Br. J. exp. Path. (1975) 56, 363
PRODUCTION OF DEFECTIVE INTERFERING VIRUS IN THE BRAINS OF MICE BY AN AVIRULENT, IN CONTRAST WITH A VIRULENT, STRAIN OF SEMLIKI FOREST VIRUS C. G. WOODWARD A:m H. SMITH From the Department of Microbiology, Univerfity of Birmingham, Birmingham B15 2TT Received for publication March 25, 1975
Summary.-An avirulent strain (A7) of Semliki Forest virus formed nearly as much haemagglutinating and complement fixing antigen in the brains of adult mice as a virulent (V13) strain, yet the infectivities of the brain tissues were different by about 100-fold. It appeared therefore that defective virus particles were formed by A7 but these were not demonstrated by fluorescent antibody studies. In short-term organ cultures of adult mouse brain, A7 derived from mouse brain showed a typical interference pattern in inoculum infectivity response curves. Furthermore, when mixed suspensions of brain-grown V13 and A7 with equal infectivities were inoculated the inoculum infectivity response patterns showed significant depressions of the V13 response at higher inocula. Such interference was not detected if chick cell grown A7 and V13 were substituted for the mouse grown virus. The avirulence of A7 in adult mice and its rapid protective effect against lethal V13 infection could be due to the production of defective interfering virus particles in the brain.
IN MIcE after intraperitoneal inoculation an avirulent (A7) strain of Semliki Forest virus replicated in muscle and produced a viraemia as well as, if not better than, a virulent (V13) strain. In the brain, however, V13 was detectable about 12 h earlier than A7, increased to a high infectivity, and killed the mice in 4-5 days. A7 was detectable at about 48 h but the infectivity remained at levels approximately 100-fold lower than V13 for 4-5 days and then receded (Pusztai, Gould and Smith, 1971; Smillie, Pusztai and Smith, 1973). A similar pattern of infection was also obtained when the 2 strains were inoculated directly into the brain (Pusztai et al., 1971). One explanation for the above result was that A7 either induced stronger host defence mechanisms than V13 or was more prone to destruction by them. However, defence mechanisms differentially induced or resisted by V13 or A7, which might explain the different patterns of infection in mice were not detected in virus-free extracts of muscle, blood and spleen from 25
mice infected with either strain using inhibition tests in vitro and in vivo (Smillie et al., 1973). Interferon production in mouse brains was poor and greater with V13 (Bradish and Alner, 1972), nor could other differentially induced defence mechanisms be detected in brain homogenates. Infections of either strain in mouse brain organ culture (see below) were slightly inhibited by ultraviolet light irradiated homogenates of mouse brains from A7 and V13 infections, but only to the same extent as by homogenates from uninfected brains (Woodward and Smith, unpublished observations). The specific immune defence mechanisms probably have little bearing on the differences between A7 and V13 in the brain since these differences occur within 24-48 h in this immunologically privileged site. This was supported by observations on X-irradiated mice. The pattern of A7 infection was similar to that in normal mice and the extent of the brain infection did not resemble the fatal infection of V13 in normal animals
364
C. G. WOODWARD AND H. SMITH
(Smillie et al., 1973). The lack of influence of antibody production on the differential behaviour of the strains was also indicated by a recent study (Allner et al., 1974) showing the potentiation of A7 infection by near-toxic levels of aurothiomalate which left antibody production unaffected but may have depressed phagocytosis of the virus by macrophages. Another possible explanation for the difference in behaviour of A7 and V13 in the brain is that A7 produces defective interfering particles in this site. Many viruses produce defective particles that interfere with virus infections in animals as well as in tissue culture (Huang, 1973; Holland and Doyle, 1973; Doyle and Holland, 1973). The production of defective interfering particles by A7 might also explain the extremely rapid (within 12-24 h) and therefore probably nonspecific protection that an injection of A7 can provide against an otherwise lethal inoculation of V13 (Pusztai et al., 1971; Bradish et al., 1972); such rapidly arising nonspecific protection has also been noticed between arboviruses of different antigenic groups, including Semliki Forest virus, although it was evoked by formalin treated virus and may have been associated with an interferon-like activity in the serum (Casals, Buckley and Barry, 1973). Recently, defective interfering particles were detected in murine cells infected with high multiplicities of Semliki Forest virus (Levin, Ramseur and Grimley, 1973) but not in chick embryo cells. This paper describes the estimation of virus antigens in mouse brains infected with A7 and V13 by haemagglutination, complement fixation and fluorescent antibody techniques, to see if the amount of such antigens in the A7 brains approached that in V13 brains despite the 100-fold differences in infectivity. Then separate mouse brain organ cultures were infected with different concentrations of mouse brain grown A7 and V13 and with equal mixtures of the 2 strains to see if evidence of interference by A7 defective particles could be obtained in A7 titrations alone
