GROWTH OF LASSA AND EBOLA VIRUSES IN DIFFERENT CELL LINES
G. VAN DER GROEN
Instituut voor Tropische Geneeskunde, Laboratory of Bacteriology and Virology, Nationalestraat 155, B-2000 Antwerpen, Belgium.P. WEBB, K. JOHNSON, J.V. LANGE, H. LINDSAY, L. ELIOTT
Center for Disease Control, Atlanta, U.S.A.SUMMARY
Twenty two cell lines - VH 2, 8625, IgH-2, A6 TH-1, ICR-2A, Tb-1Lu, Calomys, BHK, PtK 1, PtK 2, Aedes pseudoscutellaris, A. albopictus, A. aegypti, Indian muntjac, PK 15, MOCK (ATCC), Vero, Rd and FHM - were infected with Ebola virus and Lassa virus.
In the reptile, amphibian, fish and Aedes cell lines tested no multiplication of both viruses was observed, except for Lassa where a very weak positive IFA occurred in both rattle snake and viper spleen cell lines.
For Lassa, cytopathic effect was obtained in Indian muntjac and Vero cells. In 8625, VSW, BHK, PtK 2, PK 15 and RD, Lassa could only be detected by indirect fluorescence antibody test (IFA). For Ebola cytopathic effect occurred in bat, marsupial, deer, dog monkey and human cell lines. In rat, marsupial (PtK 2) and pig cell lines Ebola was only detected by IFA.
Ebola virus shows a broader cell line susceptibility than Lassa.
INTRODUCTION
Until now only African green monkey kidney cell line (Vero) was used to isolate and assay Lassa (4) and Ebola viruses (1,2,3). The present study intends to find a more sensitive assay system in cell culture for Lassa and Ebola viruses.
MATERIALS AND MIETHODS
Virus. Lassa virus, Josiah strain pool 800593, second passage Vero 98, TCD 50/ml = 10^7 and Ebola virus Mayinga strain, pool 800590, second passage on Vero 98, TCD 50/ml on Vero = 10^6.6 were used. All work with Lassa and Ebola was carried out in CDC's maximum security laboratory.
Cell cultures. Details regarding the twenty two cell lines used are given in Table 1. Stock cultures were grown in 150 cm2 plastic tissue culture flask. For experiments tube cultures were inoculated.
TABLE I
VERTEBRATE AND INVERTEBRATE CELL LINES USED IN THE STUDY
Cell line
Obtained from ATCC (1)
Culture medium
Temp. of incubation (ºC)
1. VH-2
CCL 140
BME=NEAA in Hanks BBS (2)
+ FBS 10% Room
2. 8625
Collins lab
MEM (double conc.AA and Vitam)
+ CS 10% Room
3. VSW
CCL 129
BME (2)
+ FBS 10% Room
4. IgH-2
CCL 108
BME+NEAA in Hanks BSS
+ FBS 10% 36 ± 1
5. A 6
CCL 102
NCTC 109 (3)
+ FBS 15% Room
6. TH-1
CCL 50
BME
+ FBS 10% Room
7. ICR-2A
CCL 145
Leibovitz's L-15 (2)
+ FBS 10% Room
8. Tb-1 Lu
CCL 88
MEM+NEAA+sod.pyruvate+ Earles BSS+ HCO 3 (0.85g/1)
+ FBS 10% 36 + 1
9. Calomys
MEM+NEAA+sod.pyruvate+ Earles BSS+ HCO 3 (0.85g/1)
+ CS 10% 36 ± 1
10. BHK
Dr.Taylor
(CDC) MEM+NEAA+sod.pyruvate+ Earles BSS+ HCO 3 (0.85g/1)
+ FCS 10% 36 ± 1
11. PtK 1
CCL 35
MEM+NEAA+sod.pyruvate+ Earles BSS+ HCO 3 (0.85g/1)
+ NCS 10% 36 ± 1
12. PtK 2
CCL 56
MEM+NEAA+sod.pyruvate+ Earles BSS+ HCO 3 (0.85g/1)
+ FCS 10% 36 ± 1
13. A. pseudoscutellaris
Mitsuhashi & Maramorosch (2)
+ FBS 20% Room
14. A. albopictus
CCL 126
Mitsuhashi & Maramorosch
+ FBS 20% Room
15. A. aegypti
CCL 125
Mitsuhashi & Maramorosch
+ FBS 20% Room
16. Indian muntjac
CCL 157 Ham's F 10 (3)
+ FBS 20% 36 ± 1
17. PK 15
CCL 33
MEM+NEAA+sod.pyruvate+ Earles BSS+ HCO3- (0.85g/1)
+ NCS 5% 36 ± 1
18. MDCL
Pfau
MEM+NEAA+sod.pyruvate
+ CS 10% 36 ± 1
19. MDCK
CCL 34
Lactalbumin hydrolysate (0.5%)+Earles BSS+ HC03- (0.85g/1)
+ NCS5% 36 ± 1
20. VERO 98
CDC
MEM
+ CS 10% 36 + 1
21. RD
Dr.J.Esposito (CDC)
MEM+double conc.AA+vit+ Hanks BBS
+ FBS 10% 36 + 1
22. FHM
CCL 42
MEM
+ CS 10% 36 + 1
1. ATCC American tissue culture Collection with corresponding tissue culture number ced salt solution; Abbreviations used BME: Basal medium Eagle; NEAA: Non Essential amino acids; BSS: Balan MEM: Minimum Essential Medium; FBS: Foetal bovine serum; CS: Calf serum; NCS: New born calf serum; FCS: Foetal calf serum.
2. Purchased commercially by GIBCO
3. Purchased commercially by microbiological Associates
Infection of cell lines. Supernatants of cultures growing in monolayers were decanted and the tubes refreshed with maintenance media (= culture media with 2% serum). For Aedes pseudoscutellares Laibovitz L-15 was used as a maintenance medium.
Three tubes were each inoculated with 0.1 ml 10 ^-1 and 10^-3 dilutions (in maintenance medium) of Ebola and Lassa, for each cell line investigated. Each time Vero cells were inoculated as controls. Infected cells were investigated daily on occurrence of CPE and on the 5th and 14th day cells were screened on presence of antigen by indirect fluorescence antibody test (IFA). Cells were scraped off and suspended in 0.2 ml supernatant. After addition of 0.2 ml phosphate buffered saline a mutispot slide is filled with cell suspension drops.
After drying and fixation in aceton (10') IFA is done as previously described (5).
RESULTS AND DISCUSSIONResults are summarized in Table 2.
Reptile, amphibian, fish and Aedes cell lines supported no multiplication of both viruses except for Lassa where a very weak positive IFA occurred in both rattlesnake and viper spleen cell lines.
With Ebola a cytopathic effect occurred in Vero 98 (395), PtK 1 (3,7), Tb I Lu (3,10), MOCK (ATCC) (7,10), Indian muntjac (10,11) RD (8,13) and MOCK (10, no CPE). Figures between brackets indicate first appearance (days post inoculation) of CPE in 10^-1 and 10^-3 inoculated cell cultures. The CPE with Tb 1 Lu was very pronounced and the easiest to observe.
With Lassa a cytopathic effect occurs only in Vero 98 (4,4) and Indian muntjac (10,12) cell lines.
The ease in detection of Lassa antigen by IFA decreased in the following order: RD, Vero 98, PK 15, BHK, Indian muntjac, PtK 2. With RD cells, very bright fluorescence occurred in the presence of Lassa antigen.
Ebola antigen detection by IFA was the best in Vero 98 and decreased in the following order: PK 15, RD, Calomys, MOCK (ATCC) Tb 1 Lu. PtK 1, Indian muntjac, PtK 2, MDCK (pfau), BHK.
Finally we can state that for both viruses Vero 98 is still the most sensitive cell line. The. characteristic CPE in Tb 1 Lu for Ebola and the pronounced fluorescence in RD cells with the IFA test for Lassa can be helpful.
ACKNOWLEDGEMENTS
The authors thank Mrs. Engelman, H. for technical assistance.
TABLE 2
CYTOPATHIC EFFECT AND POSITIVE IFA OF LASSA AND EBOLA IN DIFFERENT CELL-LINES
Ebola
Lassa
Cell-Lines
-1
-3
-1
-3
1. VH-2
Viper
-
-
-
-
2. 8625
Rattlesnake
-
-
C1O F7
-
3. VSW
Viper spleen
-
-
F14
4. IgH-2
Iguana
-
-
-
-
5. A6
Toad
-
-
-
-
6. TH-1
Turtle
-
-
-
-
7. ICR-2A
Frog
-
-
-
-
8. Tb-lLu
Bat
CF4
-
-
-
9. Calomys
Rat
F5
F14
-
-
10. BHK
Hamster
F10
-
F10
F10
11. PtK1
Marsupal
CF5
-
-
-
12. PtK2
Marsupal
F14
-
F4
13. Aedes ps
-
-
-
-
14. Aedes albopictus
-
-
-
-
15. Aedes aegypti
-
-
-
-
16. Indian muntiac
Deer
C10 F5
-
CF14
CF14
17. PK15
Pig
F8
F8
F6
F6
18. MOCK (pfau)
Dog
CF14
-
-
-
19. MOCK (ATCC)
Dog
CF6
CF14
20. Vero 98
Monkey
CF5
CF5
CF5
CF5
21. RD
Human
C7F4
CF14
F4
F4
22. FHM
Fish
-
-
-
-
CF cytopathic effect and positive FA on indicated day. C only cytopathic effect on day tested. F. positive FA on day tested. negative result.
If at the indicated day FA is positive and CPE occurs later, then the first day at which CPE occurs, is indicated as the number behind C. f.e. ClOF5 : at day 5 FA was positive and CPE occurred on the 10th day.
REFERENCES
1. Bowen, E.T.W., Platt, G.S., Lloyd, G., Baskerville, A., Harris, W.J., Vella, E.E. (1977) Viral hemorrhagic fever in Southern Sudan and Northern Zaire. Preliminary studies on the aetiologic agent. Lancet, 1, 571-573.
2. Johnson, K.M., Webb, P.A., Larige, V.E., Murphy, F.A. (1977) Isolation and partial characterization of a new virus causing acute hemorrhagic fever in Zaire. Lancet, 1, 569-571.
3. Pattyn, S.R., Jacob, W., Van der Groen, G., Piot, P., Courteille, G. (1977) isolation of Marburg-like virus from a case of hemorrhagic fever in Zaire. Lancet, 1, 573-574.
4. Wulff, H. et al. (1975) Recent isolations of Lassa virus from Nigerian rodents. Bull. Wld. Hlth. Org., 52, 609-612.
5. Wulff, H. Lange, J.W. (1975) Indirect immunofluorescence for the diagnosis of Lassa fever in infection. Bull. Wld. Hlth. Org., 52, 429-435.
