to inoculate
each well of a 96-well microtiter plate). The cells were allowed to
attach to the well surface by gravity for 30-60 minutes, and the
inoculated microtiter plates were incubated for up to three days at
36.degree. C. in 5% CO.sub.2 with 95% relative humidity.
[0133] Periodically during incubation, single and mixed monolayers
were checked for overall viability. The mixed cell culture
monolayers were also checked for the ability of the cell lines to
co-exist and develop as a single cell sheet (i.e., a single
monolayer), with two distinct cell morphologies (i.e., dimorphic
cell sheets), at an approximately equal density of each cell type.
At confluency, the cells were treated with a methylene blue
staining solution to fix the cells and stain them a light blue in
order to provide contrast for visualization using light
microscopy.
[0134] Some of the mixed monolayers successfully grew as a mixed
cell monolayer adhered to the well surfaces, exhibiting a smooth,
evenly distributed monolayer. These mixed cultures were designated
as "morphologic category 1." In these cultures, each cell type
could be easily distinguished and appeared to survive well in a
mixed monolayer, giving the appearance of a single cell
distribution. Mixed monolayers composed of HEp-2 and McCoy cells
displayed this morphology.
[0135] Some of the mixed monolayers successfully grew as a mixed
monolayer adhered to the well surfaces, but exhibited two distinct
morphologies at confluency. These mixed cultures were designated as
"morphologic category 2." In these cultures, separate, distinct
patches of each cell line co-existed within the monolayer, giving
the appearance of oil mixing with water. Although an understanding
of the mechanism is not necessary in order to use the present
invention, it is likely that this appearance is most likely the
result of contact inhibition between two specific cell types. The
relative sizes of the patches was found primarily to be a function
of how evenly the cells were distributed at cell planting. The more
even the cell distribution at planting, the patches or islands were
smaller as the monolayer reached confluency. Examples of monolayers
that produced this appearance were mink lung cells co-cultivated
with NCI-H292 cells, mink lung cells co-cultivated by buffalo green
monkey kidney (BGMK) cells, and human lung carcinoma A549 cells
co-cultivated with NCI-H292 cells.
[0136] However, some cells types could not produce a mixed cell
monolayer, when mixed at relatively equal cell numbers at planting
in the same culture medium. In some of these cultures, only one of
the cell types was found to be viable (i.e., the culture was
effectively a single cell type). Examples of mixed cell cultures
that were found to be unsuitable for the production of mixed
monolayers include human embryonic lung fibroblasts (MRC-5 cells)
co-cultivated with BGMK cells. In this mixture, the MRC-5 cells
become toxic and form aggregates of dead cells soon after planting.
Thus, at confluency, the monolayer only contains one functional,
viable cell type, the BGMK cells. Thus, this cell mixture was found
to be unsuitable for producing mixed cell monolayers as the cells
failed to form mixed cell monolayers of either a smooth or
dimorphic morphologic type.
EXAMPLE 2
Detection of Respiratory Viruses in Mixed Cell Cultures
[0137] In this Example, mixed cell cultures were used to detect
various respiratory viruses including Influenza A, RSV,
adenoviruses, parainfluenza viruses, and Influenza B, present in
clinical specimens. The mixed cells used in these experiments were
Mv1Lu (mink lung cells) and NCI-H292 (human mucoepidermoid
cells).
[0138] Cell Lines
[0139] Confluent T-225 flasks of Mv1Lu and H292 cells were prepared
in EMEM with HEPES, 10% FBS, 2 mM L-glutamine, and 50 .mu.g/ml
gentamicin. The cells were harvested by first rinsing them in 30 ml
HBSS without magnesium and calcium. The cells were then dissociated
from the flask by brief exposure (i.e., until the cells lifted from
the bottom of the flask) to 7 ml trypsin-EDTA solution as described
in Example 1. Then, 30 ml media was added to the cells to prepare a
cell suspension concentrate. The optical density of each cell
suspension was determined at 500 nm, using 3 ml of cells.
Typically, the OD reading was 0.2/ml for both the Mv1Lu and H292
cells. In addition to the Mv1Lu and H292 cells, rhesus monkey
kidney cells (PRMK), A549 cells, and MDCK cells were used in the
present Example. These additional cell lines were prepared in
single cell cultures as known in the art.
