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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 DISCUSSION

Results 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.

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