Bull. Org. mond. Bull. Wid Hith Org.Sant) 1968, 39, 419-424

Material for the Study of Variation in the Malayan Langat (TP-21 Strain)*

V. I. IL'ENKO, V. G. PLATONOV, I. N. PROZOROVA & A. A. SMORODINCEV

The lack of genetic uniformity of the Malayan Langat virus, TP-21 strain isolated from Ixodes granulatus ticks in Malaya, was demonstrated. Increased pathogenicity of the virus for white mice and rhesus monkeys after passage through chick-embryo fibroblast cultures at temperatures of 36°C and 40°C is due to the selection of the pathogenic virus particles which are always present in the initial brain- tissue suspension of the TP-21 Langat strain. Thefrequency ofthe occurrence ofpathogenic clones and their selection rate was studied in relation to the conditions of cultivation. A study was also made of the genetic stability and basic biological properties ofpathogenic and non-pathogenic clones of the Langat virus.

The of the tick-bome encephalitis group circulation in a natural focus (the alternation of are characterized by the high degree of stability hosts with different body temperatures, in whose of their hereditary characters. Numerous attempts bodies the virus maintains the ability to multi- to obtain variants with changed pathogenicity have plying-ticks (with a body temperature equal to either not been successful or else the variants that that of the environment), small rodents and birds). have arisen have proved extremely unstable.' In a All this has led to the establishment in phylo- number of cases the results claimed were not con- genesis of a wide range of temperature conditions firmed by further experiments (Molnar, 1956); 2 under which reproduction can take place. In this the work of Mayer (1963) provides the only connexion such rapid variation in the Langat virus exception. Mayer succeeded, after making numer- -one of the tick-borne encephalitis group-after ous passages, and subsequent cloning, of a Czecho- exposure to different temperature conditions slovak strain of tick-borne encephalitis virus, in seemed scarcely probable. obtaining completely convincing differences be- Our interest in the TP-21 Langat virus is due tween the initial and passaged strains. to the possibilities for using it to obtain harmless The special position of the Malayan virus is due and highly immunogenic strains for the production not only to the fact that it is a naturally attenuated of vaccines to protect against tick-borne encephal- agent but also to the ease with which its patho- itis. genicity for white mice and rhesus monkeys can The aim of the present research was to study be enhanced by passage through a chick fibroblast the range and mechanism of the variation that has tissue culture (Price et al., 1963). been reported, the genetic composition of the Studies of the variation of tick-borne ence- initial population and the effect of a rise in the phalitis virus under experimental conditions made temperature of cultivation on the rate of establish- over a period of many years have, according to ment of pathogenic variants. We were also inter- the reports, given uniformly negative results. The ested in the stability of the pathogenic and non- high degree of stability of the genetic characters pathogenic clones obtained. of the virus can be ascribed to the features of its

* From the Department of Virology, Institute of Experi- MATERIAL AND METHODS mental Medicine of the Academy of Medical Sciences of the USSR, Leningrad. The virus used in the study was a sample of 1 Tjusnjakova, M. K. (1962) Paper read at a conference the Malayan Langat TP-21 virus isolated from at Minsk on tick-borne encephalitis. Ixodes granulatus in Malaya in 1956 by Dr C. E. 2Also Jakovlev, A. 1. (1953) Author's summary of a thesis, Moscow. Gordon Smith and obtained from him in 1960.