[0140] Mixed Cell Cultures
[0141] When each cell suspension concentrate was determined to be
0.2 OD units/ml, 5.2 ml of the Mv1Lu, and 8.7 ml H292 cell
suspensions were added to 86.1 ml of culture medium, in order to
provide an acceptable working ratio of each cell type (i.e., it was
a preparation of diluted mixed cells). This ratio was devised in
order to achieve a confluent monolayer, in which each cell type
covered a substantially equivalent surface area within 1-3 days
post-planting of the diluted mixed cells. Prior to dispensing, care
was taken to prepare homogenous suspensions of diluted mixed cells.
The mixed cells were dispensed at 0.75 ml per glass shell vial
(i.e., glass vial containing a sterile glass coverslip). After
planting, the vials were allowed to sit for 60 minutes at ambient
temperature so that the cells could settle by gravity and produce a
more optimum cell distribution of each cell type. The mixed cells
were then incubated for 1-3 days at 36.degree. C. in 5% CO.sub.2,
at 95% relative humidity. Subsequently, the shell vials were stored
at ambient temperature to maintain each cell type at substantively
equivalent surface ratios for up to 10 days from achieving
confluency.
[0142] Samples and Processing
[0143] Nasopharyngeal specimens submitted to a diagnostic virology
laboratory were obtained from patients exhibiting influenza-like
symptoms. The specimens were centrifuged to produce a cell pellet
for direct antigen testing, and a specimen supernatant for
inoculation of various cell cultures. The cell pellet was
resuspended in phosphate buffer to prepare a cell suspension and 25
.mu.l portions of the cell suspension were spotted onto a glass
slide and dried. Each spot of cells on the slide were then fixed
with fixative (e.g., acetone), and incubated for 30 minutes with
individual antibody solutions (Bartel's) capable of recognizing
various respiratory viruses, including influenza A and RSV, as well
as other respiratory viruses. A second antibody solution containing
fluorescein (FITC) labelled goat anti-mouse antibodies and
counterstain (Bartel's) was added to cover each cell spot on the
slides, and incubated for an additional 30 minutes at 35-37.degree.
C. The counterstain in the FITC-goat anti-mouse antibody solution
contains Evans Blue, which stains the cells and appears red under
fluorescence. Slides prepared from the nasopharyngeal specimens
were observed for positive (i.e. virus-infected), apple green
staining fluorescent cells, using epifluorescence at 100-400X
magnification.
[0144] In addition, 0.2 ml aliquots of the specimen supernatant
were inoculated onto various cell cultures prepared in shell vials
containing glass coverslips. The cell cultures included primary
rhesus monkey kidney cells (PRMK; ViroMed or BioWhittaker), Mv1Lu
cells (Diagnostic Hybrids) HEp-2 cells (Diagnostic Hybrids), MDCK,
A549, and H292 cells, as single cell monolayers, as well as mixed
cell monolayers of Mv1Lu and H292 cells, produced as described
above.
[0145] Each inoculated shell vial was centrifuged for 60 minutes at
700 xg, and then incubated for 1-3 days at 36.degree. C., in
appropriate culture medium (e.g., EMEM containing 0.5 to 2% FBS, 2
mM L-glutamine, and penicillin/streptomycin [100 Units/100 .mu.g
per ml of medium each]). After incubation, the culture medium was
decanted, and the cells were fixed to the glass coverslip with a
solution of acetone and methanol (50:50, v/v). An antibody solution
(Chemicon or Bartel's) containing a pool of monoclonal antibodies
to multiple respiratory viruses, including Influenza A and RSV, as
well as other respiratory viruses was added to cover each
coverslip. The coverslips were then incubated for 30 minutes at
35-37.degree. C. The antibody solution was then removed and the
coverslips were rinsed with PBS. A second antibody solution
containing fluorescein (FITC) labelled goat anti-mouse antibodies
and counterstain (Bartel's) was added to cover each coverslip, and
incubated for an additional 30 minutes at 35-37.degree. C. The
counterstain in the FITC-goat anti-mouse antibody solution contains
Evans Blue, which stains the cells and appears red under
fluorescence. Shell vial coverslips prepared from the
nasopharyngeal specimens (i.e., inoculated cultures) were observed
for positive (i.e., virus-infected), apple green staining,
fluorescent cells, using epifluorescence at 100-400X
magnification.
