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J. gen. Virol. (1988), 69, 2199-2207. Printedin Great Britain 2199 Key words: SLE virus~mutants/persistentinfections Phenotypes of St Louis Encephalitis Virus Mutants Produced in Persistently Infected Mosquito Cell Cultures By V A L E R I E B. R A N D O L P H * t AND J A M E S L. H A R D Y Department of Biomedical and Environmental Health Sciences, School of Public Health, University of CaliJornia, Berkeley, California 94720, U.S.A. (Accepted 26 May 1988) SUMMARY Viral mutants that appeared during long-term persistent infections of mosquito cell cultures (Aedes albopictus, A. dorsalis and Culex tarsalis) with St Louis encephalitis virus were characterized. Evidence was obtained for the presence of temperaturesensitive mutants in the A. dorsalis and C. tarsalis persistently infected cultures, and small plaque mutants were predominant in all cultures except one of two cell cultures of C. tarsalis. Virus from persistently infected A. albopictus ceU cultures was growthrestricted in Vero and C. tarsalis cells. One of two persistently infected A. dorsalis cell cultures also produced viral mutants that were growth-restricted in C. tarsalis cells. Further, Western blots of persistently infected A. albopictus cell extracts showed an overproduction of capsid (C) and envelope (E) structural proteins and reduced production of an Mr 27K protein (p27) which was immunologically related to the E protein. In contrast, the production of E and C proteins in persistently infected C. tarsalis cultures was consistent with the amount of infectious virus present, whereas p27 was relatively overproduced. These observations suggest that the host cell has an important influence on both the types and relative quantities of viral mutants that accumulate during long-term persistent infections. INTRODUCTION Arboviruses, including flaviviruses (Igarashi, 1979; Ng & Westaway, 1980; Randolph & Hardy, 1988), alphaviruses (Igarashi et al., 1977; Maeda et al., 1979; Stollar, 1980; Eaton, 1981 ; Stalder et al., 1983) and bunyaviruses (Newton et al., 1981; Elliott & Wilkie, 1986), readily establish persistent infections in mosquito cell cultures. Viral mutants invariably appear in mosquito cell cultures persistently infected with these viruses, but usually only after the persistent infections have been established. It is therefore unlikely that these mutants are important in the establishment of the state of persistence. Arbovirus mutants arising in the course of persistent infection have been most extensively characterized in infected Aedes albopictus cell cultures. Small plaque (sp) and temperature-sensitive (ts) mutants are generally found within 3 weeks post-infection (p.i.) and eventually become the predominant viral phenotypes. In addition, defective interfering (DI) particles and truncated viral RNAs have been observed in A. albopictus cell cultures persistently infected with alphaviruses (Igarashi et al., 1977; Tooker & Kennedy, 1981 ; Stalder et al., 1983). DI particles have not been reported in mosquito cell cultures infected with flaviviruses, although defective 'non-plaquing' viral particles were reported in vertebrate cell cultures persistently infected with West Nile (WN) and Japanese encephalitis (JE) viruses (Brinton, 1982; Schmaljohn & Blair, 1977). Persistent infections of three mosquito cell lines (A. albopictus, A. dorsalis and Culex tarsalis) with St Louis encephalitis (SLE) virus, a flavivirus, have been described (Randolph & Hardy, 1988). Observations suggested that the characteristics of the persistent infections and the viral populations that predominated were strongly influenced by the host cell. To compare further the t Present address: Viral Vaccine Research and DevelopmentDepartment, LederleLaboratories,Pearl River, New York 10965, U.S.A. 0000-8177 © 1988 SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 08:01:56 2200 V. B. RANDOLPH AND J. L. HARDY differences in viral phenotypes found in these persistently infected mosquito cell