2235 - 419 420 V. I. IL'ENKO AND OTHERS

White mice weighing 10 g-12 g each, obtained their reactions, their appetite, mobility, etc.; and from the Rappolova Breeding Station of the Aca- (3) their neurological condition. demy of Medical Sciences of the USSR, and rhesus monkeys (Macaca mulatta) weighing Tissue culture 1.5 kg-2.5 kg were used in the experiments. In all the experiments use was made of a mono- Mice were infected by an intracerebral (0.03 ml), layer culture of chick-embryo fibroblasts. The an intraperitoneal (0.25 ml) or a subcutaneous growth and maintenance media consisted of Gey's injection (0.25 ml) of virus-containing material. salt solution with the addition of 5% of bovine Monkeys were infected by an intracerebral injec. serum inactivated by heating for 20 minutes at tion (0.5 ml) of virus-containing material. 56°C. The serum was previously checked to ensure that it contained no antibodies to the Period of observation and reading of the results Langat virus. The infected mice were kept under observation Cloning for 15 to 20 days. The number of sick and dead animals was counted after an incubation of 4 to This was done by the method of limit dilutions 5 days. The specific cause of death in the mice with subsequent checking of the population of was determined from the presence of typical each individual clone for uniformity. clinical features, the incubation period and, on a RESULTS sample basis, by checking the cause of death by The Malayan virus is the only representative of supplementary intracerebral passages in mice or the carrying out of the biological neutralization the tick-borne encephalitis group that has not been cause disease in natural test. found to human foci. The Langat TP-21 virus also differs from other repre- The condition of the infected monkeys was sentatives of the group in its lack of pathogenicity assessed on the basis of the following features: for white mice following upon intraperitoneal and (1) body temperature-measurement of the subcutaneous infection or intracerebral infection rectal temperature 3-5 days before infection and of rhesus monkeys. The Malayan virus possesses a daily after infection; sharply reduced capacity for reproduction in tissue (2) general condition of the monkeys-daily culture at 40°C and is quite unable to multiply at examination of their behaviour, the liveliness of 41.5°C (Table 1).

TABLE I BASIC CHARACTERS OF PATHOGENICITY WHICH DIFFERENTIATE VIRUSES IN THE TICK-BORNE ENCEPHALITIS GROUP FROM THE MALAYAN LANGAT VIRUS Susceptibility Susceptibility Causes of mice a of monkeys a Multi- Virus Area of distribution human illness nSubcutan- at 41.5Ca peritonealIn'tra- eoubctn Patho- Bait415Cascsypo infection infectioneoust genicity c symptom

Tick-borne encephalitis Eastern and western regions of the USSR Yes + + + Paralysis + Diphasic meningo- Western regions of the encephalitis USSR and countries of Western Europe Yes + + + Ataxia + paralysis + Great Britain Rarely + + + Ataxia + Omsk haemorrhagic Siberia Yes + + + Ataxia+ kidney + fever lesions Powassan Canada Yes + + + Paralysis + India Yes + + + No lesions of the + central nervous system Malayan Langat virus Malaya No

a + = Positive result; - = negative result. STUDY OF VARIATION IN THE MALAYAN LANGAT VIRUS 421

TABLE 2 ,CHANGES IN THE PATHOGENICITY FOR MICE AND MONKEYS OF THE MALAYAN LANGAT VIRUS AFTER VIRUS PASSAGES IN CHICK-EMBRYO FIBROBLAST CULTURES AT DIFFERENT TEMPERATURES

Virus cultivated at 36°C Virus cultivated at 40°C Concentration of the virus Concentration of the virus No. of (in log LDso) Patho- Multipli- (in log LDso) Patho- Multipli- I ntra- I ntra- Subcutan- for t 41e5iCc I ntra- I ntra- Subcutan- for at41c5oCc cerebral peritoneal eous monkeys c a cerebral peritoneal eous monkeys c infectiona infectionb infectionb infectiona lnfectionb infectionb

7.5 0 0 6.5 0 0

3 6.3 0.5 0 7.2 3.7 0.7 5 7.2 3.6 1.4 7.5 3.5 1.8 + + 7 7.1 4.3 1.3 + 7.3 3.8 3.0 9 7.2 5.0 2.5 7.5 4.4 3.1 30 7.0 4.8 3.0 6.7 5.5 4.2

a 0.03-ml doses. 0.25-mI doses. c + = positive result; - = negative resu:t.

