Lytic and Turbid Plaque-Type Mutants of Lymphocytic Choriomeningitis Virus as a

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1 INFECTON AND IMMUNITY, Sept. 1971, p Copyright ( 1971 American Society for Microbiology Vol. 4, No. 3 Prinited ill U.S.A. Lytic and Turbid Plaque-Type Mutants of Lymphocytic Choriomeningitis Virus as a Cause of Neurological Disease or Persistent Infection JOHN HOTCHIN, WILLIAM KINCH, AND LOIS BENSON Division of Laboratories and Research, New York State Departmenlt of Health, Albany, New York Received for publication 31 March 1971 Mouse-passaged lymphocytic choriomeningitis (LCM) virus was found to contain a mixture of two different plaque-type mutants when plated on BHK-21/13S cells in agarose suspension. One mutant gave rise to clear plaques, with death of the cells, whereas the other produced turbid plaques which were sometimes very difficult to see. The clear-plaque variant caused a pronounced cytopathic effect on BHK- 21 cells, but the turbid variant caused none; it also interfered with the cytopathic effect due to the clear variant. Brain-passed LCM virus was found to consist mainly of the clear-plaque-type, whereas liver-passed virus was mainly turbid-plaque-type. The clear type induced convulsions and early death after intracerebral inoculation of adult mice; the turbid variant caused no convulsions and late deaths. In newborn mice, the clear-plaque-type was uniformly fatal, whereas the turbid variant caused no deaths but instead induced persistent tolerant infection. The effects of lymphocytic choriomeningitis (LCM) virus upon mice have long suggested (6, 8, 12, 14, 15) that two different variants were involved. These variants were referred to as viscerotropic or neurotropic according to their passage history, and the viscerotropic type was found to interfere with the effects of the neurotropic type (10, 16). The two types were recently reexamined (S. Suzuki and J. Hotchin, J. Infec. Dis., in press); the neurotropic variant was found to cause early death with convulsions, and the viscerotropic variant caused later deaths without convulsions. Tissue culture of these strains in BHK-21 cells revealed the presence of lytic LCM plaques (J. Hotchin and L. Benson, in preparation) by using a liquid medium (no solid overlay). Immunofluorescent tests of the monolayers showed that essentially all of the cells were infected with virus at the time of the plaque appearance, and it was concluded that two types of virion were present in the inoculum; these were named "lytic" and "interfering," respectively. The following study describes the isolation and pathogenic behavior of these two plaque-type mutants of LCM virus by using solid overlay plaque techniques. agarose overlays. All types produced plaques, but best results were obtained with BHK-21/13S cells in suspension by the method of Sedwick and Wiktor (11). Cells were grown in monolayers in Blake bottles with Dulbecco's modified Eagle's medium (5, 13) supplemented with 10% fetal bovine serum and 20% Tryptose phosphate broth. Mice. "Albany" strain mice weighing 10 to 12 g were used for the intracerebral (ic) titrations. For footpad (fp) inoculation, 12- to 14-g mice were used. "Newborn" mice included only animals born within 24 hr prior to inoculation. Response after fp inoculation was estimated on a scale of 0 to 4+ enlargement. Virus. Three strains of LCM virus were used. These included two different substrains of the WE (UBC) strain: one passed eight times in mouse brain (MB8), the other passed six times in mouse brain and then eleven times in mouse liver (MB6L18). The Traub strain (obtained from Mogens Volkert) had been passed at least twelve times in mouse spleen. The virus strains were prepared as 20% suspensions. Titrations were prepared in GTH [0.05% gelatin in tris(hydroxymethyl)aminomethane (Tris)-buffered Hanks solution at ph 7.2]; 0.03 ml was used as inoculum in mice, and 0.1 ml was used for plaque assay. Plates were incubated in a CO2 incubator. RESULTS MATERIALS AND METHODS Plaque characteristics of mouse-passed LCM Several cell types, including BHK-21/13S, L, and virus. When the WE MB8 and MB6L1j strains of Vero cells, were used to obtain LCM plaques under LCM were titrated by BHK-21/13S plaque assay, 281