cultures, virus was characterized with respect to plaque size, temperature sensitivity, host range and relative amounts of viral protein produced. Significant differences were noted in the viral phenotypes, further emphasizing the important role of the host cell in influencing the evolution of viral mutants in long-term infections. METHODS Cell cultures and viruses. The maintenance of cell lines and initiation of persistently infected cultures have been described previously (Randolph & Hardy, 1988). Uncloned SLE (LAV5-156) virus, originally isolated from fieldcollected mosquitoes, was used to initiate persistent infections in duplicate cultures of A. albopictus, A. dorsalis and C. tarsalis cells, designated Aal, Aa2, Adl, Ad2, Ctl and Ct2 respectively. Viral titres were determined by plaque assay in Vero cells or by immunoperoxidase focus assay (IPFA) in Vero and mosquito cells (Randolph & Hardy, 1988). Immune sera. SLE (LAV5-156) immune mouse ascites fluid (IMAF) was developed in adult NAMRU mice (Naval Biosciences Laboratory, Oakland, Ca., U.S.A.) as described elsewhere (Sartorelli et al., 1966). A hybridoma (16-A-7) producing monoclonal antibody (MAb) to the envelope (E) protein of SLE virus was obtained following fusion of P3-X63-Ag8.653 myeloma cells to spleen cells obtained from BALB/c mice immunized with SLE (BFS-1750) virus. The specificity of the antibody for the E protein was shown by Western blot analysis and radioimmunoprecipitation of SLE viral proteins (Grove, 1985). PAGE and Western blotting. Virus was precipitated from the culture medium with polyethylene glycol (McSharry & Benzinger, 1970). The precipitate was resuspended in TNE buffer (10 mM-Tris-HC1, 1 mM-EDTA, 150 mM-NaC1, pH 8.2) and centrifuged for 3 h at 90000g through a 25~ sucrose cushion. The resulting pellets were resuspended in 0-2 ml of sample buffer (0.0625 M-Tris-HCI pH 6.8, 10~ glycerol, 3 ~ SDS, 5 ~ 2mercaptoethanol). To prepare samples of cell extracts, cells grown as monolayers in T-25 flasks were rinsed carefully with cold phosphate-buffered saline pH 7-5, removed by pipetting and centrifuged at 800 g for 10 min at 4 °C. Cell pellets were resuspended in 0.5 ml sample buffer, then vortexed and passed several times through a 26gauge needle to reduce viscosity. Discontinuous PAGE was carried out as described by Laemmli (1970) using 12.5~ acrylamide gels. Samples were incubated 3 rain at 100 °C before electrophoresis. Proteins were electrophoretically transferred (Bio-Rad Trans-Blot Cell) to a nitrocellulose membrane (0-45 p.m pore size; Bio-Rad) for 2 h at 50 V in transfer buffer (20 mM-Tris-HC1, 150 m~-glycine, pH 7.5, 0.08 % SDS, 20 % methanol). Blots were incubated overnight at 4 °C in blocking buffer (PBS pH 7-5, 0.25 ~ gelatin, 20 ~ goat serum, 3 ~ bovine serum albumin, 0.05 ~ Triton X- 100) and then incubated for 2 h with either a 1:100 dilution of SLE (LAV5-156) IMAF or MAb 16-A-7 in hybridoma culture supernatant fluid. This was followed by sequential incubation with biotinylated horse anti-mouse IgG (BHAM) (1:250; Vector Laboratories, Burlingame, Ca., U.S.A.) (30 rain), avidin-biotin complex (ABC) reagent (Vector Laboratories) (60 min), and 0.02% 4-chloro-I-naphthol, 0.01% H20 2 in PBS (20 rain). Blots were washed for 30 to 60 rain with blocking buffer after the initial incubation with mouse antibody and with PBS-0.05 % Triton X-100 or PBS after each subsequent incubation step. In later assays, BHAM and ABC reagents were replaced with afffinity-purified biotinylated goat anti-mouse IgG (1:250 dilution; Cooper Biomedical, Malvern, Pa., U.S.A.) and peroxidase-conjugated avidin (1:400 dilution; Cooper Biomedical). RESULTS S m a l l plaque p h e n o t y p e The plaques produced in Vero cell cultures by virus obtained from the various persistently infected mosquito cell cultures 41 weeks p.i. varied considerably in size. Also, the smaller plaques took from 9 to 14 days to appear whereas wild-type (wt) plaques appeared in 7 days. Plaques produced by viruses from both of the persistently