Passages through chick-embryo fibroblast cul- from the initial brain-tissue strain of the virus tures lead to quite speedy enhancement of the which had not been passaged through tissue cul- pathogenicity of the virus for mice and monkeys ture. and also to changes in the temperature threshold By the limit-dilution method of cloning of the for the reproduction of the virus in tissue cultures. original virus we isolated in a certain percentage These changes were found to occur more quickly of cases, in addition to non-pathogenic clones, if the virus was passaged at 40°C (Table 2). virulent variants of the virus which do not differ The reason for this variation can be ascribed to from the pathogenic virus obtained after multiple mutation of the virus in its passages through the passage through chick-embryo fibroblast cultures. tissue culture or to the selection of pathogenic Thus it was shown that enhancement of the virus particles present in the initial strain. Analy- pathogenicity of the Malayan virus after passages sis of our material suggested that the underlying through tissue culture is not due to the appearance cause of the variations is the selection of patho- of new properties but to the selection of the genic particles from the genetically non-uniform pathogenic particles of virus which are always virus. The evidence for this suggestion is the present. In the initial material there were very few regularity and reproducibility of the changes such pathogenic particles and they were not obtained and the isolation of pathogenic clones always discovered by cloning, but during supple-

TABLE 3 ISOLATION RATE OF PATHOGENIC AND NON-PATHOGENIC CLONES OF MALAYAN LANGAT VIRUS IN PASSAGES OF THE INITIAL BRAIN-TISSUE VIRUS THROUGH TISSUE CULTURES AT 360C

No. of Pathogenic VirusViruusedusefofor cloingpassagescloning | studiedNlones Non-pathogenicclones clones

Initial brain suspension - 31 29 2 After passage through tissue culture 2 12 9 3 4 8 1 7 7 11 0 11 10 10 0 10 422 V. I. IL'ENKO AND OTHERS mentary passages through tissue culture the virus used for the passages, pathogenic particles were population was enriched with pathogenic particles. detected only in a dilution of 10-3 in the first and Table 3 shows the results of cloning the Langat second tests and a dilution of 10-2 in the third virus which had been passaged through tissue cul- test. In large dilutions of the initial brain-tissue tures various numbers of times. It will be seen suspension of the virus, pathogenic particles were from the table that when 31 clones of initial brain- not detected and it can therefore be assumed that tissue virus were examined, only 2 of them proved they form about 0.001 % of the original virus to be pathogenic. After 4 passages through chick- population. embryo fibroblast culture 7 out of the 8 clones The main virulence markers in the clones de- studied were pathogenic and after 7 passages we tected were pathogenicity for adult white mice were unable to detect any non-pathogenic virus in infected intraperitoneally and subcutaneously, the population. pathogenicity for intracerebrally infected rhesus The quantitative proportion of pathogenic par- monkeys and a capacity for multiplication in ticles in the initial Langat virus mnaterial can be tissue culture at 41.5°C (Table 5). assessed from Table 4 which gives the results of 3 parallel experiments for the titration of patho- TABLE 5 genic and non-pathogenic particles. With this pur- BASIC CHARACTERISTICS TYPIFYING pose in view, 10-fold serial dilutions (from 10-2 THE PATHOGENICITY OR NON-PATHOGENICITY to 10-10) of the original brain-tissue suspension of OF CLONES OF THE LANGAT TP-21 VIRUS the virus were prepared and then a number of Characteristic Pathogenic Non-pathogenic consecutive passages of each dilution through Characteristcclone a clone a fibroblasts were made with a view chick-embryo Pathogenicity for mice: to enriching the virus population with pathogenic By intraperitoneal infection + particles if any were contained in the initial dilu- By subcutaneous infection of the virus used for passaging. tion Pathogenicity (intracerebral infection) TABLE 4 Multiplication in chick-embryo fibroblast tissue culture CONTENT OF PATHOGENIC PARTICLES IN THE INITIAL (41.5°C - 42°C) BRAIN-TISSUE SUSPENSION OF MALAYAN LANGAT TP-21 VIRUS a + = Positive result; - = negative result. Pathogenic virus detected after Dilutions of the 5 passages through tissue culture The results of study of 2 groups of Langat virus initial...... _. viruis...... --_-_--_-iused for passage Experiment Experiment Experiment clones isolated from the initial brain-tissue suspen- I a 2 a 2 a_ sion after 10 chick embryo fibroblast passages at 400C are shown in Table 6. Of the 9 clones 10-2 + + + isolated from the initial brain-tissue strain only I 10-3 + was pathogenic for mice on intraperitoneal and 10-4 subcutaneous administration and for monkeys on 10-5 intracerebral administration. This clone multiplied at After 10 10-6 41.50C in chick-fibroblast culture. passages of the initial virus in chick fibroblast 10-7 cultures all 9 clones isolated proved pathogenic 10-s for both mice and monkeys. Repeat cloning of pathogenic and non-pathogenic clones showed a +, After 5 passages the population consisted of pathogenic non- virus particles; their genetic uniformity. Passages of the -, the pathogenic virus was not detected. pathogenic clone through tissue culture did not enhance its pathogenicity and this suggests that After 5 passages (this is usually quite enough the initial brain-tissue suspension was a mixture to detect the pathogenic clones of the virus) the of pathogenic and non-pathogenic virus particles. virus obtained was checked on the basis of patho- The results of cloning such a non-virulent genicity markers. As will be seen from Table 4, variant after 10 passages through chick-embryo with a virus titre of 108 in the original suspension fibroblasts at 360C and of 5 further passages at STUDY OF VARIATION IN THE MALAYAN LANGAT VIRUS 423

