Enzyme studies for the characterization of some orthopoxvirus isolates

Bulktin of the World Health Organization, 6 (3): 377 - 380 (1982) Enzyme studies for the characterization of some orthopoxvirus isolates H. S. BEDSON 1 The thymidine kinase produced in cell cultures by several orthopoxviruses has been tested for sensitivity to inhibition by thymidine triphosphate (TTP). Thymidine kinase preparationsfrom eleven variola isolates andfrom five "whitepox" isolates were allfound to be sensitive to inhibition by 77P. Preparationsfrom other orthopoxviruses, including vaccinia, cowpox, and monkeypox, were all resistant to inhibition by 77P. There was no overlap between the two groups ofresults and no viruses werefound which gave intermedi- ate results. Tests with hybrid virusesshowed that this charactersegregated independently of other biological marker characters and is thus a valuable additional markerfor variola and variola-related viruses. The main aim of the investigation was to provide additional means for the differentiation and identifi- cation of viruses of the vaccinia-variola serological group of poxviruses. With the eradication of small- pox from West and Central Africa, a previously un- recognized poxvirus infection of man was en- countered. The virus concerned was identified as monkeypox virus because of its similarity to viruses previously isolated from institutional outbreaks of poxvirus infection occurring in primates in captivity. Searches for a natural animal reservoir of monkeypox virus have been repeatedly unsuccessful. Unexpected- ly, however, these searches have led to the detection of unusual virus isolates on four occasions, two from primate specimens and two from rodent tissues, which have been called "whitepox" viruses because of the appearance of their pocks on the chorioallan- toic membrane. Interest in "whitepox" viruses has been intense because they are indistinguishable from smallpox viruses in the laboratory and one would not, of course, expect to recover such viruses from animals. Two further variola-like viruses had previously been isolated from monkey kidney tissue cultures in Holland in 1964 a few months before an outbreak of monkeypox in the Rotterdam Zoo (1). Attempts to clarify the relationships among monkeypox virus, variola virus, and the six "white- pox" viruses have been made in several laboratories, and "whitepox" viruses have consistently behaved like variola viruses in all tests that have been applied. Our own approach has been based on virus-induced X Head, Department of Medical Microbiology, University of Birmingham, England. Professor Bedson died on 6 September 1978, and the data have been prepared for publication by K. R. Dumbell. Requests for reprints should be sent to Dr K. R. Dumbell, Virology Department, St Mary's Hospital Medical School, Paddington, London W2 IPG, England. enzymes. Poxviruses are known to induce increased activity in a number of enzymes concerned with nucleic acid metabolism. For some, notably the DNA polymerase and thymidine kinase, there is good evidence that the enzymes are new and virus-coded (8). Differences in their properties may therefore pro- vide useful markers for characterization of the virus concerned. The work has so far dealt only with the thymidine kinase. Several reasons made this an attractive first choice. The enzyme is easy to assay and high levels of activity are found in crude cell extracts. Poxvirus infection of HeLa cells is usually followed by a dramatic rise in activity, a 10-fold rise being common- place and a 50-100-fold rise not infrequent. A number of cell lines lacking thymidine kinase are available and in these the virus-induced enzyme can be examined without interference from the back- ground of host-cell enzyme. Initial studies with vaccinia, cowpox, variola major, and alastrim viruses showed that the enzymes cross-reacted and could not be distinguished in sero- logical tests with rabbit hyperimmune vaccinia and cowpox antisera. Various physico-chemical proper- ties of the enzyme were examined. These included optimum pH, sensitivity to magnesium ion concen- tration, thermal stability, ability to use uridine tri- phosphate as phosphate donor, apparent Km for thymidine, and electrophoresis of undenatured enzyme. None of these investigations suggested any properties of the thymidine kinase enzyme that might be used as a means of differentiating between the viruses. There was, however, a difference in the sensitivity of the enzymes to feed-back inhibition by thymidine triphosphate (TTP) (2) and further studies concentrated on this property. 