2 282 HOTCHIN, KINCH, AND BENSON INFEC. IMMUN. TABLE 1. Proportionz of differentt plaque types in MB8 anid MB6L,I strainis of LCM virus Clear plaques Turbid plaques plaques Virus Plaques strain counted Large 6 Tat Total~ ~ ~ ~~~~~oa Total Large ~ otal(%)1 Total Target(% MB8'... 2,386 2, (100') (96.5) (28.1) (3.46) (0.8) MB6L1,... 1, , (100) (10.0) (7.1) (89.9) (5.9) a Values in parentheses indicate per cent. Downloaded from FIG. 1. Clear plaques from a 10-6 dilution2 of tube BHK-21 cell tissute culture flliid harvested 4 days after inoculationt with a portiont ofone clear plaque from the 10-4 dilution ofa plaquie titrationt. each of the two substrains produced at least two different types of plaques. These were present in markedly different proportions in the two virus strains. One plaque type was clear with very few neutral red-containing cells in it; the other type was turbid with only slight difference in contrast from the normal cells and no visible microscopic difference. Both types were neutralized by LCMspecific convalescent human serum but not by normal human sera. Plaque titrations indicated that the MB8 virus was 95% clear plaque type and the MB6L1, was 80% turbid plaque type. To measure the proportions of the two types more accurately, appropriate dilutions of each virus were made to give about 100 plaques on each of 16 5-cm petri dishes. The results are shown in Table 1. Both strains produced clear and turbid plaques. The clear plaques varied in size, and the proportions of large ones were counted. The turbid plaques showed very little contrast between them and adjacent uninfected cells, and a few had a ringed appearance referred to as target plaques; these sometimes had red and sometimes clear centers. It is evident from Table 1 that MB8 consisted primarily (96.5 %) of clear plaques and that MBrL11 consisted mainly (89.9 %) of turbid on April 28, 2018 by guest

3 VOL. 4, 1971 MUTANTS OF LCM VIRUS 283 FIG. 2. Turbid plaques from a plaque pickedfrom the 10-4 plate of MB6L1I, treated tt,e same way as in Fig. 1. Although most of the progeny gave turbid plaques, there were a few lytic plaques preseint. The difference in contrast between adjacent lytic and turbidplaques can be seen at thejunction ofthe arrows. plaques. When larger doses of inoculum virus were used, the clear plaques in the MB6L1j virus were much smauer, becoming pinpoint in size when the dose was increased 100-fold. With a 1,000-fold increase, the clear plaques disappeared entirely and the confluent turbid plaques could not be distinguished from a control monolayer. Different plaques were seen to vary in contrast, i.e., different mutants varied in the degree of turbidity. Aftempts to obtain plaque-purified LCM strains. When individual lytic or turbid plaques were picked, relatively pure sublines were obtained (Fig. 1 and 2). The clear plaque (designated BSC) reproduced a pure population of clear plaques, but the turbid plaque (designated LT) produced a few clear plaques. This effect was well shown (Fig. 3) in another plaque-purified turbid variant designated LM2C2, which at a dilution of 10-i showed about 14 clear plaques in a field of almost confluent turbid plaques. When MB8 or MB6L1j was passed several times in BHK-21 cell tissue cultures, the resulting strains showed no detectable difference on plaque assay. Both produced the same mixture of plaque types shown in Fig. 4. In a different experiment, clear and turbid plaques were passed serially in BHK-21 cell cultures by the following procedure. A plaque was picked by aspirating a portion into a Pasteur pipette and inoculating this into a tube culture of BHK-21 cells. After 2 days of incubation, fluid from the culture was titrated by plaque assay and the same type of plaque was picked again. The procedure was repeated through four plaque passages, and 0.1 ml of fluid from the last passage was used to inoculate a 250-ml tissue culture bottle containing a monolayer of BHK-21 cells. The fluid from this culture was harvested 4 days later as a pool of the final plaque type and quickfrozen in small portions for storage. It was noticed that the clear plaque-type caused a marked cytopathic effect, whereas the turbid type caused no detectable cytopathic effect. An analysis of the plaque types present in the resulting pools of clear and turbid fourfold plaque-purified pools (designated SC2F3 and LM4, respectively) gave the results shown in the first four columns of Table