[0146] Results
[0147] Some specimens demonstrated a positive direct antigen
reaction on the cell spot incubated with Influenza A monoclonal
antibody. These specimens also demonstrated fluorescent staining on
the single cell Mv1Lu coverslip and the Mv1Lu/H292 mixed cell
coverslip, but no or very little fluorescence on the single cell
H292 coverslip. The H292 cells are either not susceptible to this
strain of Influenza A, or are significantly less susceptible, such
that infection is not detectable. Additionally, in some cases
(i.e., in specimens with low virus titers), the culture systems
were more sensitive than the direct antigen detection method. Also,
while the single PRMK cell cultures (i.e., the "gold standard"
cells used to detect Influenza A) were positive for the presence of
Influenza A, with many specimens, the numbers of infected cells and
the total of number of positive specimens were lower than those
identified as positive by the mixed cell monolayers.
[0148] In addition, both the MDCK and PRMK cells missed one low
titer specimen positive for Influenza A by direct antigen testing
(IFA), and one other specimen that was also positive for Influenza
A by IFA, while the Mv1Lu cells detected the virus in all of the
samples determined to be positive based on direct antigen detection
(IFA).
[0149] Some specimens demonstrated a positive direct antigen
reaction on the cell spot incubated with RSV monoclonal antibody.
These specimens also demonstrated fluorescent staining on the
single cell H292 coverslip and the MV1Lu/H292 mixed cell coverslip,
but no or very little fluorescence on the single cell MV1Lu
coverslip. H292 cells are susceptible to RSV infection, while Mv1Lu
cells are not susceptible (or are significantly less susceptible,
such that infection is not detectable). In addition to the mixed
cell cultures, HEp-2 cells (i.e., the "gold standard" cells used to
detect RSV) were also observed for the presence of RSV; the
performance of HEp-2 cells was generally less sensitive than that
of the Mv1Lu and H292 mixed cell monolayers, or the H292 single
cell monolayers. These results with Influenza A tested in mink lung
cells was very surprising, as the detection of Influenza A using
these cells has previously not been described.
[0150] Adenoviruses identified from five clinical specimens based
on direct antigen testing (IFA) were detected in the H292 and cell
culture mixes, while the PRMK cells missed two of the low titer
specimens (i.e., there were two false negatives). Thus, H292 and
the mixed cultures were more sensitive than PRMK for detection of
adenoviruses. While the A549 cells may provide slightly more
positive cells, the 292 cells, mixed cell cultures, and A549 cells
detected an equal number of positive specimens.
[0151] Parainfluenza viruses were also detected in the H292 and
mixed cell cultures, while the PRMK cells missed one low titer
specimen.
[0152] These results clearly show that the mixed cell cultures were
equal in sensitivity to the single cell (H292 and Mv1Lu) cultures.
Thus, the mixed cells provide savings in material, time, space, and
labor, while providing the same level of sensitivity in the
detection of respiratory viruses as single cell cultures presently
commonly used in diagnostic virology laboratories.
[0153] Influenza B Specimens
[0154] In addition to the samples discussed above, various
dilutions of multiple Influenza B strains obtained from the ATCC
were tested in MDCK, Mv1Lu, and PRMK cells. The following table
provides the results of these experiments. In this Table, "MD"
refers to the "Maryland" strain, "HK" refers to the "Hong Kong"
strain, "TW" refers to the "Taiwan" strain, and "MA" refers to the
"Massachusetts" strain.
3TABLE 3 Comparison of Influenza B Virus Detection From Prototype
Viruses by MDCK, ML, and PRMK Cells Influenza B Virus Virus Cell
Line Strain Dilution MDCK Mv1Lu PRMK MD 10.sup.-4 + + + 10.sup.-5 +
+ + 10.sup.-6 - + - HK 10.sup.-4 + + + 10.sup.-5 + + - 10.sup.-6 -
- - TW 10.sup.-4 + + + 10.sup.-5 + + + 10.sup.-6 - - - MA 10.sup.-4
+ + + 10.sup.-5 + + + 10.sup.-6 + + +
[0155] These results indicate that Mv1Lu, MDCK, and PRMK are
comparable for the detection of multiple Influenza B virus strains.
Thus, these cell lines were identified as good candidates for mixed
cell cultures, as well as single cell cultures for the
identification of this virus.
EXAMPLE 3
Detection of CMV in Mixed Cell Cultures
[0156] In this Example, mixed cell cultures of Mv1Lu and NCI-H292
cells were used to detect the presence of human cytomegalovirus
(HCMV).
[0157] The Towne strain of HCMV (ATCC #VR977) was amplified in
MRC-5 cells to a titer of greater than 10.sup.6/ml, and frozen at
-85.degree. C. in EMEM containing 10% FBS. Serial dilutions of HCMV
were prepared and inoculated into single monolayers of mink lung
(Mv1Lu) cells, MRC-5 cells, and mixed cell monolayers of Mv1Lu and
H292 cells. Each infected cell culture system was centrifuged for
60 minutes at 700 xg, and then incubated for 24 hours at 36.degree.
C. in 5% CO.sub.2, in appropriate culture medium (e.g., EMEM
containing 10% FBS). The culture medium was removed and the cells
were fixed to the glass coverslip using a solution of 80% acetone
in water. A sufficient amount of HCMV antibody solution (Chemicon)
was added to cover each coverslip and incubated for 30 minutes at
35-37.degree. C. The antibody solution was removed, and the
coverslip was rinsed with PBS. A second antibody solution
consisting of FITC-labelled goat anti-mouse antiserum was added to
cover each coverslip and incubated an additional 30 minutes at
35-37.degree. C. The specimens were then observed under
epifluorescence at 100-400X magnification for positive (i.e.,
CMV-infected), nuclear staining, fluorescent cells.
[0158] As described in previous Examples, the counterstain in the
FITC-labelled goat anti-mouse antibody solution contains Evans
Blue, which stains the cells and appears red, when excited by
fluorescent light. Fluorescent, apple green nuclear stain was
observed in the Mv1Lu single cell monolayer and in the mixed cell
monolayers, but not in the H292 cells, as the Mv1Lu cells are
susceptible to HCMV infection, while H292 cells are not (or the
H292 cells are significantly less sensitive). The MRC-5 cells
(i.e., the "gold standard" cells for detection of HCMV) performed
about the same as the mixed cell monolayer, as these cultures had a
similar number of infected cells as the cells in the mixed
monolayer.
EXAMPLE 4
Detection of Enteroviruses in Mixed Cell Cultures
[0159] In this Example, mixed cell cultures were used to detect the
enteroviruses, Coxsackie B virus and Echovirus. In these
experiments, a mixed cell monolayer of BGMK and NCI-H292 cells were
used.
[0160] Confluent T-225 flasks of BGMK and H292 cells were prepared
in EMEM with 25 mM HEPES, 10% FBS, 2 mM L-glutamine, and 50
.mu.g/ml gentamicin. The cells were harvested by first rinsing in
30 ml HBSS without magnesium and calcium, and were then dissociated
from the flasks by a brief treatment of 7 ml trypsin-EDTA solution
(as described in Example 1). Then, an additional 30 ml of culture
medium (EMEM with HEPES, 10% FBS, 2 mM L-glutamine, and 50 .mu.g/ml
gentamicin) was added to the suspension to produce a cell
suspension concentrate. The optical density at 500 nm was
determined for each suspension, using 3 ml of cells. Typically, the
OD reading was 0.2/ml for both the BGMK and H292 cell
suspensions.
[0161] Next, 3 ml of BGMK cell suspension and 8 ml of H292 cell
suspension (both suspensions were at 0.2 OD units/ml) were then
added to 29 ml of the culture medium (25 mM HEPES, 10% FBS, 2 mM
L-glutamine, and 50 .mu.g/ml gentamicin) to provide an acceptable
working ratio of each cell type in a diluted mixed cell suspension.
This ratio was intended to achieve a confluent monolayer consisting
of each cell type covering substantially equivalent surface area
within 1-3 days post-planting of the diluted mixed cells. Care was
exercised to prepare a homogenous suspension of diluted mixed cells
prior to dispensing 0.75 ml to each of 100 glass shell vials, each
of which contained a sterile glass coverslip. The vials were
allowed to sit for 60 minutes post-planting at ambient temperature
to allow the cells to settle by gravity and produce a more optimum
cell distribution. The vials were then were moved to an incubator
for incubation at 36.degree. C. for 1-3 days in 5% CO.sub.2, at 95%
relative humidity.
[0162] Stock virus suspensions and clinical specimens shown to
contain Coxsackie B virus or echovirus were used to infect
BGMK/H292 cell mixtures, as well as single cell monolayers of BGMK,
H292, MRC-5, and PRMK cells. For clinical samples, throat swab,
nasopharyngeal swab, sputum, stool, and rectal swabs were collected
from patients, placed in viral transport medium, and filtered
through 0.45 .mu.m filter to remove possible bacterial and fungal
contaminants prior to inoculation of cell cultures. Cerebrospinal
fluid (CSF) collected from patients was placed in viral transport
medium, and used directly for inoculation of cells. For inoculation
of shell vials, the media present in the vials were removed and
fresh media added. Then, 0.2 ml of specimen was inoculated into
each vial. The inoculated vials were centrifuged at 700 xg for
45-60 minutes at room temperature. Subsequently, the vials were
incubated at 37.degree. C. for 1-3 days, and viral presence was
detected using immunofluorescent staining.
[0163] For staining, the medium was removed from each vial and the
cells were fixed on the coverslip with acetone. The coverslip was
removed from each vial, and stained with 25 .mu.l primary antibody
(mouse monoclonal antibody directed against enteroviruses [Dako]),
for 30 minutes at 37.degree. C. After washing with PBS, 25 .mu.l of
the FITC-conjugated anti-mouse Ig (Dako) was used as a secondary
antibody for staining, and incubated at 37.degree. C. for 30
minutes. After another wash, the coverslips were mounted on slides
and observed under fluorescence. The presence of one or more
specific fluorescent-stained cells on the coverslip was considered
to be a positive. As described in previous Examples, the
counterstain in the FITC-labelled goat anti-mouse antibody solution
contains Evans Blue, which stains the cells, and appears red upon
exposure to fluorescent light. For Coxsackie B virus detection,
fluorescent, apple green stain was observed in many of the BGMK
cells in the BGMK single cell monolayer and in the mixed cell
monolayers primarily in the BGMK cells, but not in as many H292
cells, as BGMK cells are more susceptible to Coxsackie B virus
infection. For some types of Coxsackie B virus isolates, H292 cells
are not as susceptible (or the H292 cells are significantly less
susceptible). The "gold standard" cell line (i.e., PRMK cells) did
not exhibit the same number of infected cells as the mixed cell
monolayers.
[0164] For detection of echovirus, fluorescent, apple green stain
was observed in many H292 cells in the H292 single cell monolayer
and in the mixed cell monolayers, primarily in the H292 cells, but
not in as many BGMK cells, as H292 cells are more susceptible to
echovirus infection, while BGMK cells are not as susceptible (or
the BGMK cells are significantly less sensitive). The "gold
standard" line (i.e., MRC-5 cells) performed, but did not appear to
have as many infected cells as the mixed cell monolayers. In the
case of the BGMK/H292 mixed cell monolayers infected with high
titer samples of enteroviruses, cell-specific virus mediated
cytopathic effect (CPE) was evident (i.e., the CPE was observed in
BGMK cells when Coxsackie B virus was present at high titer, and
CPE was observed in H292 cells when echovirus was present at high
titer).
EXAMPLE 5
Detection of Herpes Simplex Virus and HCMV in Mixed Cell
Cultures
[0165] In this Example, mixed cell cultures are used to detect
herpes simplex virus (HSV) and HCMV, using a mixed cell monolayer
of genetically engineered baby hamster kidney (BHK) cells (e.g.,
ATCC #CCL-12072) and Mv1Lu cells.
[0166] The BHK and Mv1Lu cells are grown in flasks, trypsinized,
and mixed as described in previous Examples, such that a suitable
dilution of mixed cells is produced. These mixed cell dilutions are
then used to inoculate sterile glass shell vials containing
coverslips, as described above. The cells are then centrifuged and
inoculated with virus or clinical samples, incubated, and fixed, as
described above.
[0167] HCMV is detected in the Mv1Lu cells, using antibody as
described in Example 3 above, and HSV (HSV-1 and HSV-2) are
identified using a .beta.-galactosidase staining kit (i.e.,
detecting the reporter gene induced by the virus infecting the
genetically engineered BHK cells).
EXAMPLE 6
Detection of Respiratory Viruses in Mixed Cell Cultures
[0168] In this Example, mixed cell cultures are used to detect a
panel of respiratory viruses. In these experiments, three cell
types are combined so as to produce a mixed cell culture that is
capable of detecting at least three viruses.
[0169] First, A549, H292, and mink lung (e.g., Mv1Lu) cells cells
are grown in flasks, trypsinized, and mixed as described in
previous Examples, such that a suitable dilution of mixed cells is
produced. In preferred embodiments, the cells are diluted such that
the mixed cells in culture will be in approximately the same
proportions (i.e., 1:1:1). These mixed cell dilutions are then used
to inoculate sterile glass shell vials containing coverslips, as
described above. The cells are then centrifuged and inoculated with
virus or clinical samples, incubated, and fixed, as described
above.
[0170] The viruses capable of infecting these cells are detected
and identified using the methods described in Example 2 above. In
these mixed cell cultures, the 292 cells are used to detect the
presence of parainfluenza viruses and RSV, while the A549 cells are
used to detect the presence of adenoviruses, and the mink lung
cells are used to detect the presence of influenza viruses (e.g.,
Influenza A and B).
EXAMPLE 7
Detection of HSV and Chlamydia in Mixed Cell Cultures
[0171] In this Example, mixed cell cultures are provided which
allow the detection of two organisms commonly associated with
sexually transmitted diseases. In these experiments, mink lung
cells (e.g., Mv1Lu) useful for the detection of HSV are mixed with
McCoy cells useful for the detection of C. trachomatis.
[0172] First, McCoy cells and mink lung (e.g., Mv1Lu) cells cells
are grown in flasks, trypsinized, and mixed as described in
previous Examples, such that a suitable dilution of mixed cells is
produced. In preferred embodiments, the cells are diluted such that
the mixed cells in culture will be in approximately the same
proportions. These mixed cell dilutions are then used to inoculate
sterile glass shell vials containing coverslips, as described
above. The cells are then centrifuged and inoculated with samples
(e.g., clinical samples), incubated, and fixed, as described
above.
[0173] The organisms capable of infecting these cells (e.g., HSV
infects the mink lung cells, while C. trachomatis infects the McCoy
cells) are detected and identified using the methods described in
Example 2 above. As with the other mixed cell culture systems, the
presence of virus and/or C. trachomatis may be detected by other
methods, such as the observation of CPE, animal inoculation, etc.
Thus, it is not intended that the mixed cell culture assay systems
of this Example or any of the preceding examples be limited to any
particular method of microorganism detection, identification,
and/or quantitation.
EXAMPLE 8
Detection of Enteroviruses In Mixed Cell Cultures of Human
Embryonal Rhabdomyosarcoma (RD) Cells and H292 Cells, and In Mixed
Cell Cultures of BGMK and A549 Cells
[0174] In this Example, two different mixed cell cultures [Mix A
containing RD cells (ATCC #CCL 136) and H292 cells; Mix B
containing BGMK cells and A549 cells] were used to detect eighty
(80) different isolates of enteroviruses. The mixed cell cultures
were prepared essentially as described in Example 1, supra. The
eighty isolates were stored (frozen) clinical isolates which had
been harvested on MRC5 and/or PMK from cerebral spinal fluid,
throat, and feces. The results are shown in Table 4.
4TABLE 4 Percentage of Isolates with Growth in Mix A and Mix B
Virus (Number of Growth Growth in Either Isolates Tested) in Mix A
Growth in Mix B Mix A or Mix B Coxsackie (36) 64% 75% 100% Echo
(37) 95% 51% 100% Polio (7) 100% 100% 100% All Enteroviruses (80)
81% 66% 100%
[0175] The results in Table 4 show that all 80 enteroviruses grew
on shell vials of either Mix A or Mix B. Approximately 80% of all
enteroviruses grew on Mix A; 66% grew on Mix B. Over half the
Coxsackie viruses (64%) grew in Mix A; 75% grew in Mix B. Of the
Echo viruses, 95% grew on Mix A; 51% grew on Mix B. All the Polio
viruses grew on both Mix A and B.
[0176] These results demonstrate that each of Mix A and Mix B was
successful in detecting the vast majority (81% and 60%,
respectively) of all enteroviruses tested. The data also
demonstrate that the combination of Mix A and Mix B detected 100%
of all the clinical isolates of enteroviruses tested Thus, the
combination of Mix A and Mix B makes it possible to detect all
enteroviruses from clinical sources such as cerebral spinal fluid,
throat, and feces.
EXAMPLE 9
Detection of Enteroviruses In Mixed Cell Cultures Containing Human
Colon Adenocarcinoma (Caco-2) Cells
[0177] In this Example, mixed cell cultures containing human colon
adenocarcinoma (Caco-2) cells (ATCC #HTB 37) are used to detect and
identify enteric viruses, including coxsakieviruses, echoviruses,
polioviruses, rotaviruses, astroviruses, and adenoviruses.
Rotaviruses, astroviruses, and adenoviruses are responsible for 80%
of viral gastroenteritis occurring in young children [Pinto et al.
(1994) J. Medical Virol. 44:310-315].
[0178] Mixed Cell Cultures
[0179] Mixed cell cultures containing human colon adenocarcinoma
(Caco-2) cells (ATCC #HTB 37) in combination with one, two and
three other cell types selected from RD cells, H292 cells, BGMK
cells, and A549 cells (Table 5) are used to detect eighty (80)
different isolates of enteroviruses, which are prepared as
described above in Example 8. The mixed cell cultures are also used
to detect polio virus 1, coxsakievirus A24, enterovirus 70,
reovirus 3, human rotavirus, Adenovirus 5, Adenovirus 40, and
Adenovirus 41, astrovirus 1, and HAV strain HM175. These viruses
have been previously described by Pinto et al. (1994), supra. The
mixed cell cultures described in the following Table 5 are prepared
essentially as described in Example 1, supra, and are compared to
single cell type cultures which contain Caco-2, RD, H292, BGMK, or
A549 cells.
5TABLE 5 Mixed Cultures Containing Caco-2 Cells Sample Number Cell
Lines 1 Caco-2, RD 2 Caco-2, H292 3 Caco-2, BGMK 4 Caco-2, A549 5
Caco-2, RD, H292 6 Caco-2, RD, BGMK 7 Caco-2, RD, A549 8 Caco-2,
RD, H292, BGMK 9 Caco-2, RD, H292, A549 10 Caco-2, RD, BGMK, A549
11 Caco-2, RD, H292, BGMK, A549
[0180] Periodically during incubation, single and mixed monolayers
are checked for overall viability and ability of the cell lines to
co-exist and develop as a single cell sheet (i.e., a single
monolayer), with two distinct cell morphologies (i.e., dimorphic
cell sheets), at an approximately equal density of each cell type.
Successful mixed cultures are those in which mixed cell monolayers
adhere to the well surfaces, exhibiting a smooth, evenly
distributed monolayer in which each cell type can be easily
distinguished (i.e., "morphologic category 1"). Alternatively,
successful mixed cultures are those in which mixed monolayers
adhere to the well surfaces, but exhibit two distinct morphologies
at confluency (i.e., "morphologic category 2").
[0181] Samples and Processing
[0182] Enteric viruses are detected by identification of
virus-induced cytopathic effect (CPE), and/or by detection of
expression of at least one early virus-specific nucleic acid before
CPE is evident in the infected cultures. The presence of enteric
viruses is assayed by dot-blot hybridization before and after
infection of mixed cell cultures which contain Caco-2 cells as
previously described [Pinto et al. (1994) supra]. Briefly, virus
RNA and DNA is extracted from stool suspensions and at 48 hours
post-infection (p.i.) of mixed cell cultures. A probe corresponding
to the 2,300 bp of gene 4 of rotavirus Wa is used to detect human
rotavirus; a probe corresponding to 1 kb from the 3' end of human
astrovirus type 1 is used to detect human astroviruses; and a probe
corresponding to the 1,800 bp PstI fragment H of Adenovirus 40, and
a probe corresponding to the 6,500 bp PstI fragment B of adenovirus
41 is used to detect Adenovirus 40 and Adenovirus 41, respectively
[Pinto et al. (1994) supra]. All cDNA probes are labelled with
digoxigenin-11-UTP (Boehringer Mannheim) following the
manufacturer's instructions. A dot-blot hybridization assay is
carried out with the different cDNA probes following standard
procedures.
[0183] Results
[0184] The detection of CPE and/or virus nucleic acids using
dot-blot hybridization in mixed cell cultures containing Caco-2
cells demonstrates that these cells are susceptible to the enteric
viruses (i.e., coxsakieviruses, echoviruses, polioviruses,
rotaviruses, astroviruses, and adenoviruses) with which the mixed
cell cultures are inoculated.
[0185] From the above, it is clear that the present invention
provides many advantages over presently used methods in diagnostic
microbiology.
[0186] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes for carrying out the
invention which are obvious to those skilled in diagnostic
microbiology and virology, cell culture, and/or related fields are
intended to be within the scope of the following claims.
* * * * *