4186 -377- 378 H. S. BEDSON MATERIALS AND METHODS Viruses The origins of the various viruses used in this study have been detailed in a previous report (6). High-titre virus stocks were grown in HeLa cells or on the chick chorioallantoic membrane (CAM). In many instances the virus was concentrated and partially purified by differential centrifugation but crude extracts of infected cells, clarified only by low- speed centrifugation, have also been satisfactory as virus inocula. Enzyme preparations Each virus was used to prepare infected cell extracts in both HeLa cells and in BHK cells lacking thymidine kinase (BHK-TK- ). The cells were grown as mono- layers in Petri dishes and harvested 16-18 h after infection at high multiplicity.a The cells were de- tached from the glass with a silicone rubber-coated rod, suspended in 4 mmol/litre phosphate buffer, pH 7.4, at 2 x 107 cells/ml, and disrupted in a a Multiplicity = no. of virus particles/no. of cells. sonicator. The low-speed supernatant fluid was obtained and stored frozen at - 20 °C until required for assay. An additional cell line (LTK -) lacking thymidine kinase was used for enzyme preparations in some of the experiments. Enzyme assays Cell extracts suitably diluted were added to an assay mixture containing 2.1 umol of '4C-thymidine (4.63 x 103 Bq or 0.125 gCi), 1.2 Amol of ATP, 0.625 itmol of MgCl2, 12.5 itmol of phosphate buf- fer, pH 7.5, and variable amounts of TTP in a final volume of 0.25 ml. Incubation was for 10 min at 35 °C and the product was separated by application to DEAE-cellulose paper (Whatman Chromedia DE 81) and washing in 4 mmol/litre ammonium formate. RESULTS Preliminary experiments revealed that the extent of inhibition from TTP was independent of enzyme concentration and that mixtures of enzymes gave the Table 1. Percentage residual activity, at different thymidine triphosphate (TTP) concentrations, of the thymidine kinase enzymes induced by standard vaccinia-variola group poxviruses HeLa cells BHK TK- cells Virus Strain 4 Amol/litre TTP 8 Amol/litre TTP 4 Amol/litre TTP 8 ,mol/litre TTP Animal poxviruses vaccinia Lister 60 48 97 72 vaccinia Connaught Laboratories 80 63 68 31 rabbitpox Utrecht 86 65 95 81 cowpox Brighton 79 54 90 - monkeypox Washington an.m1l73 51 63 52 monkeypox Holland isolates 53 70 52 37 monkeypox Denmark i 56 65 91 65 monkeypox Congo-8 human 93 83 104 87 monkeypox Liberia-1 f isolates 73 68 54 47 ectromelia Mill Hill 85 62 85 61 buffalopox 78 75 76 84 camelpox CM-5 95 86 99 85 elephantpox EP 1 - - 90 66 elephantpox EP 2 - - 77 73 turkmenia - - 93 65 Variola viruses variola major Harvey 12 4 19 5 variola major Hinden 21 6 20 8 alastrim Butler 19 3 19 7 alastrim Brazil 1 13 4 24 10 East Africa 12/61 9 2 14 5 East Africa 17/61 12 4 14 5 West Africa Congo 5 10 5 23 10 WestAfrica Congo 6 4 2 17 12 Iran 2602 7 1 20 6 Botswana B 21 - - 15 3 Botswana B89 - - 10 3 ORTHOPOXVIRUSES: ENZYME CHARACTERIZATION Table 2. Percentage residual activity, at different thymidine triphosphate (TTP) concentrations, of the thymidine kinase enzymes induced by "whitepox" viruses and some other isolates HeLa cells BHK TK- cells Virus Strain 4 Amol/litre TTP 8 umol/litre TTP 4 rmol/litre TTP 8 Amol/litre TTP "Whitepox" viruses "whitepox" 64/7275 10 6 18 9 "whitepox" 64/7255 10 2 19 10 "whitepox" CHIMP 9 16 5 16 5 "whitepox" MK-7 14 3 23 6 "whitepox" RZ 38 11 2 22 5 Other isolates monkeypox, white variant MPD-1W 85 62 90 86 gerbilpox 90 60 95 63 "Lenny" 82 56 98 73 vaccinia-like MK-10 80 60 85 76 values to be expected from the sum of the activities of their constituents. Maximum differences between the viruses were observed in the range 4-8 umol/litre of TTP. Each enzyme preparation was therefore assayed at concentrations of 0, 4, and 8 gmol/litre of TTP. Values were obtained from replicate tubes and the inhibition at each concentration of TTP was then expressed as a percentage of the activity without added TTP. The behaviour of standard poxviruses in tests of this sort are shown in Table 1. The thymidine kinases induced by strains of vaccinia and monkeypox were relatively resistant to inhibition by TTP, giving values of 52% or more at 4 itmol/litre and of 31% or more at 8 Amol/litre. By contrast all of eleven strains of variola virus were very sensitive to TTP and gave values of 24/o or less at 4 Amol/litre and of 12% or less at 8 Amol/litre. The variola strains were taken from different parts of the world and covered a range of pathogenicity from variola major to alastrim. Samples of other animal poxviruses, rabbitpox, cow- pox, ectromelia, camelpox, buffalopox, and ele- phantpox, all gave results in the same range as those of vaccinia and monkeypox. This test appeared to differentiate variola viruses from other orthopoxviruses and it was next applied to the "whitepox" viruses, which resemble variola in other laboratory characters but which were reported to have been isolated from animal sources (1). The results are shown in Table 2, which also includes the Table 3. Percentage residual activity, at different thymidine triphosphate (TTP) concentrations, of the thymidine kinase enzymes induced by hybrid poxviruses BHK TK- cells Virus Designation 4 zmol/litre TTP 8 Amol/litre TTP Alastrim/rabbitpox hybrids AR1 76 81 AR2 88 74 AR3 89 70 AR4 92 70 AR6 93 86 AR7 107 90 Variola/cowpox hybrids VC2 89 68 VC5 89 63 VC6 87 66 VC7 21 9 VC8 92 65 VC1o 88 64 VC12 78 70 VC13 92 64 VC14 88 72 VC16 94 65 379 380 H. S. BEDSON results from a white pock variant of monkeypox Den- mark, a virus from an African gerbil (7), and two vac- cinia-like viruses from Africa: "Lenny" (5) and MKIO (9). It can be seen that all five of the "white- pox" viruses gave results in the same range as variola and that all others were in the vaccinia range. Table 3 shows the results obtained from 16 hybrid poxviruses. Six of these (AR series) were hybrids between alastrim and rabbitpox and 10 (VC series) were hybrids between variola major and cowpox (3, 4). Among these 16 viruses of which variola was one parent, all except one gave results within the vaccinia range. The variola/cowpox hybrid VC7 had a thymidine kinase that was as sensitive as that of its variola parent. This hybrid produced greyish, ulcerated pocks on the CAM which were not inhibited by incubation at 40 °C and which showed the large type A inclusions characteristic of cowpox; it was pathogenic for the rabbit and 100 times more lethal for chick embryos than was variola major (4). COMMENT AND CONCLUSIONS These results require little comment. They show clearly that variola and "whitepox" viruses are similar to each other, but quite different from all the other orthopoxviruses so far tested. The difference appears to be a virus property and not dependent upon the host cell. There is no overlap between the groups and there do not appear to be any intermediate viruses. Results of the tests on hybrid viruses show that this new marker segregates independently of other biological characters used in the identification of orthopoxviruses; it is thus a valuable additional marker for variola and variola-related viruses. It is possible that there are other minor differences among the viruses of each of the major groups. Re- peated tests, repeated specimens, and the replicate values of individual tests themselves (data not pre- sented) suggest that the assay used is insufficiently precise to make any meaningful analysis possible at present. There is in any case very little enzyme activity left to assay in those cases in which TTP inhibition is marked. The test is relatively easy to apply provided that the virus in question can be grown to high titre and is efficient in inducing thymidine kinase. Fortu- nately variola and monkeypox viruses behave well in both respects. Difficulties of one sort or another were found for some of the miscellaneous viruses shown in Table 2. The finding that all five "whitepox" viruses examined behave like smallpox viruses in this new test adds weight to the existing laboratory evidence which suggests that they must be regarded as genuine variola viruses." b Editor's footnote. All the "whitepox" viruses studied in this investigation came from areas where smallpox was not prevalent at the time. Although the five "whitepox" viruses tested were originally considered to have been isolated from animals, evidence has recently been published that two of them (64/7275 and 64/7255) almost certainly originated as the result of laboratory cross-contamination(DUMBELL, K. R. & KAPSENBERG, J. G. Bulletin of the World Health Organization, 60: 381 - 387 (1982)). RESUME ETUDES ENZYMATIQUES POUR LA CARACTERISATION DE CERTAINS ISOLEMENTS D'ORTHOPOXVIRUS En six occasions, des virus inhabituels ont 6te isoles a partir de tissus animaux et ils ont ete dtnomm6s virus <<whitepox> en raison de I'apparence des pustules qu'ils determinent sur la membrane chorio-allantoidienne. A tous egards sauf I'apparence, les virus <<whitepox>> sont iden- tiques aux virus de la variole. On sait que l'inoculation de cultures cellulaires avec des poxvirus d6termine une activitt accrue d'un certain nombre d'enzymes intervenant dans le m6tabolisme des acides nucl6iques. Dans la pr6sente 6tude, on a mesur6 l'augmentation de l'activite de la thymidine- kinase et son inhibition par l'acide thymidine-triphospho- rique dans differentes lign6es cellulaires A la suite de l'inocu- lation de la culture avec diff6rents orthopoxvirus: variole, <whitepox> et autres. Une diff6rence nette a 6t6 constat6e entre les virus de la variole et les autres orthopoxvirus. Cinq virus (<whitepox)> se comportaient comme la variole, ce qui est une nouvelle raison de consid6rer les virus <whitepox> comme 6tant de veritables virus varioliques. REFERENCES 1. ARITA, I. & HENDERSON, D. A. Bulletin of the World Health Organization, 53; 347 - 353 (1976). 2. BEDSON, H. S. International virology 3. Abstracts. Madrid, Third International Congress for Virology, p. 241. 3. BEDSON, H. S. & DUMBELL, K. R. Journal of hygiene, 62: 141 - 146 (1964). 4. BEDSON, H. S. & DUMBELL, K. R. Journal of hygiene, 62: 147- 158 (1964). 5. BOURKE, A. T. C. & DUMBELL, K. R. Bulletin of the World Health Organization, 46: 621 - 623 (1972). 6. HARPER, L. ET AL. Virology, 93: 435 - 444 (1979). 7. LOURIE, B. ET AL. Journal of infectious diseases, 132: 677-681 (1975). 8. Moss, B., In: D. P. Nayak, ed., Molecular biology of animal viruses, Marcel Dekker, 1978, pp. 849- 890. 9. SELUHINA, E. M. ET AL. British journal of veterinary medicine, 131: 746 - 748 (1975).