4 FIG. 3. Progeny ofa turbidlcmplaque variant LM2C2 showing abouit 14 ckear plaques on a background ofturbid plaques. Lower dilutionisproduced co,i'luient turbidplaques anid minutte or absenti clear plaques which were suppressed by interferenice. Downloaded from on April 28, 2018 by guest Kr-4 NP: FIG. 4. Appearance of LCM plaqiies from BHK-21 cell-passed MB8 or MB6L, 1 virits. Clear, ttirbid, and a few haloedplaques are presetit. 284

5 VOL. 4, 1971 MUTANTS OF LCM VIRUS 285 TABLE 2. Properties of tihe progeny of turbid and clear LCM plaques in BHK-21 cells No. of progeny plaque-type Mouse titrationa Parent Plaque CPE Adult Newborn infaust plaque-type titer/ml weight Peet(g) atb Clear Turbid IC fp IC Per cent day 13b talitya Turbid X < C (LM4) (7.93%) (92%) Clear X d (SC2F3) (99.96%) (0.04%) a Overall mortality for dilutions through Abbreviations: CPE, cytopathic effect; IC, intracerebral; fp, footpad. I Control uninoculated mice weighed 6.3 g at this time. c Average of undiluted through d Average of dilutions 10-i, Both plaque types were neutralized by a 1:16 dilution of human LCM convalescent serum, but not by normal human serum. Behavior of clear and turbid plaque-type LCM virus in mice. The pools of clear and turbid LCM plaque variants were tested in mice by inoculating 0.03 ml of serial 10-fold dilutions by ic, intravenous (iv), and fp routes into adult mice. Five mice were used for each dilution. In addition, each virus strain was titrated in newborn mice with three litters for each dilution. The results are summarized in Table 2. One adult mouse survived ic inoculation with undiluted turbid-plaque virus; it was found to be persistently infected. Although adult mouse mortality caused by the two virus types was similar, the average day of death for the clear plaque-type was 6.9 days and deaths were accompanied by convulsions, whereas the average day of death of the turbid plaque type was 10.9 days and there were no convulsions. The clear type caused earlier death (6.1 days) with the highest doses and later deaths (8.0 days) in the lower doses; the time before death was inversely related to virus dose. With the turbid plaque variant, the opposite effect occurred; the average day of death was 13.5 with the highest virus dose and 9.5 with the lowest dose. Time before death was directly related to virus dose, and there was some evidence of high-dose immune paralysis (some survivors after high virus doses). Inoculation of newborn mice showed marked differences between the two plaque types since the clear variant caused 100% deaths, whereas the turbid variant caused only one death (at 10 ) in 127 mice inoculated with virus. Survivors of neonatal inoculation with the turbid variant were all persistently infected. Comparisons of the median Avg TABLE 3. Summary of differences between clear and turbid LCM plaque types Property LCM plaque type Clear Turbid Cytopathogenicity + - Comet plaques + - Neurological disease + - Early death of adult mice + - Newborn mouse mortality + - Tolerance induction - + Autointerference - + Late death of adult mice - + Persistent infection of newborn - + mice mouse infective dose (MID5o) with plaque titer gave figures for the efficiency of plating [plaque titer/(mid5o)] of 0.25 for the clear plaque type and 0.40 for the turbid plaque type. Appearance of LCM plaque variants in other systems. The clear-plaque LCM variant was found to cause the comet plaques previously reported (J. Hotchin and L. Benson, in preparation) to occur in BHK-21 cells, and it also produced the same effect in Vero cells in the absence of overlay. Round clear plaques were produced by this variant in Vero- or L-cell monolayers with overlay. The turbid plaques were difficult or impossible to see on Vero- or L-cell monolayers. Clear plaques developed target-like patterns of rings of heavy and light neutral red staining by the 6th day and thereafter. The fourth or fifth day after inoculation was found to be the best for staining with neutral red; plaques could be differentiated 2 to 4 hr later.