TABLE 6 RESULTS OF STUDY OF CLONES ISOLATED FROM THE INITIAL VIRUS AND THE TISSUE-CULTfURE VARIANT AFTER 10 PASSAGES AT 400C USING THE METHOD OF LIMIT DILUTIONS

Numbers of clones Numbers of clones isolated from the Characteristic ~isolated from the initial virus tissue culture virus after 10 passages

Pathogenicity for mice 4.5 4.7 5.5 4.5 5.5 57654.7 7.3 7.5 7.5 7.2 7.8 7.5 8.2 7.5 7.7 7.5

for monkeys + + + + + + + + Multiplication +I at 41.50C - + +- + + + + +

40'C are given in Table 7. It will be seen from to selection of the pathogenic virus particles, the table that-all the 6 clones of this variant that whereas the original quantitative interrelationships were studied proved to be non-pathogenic. The of virus particles of varying degrees of patho- composition of the pathogenic strain of the vi'rus genicity are maintained in mo-use brain. It is of was also uniform. interest to study the reasons for this selection in 'The virulent and non-virulent- clones,of Langat chick-embryo fibroblast cultures and its failure to virus- obtained were very stable and preserved occur in passages through the white-mouse brain. their -properties- in the course of multiple (20-30) In the light of the data obtained from the passages thro'ugh tissue cultures (non-pathogenic experiments it is essential to reconsider existing clones) or after 10 passages through the brain of ideas regarding the almost insuperable stability of white mice (pathogenic clones) (Table 8). tick-borne encephalitis virus. In view of the fact that previous investigations were carried out with rather DISCUSSION ~~mixed, than pure, lines of virus, it was DISCUSSION ~~~difficult to ensure the necessary conditions for The material presented in this paper indicates selecting non-pathogenic virus particles. The that the Langat virus circulating in nature is Malayan virus, for example, produces swift, but genetically non-uniform and that it consists of a only apparent, " changes " in passage through mixture of pathogenic and non-pathogenic par- chick-embryo fibroblast cultures. These changes tidles which can quite easily be separated by the are in fact no more than a quantitative shift in the limit method of cloning in white mice. It is im- relative proportions of the 2 previously existing possible to use chick-embryo fibroblast cultures lines of the virus. In passages of the virus through for this kind of cloning since that procedure leads the brain of white mice, the relationship between

TABLE 7 STUDY OF THE COMPOSITION OF THE POPULATION OF THE PATHOGENIC AND NON-PATHOGENIC CLONES AFTER 15 PASSAGES THROUGH TISSUE CULTURES