infected A. albopietus cell cultures were uniformly small. In contrast, plaque phenotypes of virus obtained from the two A. dorsalis cultures differed from each other in that Adl virus produced mainly indistinct, pinpoint-size plaques and some distinct small plaques, whereas Ad2 viral plaques were distinct and varied in size. Notably, the plaque phenotype of virus from Ctl cultures (last assayed 110 weeks p.i.) was indistinguishable from wt virus in both plaque morphology and time of appearance, whereas Ct2 cultures produced distinct plaques that were somewhat variable in size. Temperature-sensitive phenotype To determine whether virus from persistently infected mosquito cell cultures was temperature-sensitive, samples obtained from cultures 81 weeks p.i. were assayed by IPFA in Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 08:01:56 SLE virus mutants 2201 Table 1. Temperature sensitivity of SLE virus obtained from persistently infected mosquito cell lines Viral titre (lOglo f.f.u./ml)* Virust Aa (wt) Aal Aa2 Ad (wt) Ad 1 Ad2 Ct (wt) Ctl Ct2 r 33 °C 7.7 2-8 < 2.0 7-1 5-4 5-1 5.4 5.7 5.7 39-5 oc' 7.6 < 2.0 < 2.0 6.8 < 3.0 4.0 5.4 5.0 4.3 Relative plating efficiency:~ 1.00 <0.19 1.00 <0.01 0.16 1.00 0.20 0.04 * Viral titres were determined by IPFA on Veto cells. t Virus was obtained from acutely infected mosquito cell cultures 5 days p.i. (wt) or from persistently infected cell cultures 81 weeks p.i. (Aal, Aa2, Adl, Ad2, Ctl, 02). $ Relative plating efficiency = [f.f.u. (persistently infected) (high temp.) x f.f.u. (wt) (low temp.)]/[f.f.u. (persistently infected) (low temp.) × f.f.u. (wt) (high temp.)]. Vero cells at 33 °C and 39-5 °C (Table 1). These titres were compared to those obtained with wt virus assayed at these same temperatures. Relative plating efficiences of virus from A d l , Ad2, Ctl and Ct2 cultures ranged from 0.20 to <0.01, thus indicating the probable presence of ts variants in these viral populations. There was some variation between virus from the duplicate cultures in that relative plating efficiencies were 20-fold lower for Adl virus than for Ad2 virus, and fivefold lower for Ct2 than for Ctl virus. When assayed in Vero cells titres of virus from Aal and Aa2 cultures were too low to determine temperature-sensitivity. None of these viral populations was significantly temperature-sensitive when assayed in the homologous mosquito cell lines at 28 °C and 36 °C (data not shown). Mosquito cells could not be maintained at temperatures above 36 °C and therefore temperature-sensitivity of virus at 39-5 °C could not be evaluated in them. Host restriction phenotype It was shown (Randolph & Hardy, 1988) that titres obtained with virus from A a l and Aa2 cultures after 81 weeks of persistent infection were 103- to 104-fold higher when assayed in A. albopictus cells (at 28 °C) than when assayed in Vero cells (at 36 °C). This contrasts with the wt virus whose titres were very similar when assayed in these two cell lines. This difference in titres was not due simply to temperature-sensitivity because virus from these cultures replicated in A. albopietus cells equally well at 28 °C and 36 °C (data not shown). To evaluate further the presence of host-range (hr) mutants in persistently infected mosquito cell cultures, and to determine which host cells were permissive for viral replication, the infectious titres of virus obtained 3, 41 and 81 weeks p.i. from each persistently infected culture were determined by I P F A in four different assay cell lines (Table 2). Virus was assayed in Vero cells incubated at 36 °C and in A. albopictus, A. dorsalis and C. tarsalis cells incubated at 28 °C. Titres obtained with virus from the six mosquito cell cultures 3 weeks p.i. varied approximately 10-fold among the four assay systems, with titres determined in A. dorsalis cultures being highest and titres determined in C. tarsalis cells being lowest. However, when measured 81 weeks p.i., titres of virus from Aal and Aa2 cultures differed significantly when measured in the four cell lines. The highest titres were observed when measured in A. albopictus and A. dorsalis cells while titres in Vero cells were 50- to 100-fold lower and were no longer detectable when assayed in C. tarsalis cells. For virus samples obtained from the persistently infected Adl and Ad2 cultures 81 weeks p.i. viral titres were lower when measured in A. albopictus and C. tarsalis cells than when measured in A. dorsalis and Vero cells. Virus taken from the persistently infected Ctl and Ct2 cultures 81 weeks p.i. produced similar titres in all four cell assay systems. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 08:01:56 2202 V . B . RANDOLPH AND J . L . HARDY Table 2. Comparison of SLE viral titres from persistently infected mosquito cell cultures assayed in four different cell lines Viral titre (logl0 p.f.u./ml)* A f Assay cell line Virus Time p.i. source (weeks) Aal 3 41 81 Aa2 3 41 81 Adl 3 41 81 Ad2 3 41 81 Ctl 3 41 81 Ct2 3 41 81 Vero A. albopictus A. dorsalis 8-0 4.8 2.6 8.0 5.1 2.1 7-6 5.7 5.2 7.2 5.5 4.9 6.0 5.6 5.5 6.2 5.9 5.5 7.7 4.8 4.7 7.7 5.8 3-4 7-0 4.4 4.3 6.5 4-4 3.8 5-6 5.0 4.8 5.5 5.4 5.3 8.3 5.5 4.1 8.2 5-9 3.4 7-4 5.5 5.2 7.1 5.4 5.1 6-6 6.0 5.8 6.5 6.4 6.2 C. tarsalis 7.0 3.8 <2.0 7.3 4-4 <2.0 6.6 4.1 3.5 6.2 4-3 3-7 5.7 5.0 5.2 5.7 5.5 5.5 * Viral titres were assayed by IPFA as described in Methods. O f particular interest was the observation that many of the foci of infection produced in A. albopictus cells by viruses obtained from Aal and Aa2 cultures 81 weeks p.i. appeared to be significantly larger than foci produced in these cells by viruses obtained after only 3 weeks (Fig. 1). This further suggests that viral phenotypes that had enhanced growth potential in the homologous cell line were selected for during long-term persistent infections in A. albopictus cells. In contrast, the size of the foci in Vero cells decreased with time after infection, and was consistent with the appearance of sp mutants in plaque assays. A n increase in the size of foci of infection in homologous cells was not observed for viruses from A d l , Ad2, Ctl and Ct2 cultures. Further evidence of host range restriction was obtained by experiments in which virus obtained from persistently infected cultures at 69 weeks p.i. was passaged once in uninfected A. albopictus, A. dorsalis and C. tarsalis cell cultures. The titres of the virus produced as determined by plaque assay on Vero cells were in each case compared to titres of virus before passage (Table 3). Consistent with the results shown in Table 2, virus from the persistently infected A. albopictus cell cultures was unable to replicate in C. tarsalis cells; thus this virus was clearly host-restricted. Replication of A d l but not the Ad2 virus was also restricted in C. tarsalis cultures. N o restriction was observed with the Ctl or Ct2 virus. Wt SLE virus replicated well in each of the cell types and in fact produced higher titres than any of the viruses from the persistently infected cultures. Viral proteins To examine whether viral protein synthesis was altered quantitatively or qualitatively in the persistently infected cultures, viral proteins were compared by Western blotting. N o changes were seen in the migration patterns of the E proteins of virions obtained from the culture fluids of the persistently infected cultures (data not shown). Capsid (C) and membrane proteins from virions in samples obtained from persistently infected cultures were not visible due to limits of sensitivity of the assay, and therefore could not be compared. The largest amounts of viral proteins (E, C, p98, p71 and p27) were found in cell extracts of the acutely infected mosquito cells (Fig. 2a) which is consistent with the fact that these cells produced higher titres of infectious virus than the persistently infected cultures. However, the Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 08:01:56 S L E virus mutants 2203 Fig. 1. Fociof infection produced in Vero and A. albopictus assay cell lines by SLE virus obtained from a persistently infected A. albopictus cell culture (Aal). Foci were visualized by immunoperoxidase staining with virus-specificantibody. Virus was obtained 3 weeks (a) or 81 weeks (b) p.i. and assayed in A. albopictus cells. Virus was obtained 3 weeks (c) or 81 weeks (d) p.i. and assayed in Vero cells. amounts of viral proteins in cell extracts of the three persistently infected cell lines did not always correlate with viral titres. In Aal and Aa2 cultures titres of infectious virus had decreased about 1000-fold, yet the decrease in the synthesis of the E and C proteins was clearly much less. P27 was only detected in acutely infected cultures. The significance of this protein band is uncertain. It reacted with the E protein-specific MAb (Fig. 2b) and might therefore be a proteolytic fragment of the E protein. In the persistently infected Adl and Ad2 cultures where viral titres had decreased about 100fold, E and p27 proteins were clearly seen in most cases, but the C protein was not detected. (These proteins in Ad2 cultures 81 weeks p.i. were difficult to see in the photographic reproduction but were present in the original Western blot.) In Ctl and Ct2 cultures, the decreases in the synthesis of the E and C proteins (the C protein could no longer be seen) were consistent with the slight ( < 10-fold) decrease in viral titres, although the p27 protein appeared to be relatively overproduced. To determine whether the staining patterns of E, C and p27 proteins found in the persistently infected cultures would be maintained after passage in uninfected cultures, normal mosquito cell Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 08:01:56 2204 V. B. RANDOLPH AND J. L. HARDY (a) 1 (b) 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 1 2 Fig. 2. Western blots ofSLE viralproteins from extracts of acutely and persistently infected mosquito cell cultures. Samples were obtained from acutely infected (ai), mock-infected (mi) and persistently infected A. albopictus,A. dorsalisand C. tarsaliscell cultures. Persistently infected cell cultures were 42 and 81 weeks p.i. (indicated after cell culture line, see below). Blots were reacted with (a) SLE (LAV5156) IMAF or (b) MAb 16-A-7 (specific for E protein). (a) Lane 1, Aa mi; lane 2, Aa ai; lane 3, Aa 1 42; lane 4, Aa2 42; lane 5, Aal 81 ; lane 6, Aa2 81 ; lane 7, Ad mi; lane 8, Ad ai; lane 9, Adl 42, lane 10, Ad2 42; lane 11, Ad 1 81 ; lane 12, Ad2 81 ; lane 13, Ct mi; lane 14, Ct ai; lane 15, Ct 1 42; lane 16, Ct2 42; lane 17, Ctl 81; lane 18, Ct2 81. (b) Lane 1, A2 ai; lane 2, Ad ai; lane 3, Ct ai. E, p27 and C proteins are indicated by closed arrowheads, open arrowheads and arrows, respectively. T a b l e 3. Viral yields of SLE virus from persistently infected mosquito cell cultures after one passage in uninfected homologous and heterologous cell cultures Viral titre (log10 p.f.u./ml)t ,A Passage cell line Virus SLE wt Aal Aa2 Adl Ad2 Ctl Ct2 Before passage 4.0 3-5 4.6 5-4 6.5 6.4 A. albopictus 8.2 5.5 6.7 4.2 6.7 6.6 6-3 A. dorsalis 7-1 4-1 5.0 5-0 6.4 5.9 6.0 C. tarsalis 7.0 1.8 < 1.0 < 1.0 6-3 6.3 6.2 * Mosquito cell cultures were inoculated with stock SLE virus or virus obtained from persistently infected mosquito cell cultures 69 weeks p.i. and then incubated at 28 °C for 5 days. ? Viral titres were determined by plaque assay in Vero cell cultures. cultures w e r e newly infected w i t h virus o b t a i n e d f r o m the persistently i n f e c t e d h o m o l o g o u s cultures, a n d cell |ysates w e r e analysed by W e s t e r n b l o t t i n g (Fig. 3). T h e c o r r e s p o n d i n g titres o f virus in culture s u p e r n a t a n t fluids are p r e s e n t e d in T a b l e 3. T h e p a t t e r n o f viral proteins seen in these cells showed no differences f r o m those seen in cultures acutely infected w i t h wt virus. M o s t notably, w h e r e a s p27 was not seen in the persistently i n f e c t e d A a l or A a 2 cell cultures, it was readily d e t e c t e d in A. albopictus cell cultures acutely i n f e c t e d w i t h virus f r o m these cultures. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 08:01:56 2205 SLE virus mutants (a) 1 (b) 2 3 4 (c) 1 2 .... 3 4 1 2 3 i. p27 D C-.I~ / • " , :Z¸,¸i~ : :? i ¸ Fig. 3. Western blotofcellextractsof'normal'mosquitocellculturesinfectedwithSLEvirusobtained from persistently infected mosquito cell cultures. Cell lines used were (a) .4. albopictus, (b) A. dorsalis and (c) C. tarsalis. Cultures were mock-infected (lanes 2) or infected with SLE wt virus (lanes 1) or virus obtained from the persistently infected cultures of the homologous cell lines 69 weeks p.i. (a) lane 3, Aal virus; lane 4, Aa2 virus; (b) lane 3, Adl virus; lane 4, Ad2 virus; (c) lane 3, Ctt virus; lane 4, Ct2 virus. Cell extracts were obtained 5 days p.i. Blots were reacted with SLE (LAV5-156) IMAF. E, p27 and C proteins are indicated by closed arrowheads, open arrowheads and arrows, respectively. DISCUSSION The virus generated by mosquito cells persistently infected with SLE virus displayed several different phenotypes. As has been described for A. albopictus cells persistently infected with alphaviruses and other flaviviruses (Igarashi et aL, 1977; Igarashi, 1979; Ng & Westaway, 1980), sp mutants were prominent in all but the Ctl cultures, and there was evidence for the presence of ts mutants in the persistently infected cell cultures of A. dorsalis and C. tarsalis. In addition, evidence was obtained pointing to the presence of hr mutants in the Aedes cultures. Specifically, the Aal, Aa2 and Ad2 viruses were restricted in both a vertebrate line (Vero) and in the C. tarsalis cells. There have been no previous reports of hr mutants in flavivirus-infected mosquito cell cultures; however, such mutants have been generated in A. albopietus cells following serial transfer of Sindbis virus, an alphavirus (Durbin & Stollar, 1984; Steacie & Eaton, 1984). Also, Brown & Condreay (1986) reported that Sindbis virus isolated from persistently infected A. albopictus cells was growth-restricted in baby hamster kidney cells. It should be noted that many different types of viral mutants may be present in the persistently infected cultures described here and that cloning of the hr mutants would be necessary to determine the basis of restriction in replication. Another fact of interest is that the foci of infection produced by Aal virus in normal A. albopictus cells were larger than those produced by wt virus (Fig. 1). Thus the replication of this Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 08:01:56 2206 V. B. R A N D O L P H A N D J. L . H A R D Y virus was not only restricted in certain cells but was actually enhanced in the homologous cell line. A similar finding was reported by Simizu & Maeda (1981) who showed that several viral mutants isolated from A. albopictus cells persistently infected with western equine encephalomyelitis virus (an alphavirus) replicated more efficiently than wt virus in A. albopictus cells. Differences that appeared to be host-related were seen in the relative amounts of viral proteins produced in the persistently infected cultures. Of particular interest was the presence of a protein, p27, which seemed to be associated with the production of high levels of infectious virus. P27 disappeared in A. albopictus cells persistently infected with SLE virus, despite the continued production of E protein, but it subsequently reappeared when Aal or Aa2 virus was passaged once in normal A. albopictus cells and high titres of infectious virus were once again produced. Also, p27 continued to be produced in persistently infected C. tarsalis cell cultures in which the titre of infectious virus decreased relatively little during the course of persistent infection. The significance of p27 is unknown. Since it reacts with an E protein-specific MAb (Fig. 2), it would appear to be a degradation or cleavage product of the E protein and may not play any role in viral replication. Proteins of this size have been previously reported in fiavivirus-infected cultures (Westaway et al., 1977). Also, Gould