6 286 HOTCHIN, KINCH, AND BENSON INFEC. IMMUN. DISCUSSION Although several plaque assays for LCM virus have been reported by using chick embryo tissue (1, 3, 4, 17), L cells (2), human cell lines (17), and BHK-21/13S (11), none of these has differentiated significant genetic variants. Pulkkinen and Pfau (9) reported large- and small-plaque types but these failed to breed true, and the authors concluded that plaque size heterogeneity was a genetic trait of LCM virus. The results reported here show that genetically distinct LCM plaquetype mutants can be detected by standard procedures and that the plaque-type variability is associated with highly significant pathogenic differences (Table 3). The clear plaque type causes lysis of cells in vitro and produces convulsive neurological disease and early death after inoculation of adult mice. It is also lethal for newborn mice. This variant appears to be identical to the "aggressive" LCM variant previously postulated (7). The turbid variant is responsible for tolerance induction, persistent infection, and autointerference with the clear plaque variant. The clear and turbid variants are the same as the lytic (L) and interfering (I) variants previously mentioned (J. Hotchin, and L. Benson, in preparation). The fact that both of these variants are present in all of the UBC-LCM substrains and also in the Traub (Volkert) strain suggests that their presence is a general property of al LCM strains. LITERATURE CITED 1. Benson, L Tissue culture experiments and plaque assay with lymphocytic choriomeningitis virus, p N.Y. State Dept. Health Annu. Rep. Div. Lab. Res. 2. Benson, L The use of strain L mouse cells in an improved plaque assay of lymphocytic choriomeningitis virus, p N.Y. State Dept. Health Annu. Rep. Div. Lab. Res. 3. Benson, L. M., and J. E. Hotchin Cytopathogenicity and plaque formation with lymphocytic choriomeningitis virus. Proc. Soc. Exp. Biol. Med. 103: Chastel, C Technique des plages de l'inhibition des plages en cultures cellulaires pour l'identification du virus de la choriomedningite lymphocytaire. Ann. Inst. Pasteur 109: Dulbecco, R., and G. Freemian Plaque production by the polyoma virus. Virology 8: Hotchin, J., and L. Benson The pathogenesis of lymphocytic choriomeningitis in mice: the effects of different inoculation routes and the footpad response. J. Immunol. 91: Hotchin, J., L. M. Benson, and J. Seamer Factors aflecting the induction of persistent tolerant infection of newborn mice with lymphocytic choriomeningitis. Virology 18: Hotchin, J., and H. Weigand Studies of lymphocytic choriomeningitis in mice. I. The relationship between age at inoculation and outcome of infection. J. Immunol. 86: Pulkkinen, A. J., and C. J.Pfau Plaquesize heterogeneity: a genetic trait of lymphocytic choriomeningitis virus. Appl. Microbiol. 20: Rowe, W. P Studies on pathogenesis and immunity in lymphocytic choriomeningitis infection of the mouse. Res. Rep. Naval Med. Res. Inst. (Bethesda, Md.) 12: Sedwick, W. D., and T. J. Wiktor Reproducible plaquing system for rabies, lymphocytic choriomeningitis, and other ribonucleic acid viruses in BHK-21/13S agarose suspensions. J. Virol. 1: Shwartzman, G Alterations in pathogenesis of experimental lymphocytic choriomeningitis caused by prepassage of the virus through heterologous host. J. Immunol. 54: Smith, J. D., G. Freeman, M. Vogt, and R. Dulbecco The nucleic acid of polyoma virus. Virology 12: Traub, E Factors influencing the persistence of choriomeningitis virus in the blood of mice after clinical recovery. J. Exp. Med. 68: Traub, E Ulber die immunologische Toleranz bie der lymphocytaren Choriomeningitis der Mause. Zentralbl. Bakteriol. Parasitenk. Infektionskr. Hyg. Abt. Orig. 177: Traub, E Observations on immunological tolerance and "immunity" in mice infected congenitally with the virus of lymphocytic choriomeningitis (LCM). Arch. Gesamte. Virusforsch. 10: Wainwright, S., and C. A. Mims Plaque assay for lymphocytic choriomeningitis virus based on hemadsorption interference. J. Virol. 1: