Isolation and characterization of temperature sensitive mutants of Broadhaven virus, a Kemerovo group orbivirus (family, Reoviridae)

331

Virus Research, 4 (1986) 331-336 Elsevier

VRR 00246

Isolation and characterization of temperature sensitive mutants of Broadhaven virus, a Kemerovo group orbivirus (family, Reoviridae) Stephen Natural Environment

R. Moss and Patricia A. Nuttall

Research Council, Institute of Virology, Mansfield Road, Oxford OX1 3SR, 7J.K (Accepted

20 December

1985)

Summary

Eighteen stable temperature sensitive (ts) mutants of Broadhaven virus were isolated without the aid of mutagens. Spontaneous mutants were detected using 41’C as the nonpermissive temperature and 36°C as the permissive temperature. High frequency pairwise recombination defined five recombination groups. Four mutants belonged to group I, three to group II, six to group III, two to group IV, and two to group V. ts 7 was a possible double mutant representing lesions corresponding to those of groups III and V mutants. This is the first reported isolation of temperature sensitive mutants of a tick-borne orbivirus. Broadhaven

virus, orbivirus,

temperature

sensitive

mutants,

recombination

groups

Four subgroups are distinguished within the Kemerovo serogroup of the Orbivirus genus in the family, Reoviridae (Gorman, 1983). Kemerovo virus (Kemerovo subgroup) was originally isolated from ticks and from cerebrospinal fluid of human patients with encephalitis (Chumakov et al., 1963). Members of the Great Island subgroup are tick-borne viruses associated with seabirds. Although members of a subgroup are related serologically, they show a high degree of antigenic variation, as demonstrated by neutralization tests (Libikova and Buckley, 1971; Main et al., 1973; Main, 1978; Nuttall et al., 1981, 1984). In order to study this antigenic variation, spontaneous ts mutants of a derivative of FT363 virus (Great Island subgroup) were selected. This is the first report of ts mutants of a tick-borne orbivirus. FT363 virus was originally isolated from ticks collected at St. Abb’s Head, Scotland (Nuttall et al., 1981). The virus (plaque-purified 3 times at 37°C) was 0168-1702/86/$03.50

0 1986 Elsevier Science Publishers

B.V. (Biomedical

Division)

332 shown to have a genome comprising 10 segments of double-stranded RNA, and 9 major virus-induced proteins were detected in infected Vero cell cultures (Eley et al., 1985). After plaque-picking once at 41°C a derived isolate was obtained that differed from FT363 virus and, therefore, was named Broadhaven virus after the name of the site from which the FT363 ticks were originally collected. Broadhaven and FT363 viruses showed one-way cross-neutralisation (Broadhaven virus 8/32, FT363 virus 32/256; reciprocal of heterologous/homologous 50% neutralising titre of the ascitic fluid), and differed in the migration of 6 RNA segments (segments 3, 4, 5, 7, 8, and 9) in 10% SDS-free polyacrylamide gels run at 220 V for 3 h. Spontaneous ts mutants of Broadhaven virus were isolated using the following protocol. Firstly, plaque assays were carried out at various temperatures: 36°C and 41°C were chosen as the permissive and non-permissive temperatures, respectively, since virus growth rate was slow below 36°C and virus yields were greatly reduced above 41°C. Wild type virus titres were reduced by 50% at 41’C compared with 36°C. Virus stocks originating from seven different plaques picked at 41°C were diluted to give 20 to 30 plaques per plate and inoculated onto confluent monolayers of Vero cells in 90 mm Petri dishes. The cells were overlayed with a mixture of 1.2% Seakem agarose and 2 x Eagle’s minimum essential medium with Earle’s salts (EMEM) supplemented with 4% foetal calf serum (FCS). The plates were incubated for 4 days at 36°C in an atmosphere of 5% CO,, and then stained for 1 h with 0.05% neutral red. Discrete plaques were picked and stored at 4°C in 1 ml EMEM-20% FCS. 100 ~1 of these suspensions were inoculated onto Vero cells in 60 mm Petri dishes, overlayed, and incubated at 41°C for 3 days. The plaques were then stained with neutral red and the original virus suspensions that gave less than 10 plaques at 41°C were used to inoculate duplicate Petri dishes which were then incubated at 36°C and 41°C. The plates were stained after 4 days and two plaques were picked, from plates incubated at 36°C provided that they had greater than 50 plaques at 36°C and that the corresponding 41°C plate contained less than 10 plaques (usually there were no plaques). The secondary plaques were stored in 1 ml EMEM-20% FCS at 4°C and 500 ~1 of this virus was inoculated onto Vero cells in a 25 cm2 flask. The virus was allowed to adsorb for 30 min, after which 5 ml EMEM-2% FCS were added. When a cytopathic effect appeared, the cells were frozen and thawed and cell debris removed. These stocks were titrated at 36°C and 41°C and any showing an efficiency of plating (EOP = titre 41”C/titre 36°C) of less than 0.002 were considered to be rs mutants. Such mutants were inoculated into 75 cm2 flasks of Vero cells, to give a working stock. Eighteen stable ts mutants were isolated by these procedures from 2800 plaques initially grown at 36°C. They all had an EOP value less than 0.002. The lowest titre was 7 X lo6 pfu/ml. Recombination frequencies for any two strains, eg. A and B, were calculated using the following formula. Since reciprocal double mutants cannot be detected, the calculated frequency was doubled: %R =

(yield A x B),,-(yield

A + yield B)41

(yield A X B)36

x 100 x 2 I

333 Percentage recombination values of less than 1 were taken to indicate that the two ts mutants in question belonged to the same recombination group; values greater than 1 were taken to indicate that they were of different groups. Recombination assays were carried out using confluent monolayers of Vero cells (4 x lo5 cells) in 30 mm Petri dishes. Virus mixtures were inoculated at a multiplicity of infection of 4 pfu/cell. After adsorbtion for 30 min, the virus was removed and 1 ml EMEM-2% FCS added. The cells were incubated at 36°C and then frozen and thawed and titrated at 36°C and 41°C. The appropriate time to harvest the cells was ascertained from growth curves using the method of infection described above for recombination assays. Growth curves of ts 4, ts 10, and a mixture of the two were compared (Fig. 1). Recombination frequencies were less than 1% for the first 2 h increasing to 6.8% by 18 h, and were maintained at this level until 22 h. Eighteen hours post-infection was selected as the optimum time to harvest the cells. Recombination assays showed that the eighteen ts mutants comprised five different recombination groups (Table 1). Each ts mutant was a member of a single recombination group apart from ts 7 which appeared to belong to both groups III

8

2

4

6

8

18 12 14 16 18 28 22 HOURS PI.

Fig. 1. Growth curve of recombination between ts 4 and ts 10. Titrated 41°C (- - -); 0. ts 4x ts 10; -, ts 4; x, IS 10.

at 36°C (-

); titrated

at

1

’ Recombination

a Assignments:

25 26

24

23

22

21

20

17

15

14

12

10

8

I

6

5

4

3

TVmutant

BETWEEN

0 14

20 0 74 21 0 21

0 0 71 6 45

35 8

0

1 3 0

18 10

III/V:

Group

less than 1 are recorded

fs 7.

IV: IS 22, fs 25.

V: ts 12, fs 15.

Group as 0.

III: fs 3, IS 6, 1s 14, ts 17, TV 23, ts 26.

Group

II: fs 5, IS 8, fs 10.

Group

frequencies

69

21

7

22

42

29

0

0

8

0 70 49

36 46 116

38 79 55 38 46

19 40 40 0

46 24

0

21

0

48

0

39

33

22

76

12

40

63

17

13

82

108

82

29

8

8

37

29

0

35

24

16

0

0

11

8

0

23

8

15 32

11

7 10

4

39

16

0

22

36

21

10

71

10

23

0

34

20

25

48

0

0

43

0

17

15

15 14

a

8

14

VIRUS

0

4

4

0

7

I2

8

3 7

16

10

25

0

8

4

0 ’

7

3

6

I: IS 4, ts 20, IS 21, rs 24.

2

7

5

Group

5

4

Group

3

OF BROADHAVEN

with IS mutants

ts MUTANTS

Percentage recombination

RECOMBINATION

TABLE

48 33 0 23

31 0 34 36

28

0 31

20 49

0

31

59

23

71

2

18

20

12

89

0 0

98

5 24

8 14

5

0

26 4

25

335 and V and hence was a possible double (multiple) mutant. There was no correlation between recombination group and the parent virus stock from which each mutant was derived thus indicating that each recombination group consisted of different mutants. Temperature sensitive mutants have been used to study the genetics of several families of segmented genome RNA viruses, including the Reoviridae (Cross and Fields, 1977) Orthomyxoviridae (Mahy, 1983) Arenaviridae (Compans and Bishop, 1985) and Bunyaviridae (Bishop, 1979; Iroegbu and Pringle, 1981). Most of these studies have utilised IS mutants generated by the use of mutagens, such as nitrous acid, nitrosoguanidine, proflavin and 5-fluorouracil. The frequency of ts mutants resulting from treatment with mutagens is generally 1 to lo%, depending on the type of mutagen and the virus, whereas the frequency of spontaneous ts mutants is lower, in the region of 0.1 to 0.3% (Fields and Joklik, 1969; Ramig, 1982, 1983; Gorman et al., 1983). However, mutagenesis increases the probability of generating more than one point mutation within a single gene. Temperature sensitive mutants of Broadhaven virus were selected without the use of mutagens in order to increase the probability that the mutants contained a single point mutation. The frequency of stable spontaneous ts mutants of Broadhaven virus was relatively high, at 0.6%. Co-infection of cells with two mutants, at the permissive temperature, resulted in the production of wild type (ts’) virus. Generation of ts+ virus presumably resulted from reassortment of genomic segments of two mutants that had ts lesions in different segments. Since there are 10 RNA segments comprising the genome of orbiviruses, there are 10 possible recombination groups. Pairwise recombination assays between the 18 ts mutants of Broadhaven virus delineated 5 recombination groups. Interestingly, 7 of the mutants (including one possible double mutant) belonged to the same group (group III). Further studies using the ts mutants of Broadhaven virus are in progress to define the genomic segment that codes for the neutralizing epitope(s) of this virus.

Acknowledgements The authors would like to thank Dr. D.H.L. Bishop ment, and Ms. J.M. Murdock for typing the manuscript.

for advice and encourage-

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