# US 4,447,537 A · Tick cell lines

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- Number: US 4,447,537 A
- Document ID: US4447537A
- Status: Granted
- Kind: A
- Application number: 06/227,166
- Filed: 1981-01-22
- Granted: 1984-05-08
- Earliest claimed: 1981-01-22
- Assignees: The United States of Americas as represented by the Department of Health and Human Services
- Inventors: Conrad E. Yunker; John C. Cory; Harold R. Meibos
- Web page: https://patentgrep.com/patent/US4447537A

## CPC

- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 2770/24064: by serial passage
- Y10S 435/948: using viruses or cell lines
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS

## Abstract

Six new cell lines of Acari: Ixodidae are provided, four from embryonic tissue of Dermacentor variabilis and two from embryonic tissue of Dermacentor parumapertus; and the use of these cell lines for replicating representative microorganisms is shown.

## Claims

### Claim 1

A continuous cell line of embryonic cells of Acari: Ixodidae of the species Dermacentor variabilis designated RML-15 and having ATCC No. CRL 8052.

### Claim 2

A continuous cell line of embryonic cells of Acari: Ixodidae of the species Dermacentor variabilis designated RML-18 and having ATCC No. CRL 8055.

### Claim 3

A continuous cell line of embryonic cells of Acari: Ixodidae of the species Dermacentor variabilis designated RML-19 and having ATCC No. CRL 8056.

### Claim 4

A continuous cell line of embryonic cells of Acari: Ixodidae of the species Dermacentor variabilis designated RML-20 and having ATCC No. CRL 8057.

### Claim 5

A continuous cell line of embryonic cells of Acari: Ixodidae of the species Dermacentor parumapertus designated RML-16 and having ATCC No. CRL 8053.

### Claim 6

A continuous cell line of embryonic cells of Acari: Ixodidae of the species Dermacentor parumapertus designated RML-17 and having ATCC No. CRL 8054.

### Claim 7 (depends on 1, 2, 3, 4)

The cell line of claims 1, 2, 3 or 4 adapted to an incubation temperature of from about 25.degree. C. up to about 39.degree. C.

### Claim 8 (depends on 7)

The cell line of claim 7 adapted to an incubation temperature of about 37.degree. C.

### Claim 9 (depends on 5, 6)

The cell line of claim 5 or 6 adapted to an incubation temperature of from about 25.degree. C. to about 30.degree. C.

### Claim 10 (depends on 9)

The cell line of claim 9 adapted to an incubation temperature of about 27.degree. C.

### Claim 11 (depends on 1, 2, 3, 4, 5, 6)

A composition consisting essentially of the cell line of any one of claims 1 to 6 maintained in a sterile medium consisting essentially of: modified Liebovitz-15 medium with glutamine and Eagle's minimal essential medium with Hank's salts and glutamine in a ratio of about 0.5-2:1; to which have been added: about 20% fetal bovine serum inactivated at 56.degree. C. for 1 hr; about 10% tryptose phosphate broth; and about 0.1% bovine plasma albumin-fraction V; all adjusted to a pH of about 6.8.

### Claim 12 (depends on 11)

The composition of claim 11 wherein after establishment, the cell line is maintained in a culture medium in which the ratio of modified Liebovitz-15 medium with glutamine to Eagle's minimal essential medium with Hank's salts and glutamine is about 1:1.

### Claim 13 (depends on 1, 2, 3, 4, 5, 6)

A method of replicating a microorganism selected from one of the group consisting of viruses, rickettsias, and spiroplasms, comprising infecting the cell line of any one of claims 1 to 6 with said microorganism and incubating the infected culture.

### Claim 14 (depends on 13)

The method of claim 13 wherein the microorganism is a virus selected from the group consisting of Chikungunya (23161), O'nyong-nyong (MP30), St. Louis encephalitis (798-55), Yellow fever (17D), Langat (TP21), Powassan (794), Modoc (M544), Kemerovo (R10), Colorado Tick Fever (SS18), Sawgrass (PR96406), and Cascade Virus (109231D).

### Claim 15 (depends on 13)

The method of claim 13 wherein the cell line infected consists essentially of cells of the species Dermacentor variabilis.

### Claim 16 (depends on 13)

The method of claim 13 wherein the microorganism is a rickettsia selected from the group consisting of R. Rickettsii (R), R. akari (Kaplan), R. typhi (Wilmington), and argasid tick isolate (113704-14).

### Claim 17 (depends on 13)

The method of claim 13 wherein the microoganism is a virus selected from the group consisting of Togaviridae, Reoviridae, Rhabdoviridae, Bunyaviridae, and taxonomically unclassified Arboviruses.

### Claim 18 (depends on 1, 2, 3, 4, 5, 6)

A method of isolating temperature sensitive variants of arboviruses comprising replicating the arboviruses in the cell line of any one of claims 1 to 6 at an incubation temperature set at from about 22.degree. C. to about 36.degree. C. and isolating the replicated variants.