et al. (1985) reported that a MAb specific for the E protein of yellow fever virus immunoprecipitated a 26K protein from infected Vero cells. It should be noted that the virus used to initiate the persistent infections was uncloned and had been passaged only twice in mosquito cells after isolation from a mosquito pool. Virus was not plaque-purified as this would have required additional passages in a vertebrate cell line, and thus would introduce another selection factor, i.e. virus able to form plaques in vertebrate cell cultures. The virus phenotypes observed in the persistently infected cultures could have been present in the original inoculum, and then selected for during long-term culture. However, given the high mutation rate of R N A viruses (Holland et al., 1982) and the length of time before these phenotypes were observed, it is likely that at least some mutants were generated during the course of infection. The observed phenotypic alterations in the virus from persistently infected cultures differed depending on the cell line, thus emphasizing the importance of the host cell in evolution of virus. Viral mutants (sp, ts and hr) were much more prominent in the persistently infected Aedes cell cultures, particularly the A. albopictus cultures, than in the C. tarsalis cultures. The amount of viral protein produced in the Aedes cells was also much greater than would be expected from the low titres of infectious virus. Although DI particles were not specifically looked for in this study, truncated viral RNAs were observed in Northern blots of the R N A from the persistently infected A. albopictus cells (data not shown). The host cell may influence the evolution of virus in several ways. Brinton (1983) and Brinton et al. (1985), have presented evidence that some vertebrate cells preferentially amplify W N virus ts mutants and defective viral RNAs and have suggested that the enhanced production of viral mutants may be related to the interaction of host proteins with the viral replicase (see Brinton, 1987). Additionally, selective pressures for certain viral phenotypes may be different in the various host ceils. In this respect, the ability of antibody to cure persistently infected cultures may be a relevant factor. Antibody cured one of the persistently infected C. tarsalis cultures but had no effect on the persistently infected Aedes cell cultures (Randolph & Hardy, 1988). The fact that antibody can cure some persistently infected cell cultures suggests that extracellular virus is vital for the maintenance of infection as was the case in the alphavirus-mosquito cell systems described by Riedel & Brown (1977) and Igarashi et al. (1977). In the Aedes cultures, antibody had no effect, suggesting that extracellular virus is not required for the maintenance of infection and that the persistent infection is maintained by partitioning of virus genome into daughter cells during cell division. In such a case there may be reduced selective pressure to produce infectious virus particles. We thank the members of the arbovirus research staff for their technical support, Dr L. E. Volkman for helpful discussions, and Dr V. Stollar for his critical reading of the manuscript. This research was funded by research grant AI-3028 from the National Institute of Allergy and Infectious Diseases and biomedical research support grant 5-507-RR-05441 from the National Institutes of Health. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 04 May 2017 08:01:56 S L E virus mutants 2207 REFERENCES BRI~ON, M. A. (1982). Characterization of West Nile virus persistent infections in genetically resistant and susceptible mouse cells. I. Generation of defective nonplaquing virus particles. Virology l l 6 , 84-98. BRItCrON,S~. A. (1983). Analysis of extracellular West Nile virus particles produced by cell cultures from genetically resistant and susceptible mice indicates enhanced amplification of defective interfering particles by resistant cultures. 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