Pathogenic clone a Non-pathogenic clone a Virulence markers ______~~~~~~~~123] 4 [5 6 1213 [41516

Pathogenicity for mice: By intracerebral infection 5.5 6.2 7.2 7.5 7.7 8.2 4.2 5.6 4.2 6.2 4.2 5.5 By intraperitoneal Infection 4.0 4.6 5.5 5.5 4.5 4.4 0 0 0 0 0 0 Pathogenicity for monkeys + + + + + + ------Multiplication at 41.50C + + + + + + ------

a + = positive result; - = negative result. 424 V. I. IL'ENKO AND OTHERS

TABLE 8 CHANGES IN THE PATHOGENICITY OF THE LANGAT VIRUS AFTER PASSAGES THROUGH CHICK-EMBRYO FIBROBLAST CULTURES AT DIFFERENT TEMPERATURES

Concentration of virus 1 (log LDso) Pathogen- Multipli- No. of Type of virus Type ofWhenvirusinvestigated pasontrapassages I ntra- ~Inta-Intra- Subcutan- mnesCa415Cicity' for cation cerebral peritoneal eous infection a infection b infection b

Mixed (initial brain- Initial - 7.5 0 0 - tissue suspension) After passages at 36°C 9 7.2 5.0 2.5 + + After passages at 40°C 9 7.5 4.4 3.1 + +

Non-virulent clone Initial - 7.5 0 0 _ After passages at 36°C 30 6.3 0 0 _ After passages at 40°C 20 6.5 0 0 _

Virulent clone Initial - 7.3 5.2 3.4 + + After passages at 40°C 10 7.0 4.8 3.5 + After passage through the brain of white mice 8 7.5 4.0 3.2 + +

a 0.03-mi doses. b 0.25-ml doses. c + = positive result; - - negative result. the pathogenic and non-pathogenic variants is The uniformity and genetic stability of the solidly maintained since it exists in the initial virus avirulent clone of the Langat virus makes it strain- used for passage, while in passages through possible to recommend it as a promising candi- chick-embryo fibroblast cultures the pathogenic date for the preparation of a live vaccine against variant is selected. tick-borne encephalitis.

RESUMI Parmi les virus du groupe de 1'encephalite a tiques, le cerebral infect6) genetiquement non uniforme. Les virus Langat (souche TP21) est le seul qui ne soit pas clones virulents different nettement des clones avirulents: pathogene pour 1'homme dans les conditions naturelles et ils sont pathogenes pour la souris en inoculation intra- qui soit depourvu de virulence pour la souris et le singe peritoneale ou sous-cutan6e et pour le singe rhesus en rhesus. Sa multiplication en culture cellulaire est for- inoculation intracerebrale; ils sont capables de se mul- tement r6duite A 40° C et completement inhibee A 41,50 C. tiplier a 41,5° C. Les recherches decrites dans le present article mettent Les lignees pures de variants pathogenes ou non en evidence le manque d'uniformite genetique du virus pathogenes du virus Langat sont stables et leurs caracte- Langat circulant et 1'existence, dans une meme souche, ristiques ne se modifient pas apres de multiples passages de particules virulentes et non virulentes. La culture sur sur fibroblastes d'embryon de poulet ou tissu cerebral fibroblastes d'embryon de poulet permet de deceler les de souris. L'uniformitM et la stabilite genetique des clones pathogenes; en repetant les passages sur ce milieu, clones avirulents permet d'envisager leur utilisation en on obtient des lignees pures de particules de pathogenicit6 vue de la prdparation d'un vaccin vivant contre 1'encd- elevee selectionn6es A partir du materiel initial (tissu phalite a tiques.

REFERENCES Mayer, V. (1963) Virology, 20, 2, 372 Price, W. H. et al. (1963) Amer. J. trop. Med. Hyg., 12, 5 Molnar, E. (1959) Acta microbiol. Acad. Sci. hung., 6, 23 782