and when mixed with V13. A preliminary report of some of this work has been published (Woodward and Smith, 1974). MATERIALS AND METHODS
Mice.-Porton white mice were used, usually aged 25-30 days. Viru8.-Semliki Forest virus (SFV) strains A7 (avirulent) and V13 (virulent) were those described as A7.C1 and V13.C1 by Bradish et al. (1971) or A7 and V13 by Pusztai et al. (1971). Chick cell grown virus was obtained from chick embryo cells as described by Pusztai et al. (1971). Brain grown virus was produced by inoculating 104 pfu of the chick cell grown virus intraperitoneally into mice and removing the brains 4 days later. After homogenization in Parker's medium 199 containing 5% v/v calf sertum and centrifugation (2000g, 15min, room temperature) the virus was stored at _70°. Infectivity assay.-This was done by plaque counts on chick embryo cells as described by Bradish et al. (1971). Samptel of peritoneal mu8cle, brain and blood from infected mice.-These were obtained and processed for virus assay as described by Pusztai et al. (1971). Haemagglutination as8ay.-Brains and occasionally other tissues taken from groups (5-10) of mice were homogenized and extracted by the sucrose/acetone method of Clarke and Casals (1958). The haemagglutinin was titrated against goose or day-old-chick red blood cells (Tissue Culture Services Ltd) using the method of Clarke and Casals (1958) in Perspex WHO plates with 0-2 ml red blood cells and 0-2 ml antigen; the end point was taken as the last well to give complete agglutination and replicate assays agreed within one two-fold dilution. The pH optimum for assay of antigen from either chick cells or mouse brain was shown to be pH 6-2. To check the validity of the assay a suspension of chick cell grown virus (V13 or A7) was assayed and titres of 1/256-1/512 were obtained for an infectivity of 1 x 109 pfu/ml; this agreed with the figures of uthers for SFV (Cameron and Bradish, 1972, 1973; personal communication from C. J. Bradish). Then suspensions of the chick cell grown virus (V13 and A7) and virus (V13) harvested from the brains of suckling mice and having known infectivities and haemagglutination titres were added to normal adult mouse brain and assayed after homogenization and extraction as described above. In 5 tests, a four-fold reduction in haemagglutination titre was observed after extraction and assay. Thus, suspensions of chick cell grown virus and suckling mouse brain grown virus each with an infectivity of 1 x 109 pfu/ml and haemagglutination titres of 1/256-1/512 and 1/1024
PRODUCTION OF DEFECTIVE INTERFERING VIRUS IN THE BRAINS OF MICE 365
respectively showed titres of 1/64-1/128 and 1/256 respectively after mixing and extraction from adult mouse brain tissue. These figures are in good agreement with those obtained for the brains of adult mice infected with V13 (Table I); 20% w/v suspensions of adult mouse brains infected with V13 with infectivities of about 5 x 107 pfu/ml had haemagglutination titres of 1/4-1/8, that is 1/80-1/160 for an infectivity of 1 x 109 pfu/ml. To check the specificity of the haemagglutinin in the brains, haemagglutination inhibition tests were made with a rabbit antiserum raised against live A7. This had a titre of 1/1024 when measured against amounts of A7 or V13 suspensions having haemagglutination titres of 1/8. In the inhibition tests it was used at a final dilution of 1/100 with nonimmune rabbit serum (1/100) as control. Conmplement fixation. Chick cell grown virus was assayed directly but brain grown virus was extracted as for the haemagglutination tests. The assay was carried out with 01 ml volumes of reagents (complement and horse haemolytic serum from Wellcome Reagents Ltd, sheep blood from Tissuie Culture Services Ltd) in Perspex WA,'HO plates and the end point was taken as the dillution showing 50% haemolysis. In replicate assays end points varied by one two-fold dilution. The rabbit antiserum against live A7 was treated for anticomplementary activity (Lennette and Schmidt, 1964) and adsorbed 3 times with homogenates of normal adult mouse brains before use. The dilutions of antiserum and complement used with the mouse brain grown viruses were determined by initial titrations w ith chick cell grown SFV (A7 and V13). In these titrations complement fixing titres of 1/80-1/160 were obtained for A7 and V 13 corresponding to infectivities of 1 x 109 pfu/ml. This is in reasonable agreement with the figure obtained for mouse brains infected with V13 (Table II); 20% w/v suspensions with infectivities of about 5 x 107 pful/ml had titres of 1/16, that is 1/320 for an infectivity of 1 x 109 pfu/ml. It should be noted that suspensions of uninfected brains showed significant complement fixing titres although they were always less than suspensions of infected brains (see Table II). Attempts to reduce the effects of this nonspecific complement fixing activity by titrating the complement in the presence of uninfected brain antigens were not successful. Fluorescent antibody studies.-Brains were removed from control mice or infected (104 pfu, intraperitoneally, A7 or V13, 4 days previously) mice, frozen immediately in solid carbon dioxide and sectioned on a cryostat. The specific antiserum was prepared in rabbits against A7 and non-immune rabbit serum was the control. Sera were adsorbed (2 x) with homogenates of
normal mouse brain or whole mice. Direct and indirect staining was used (Nairn, 1969). For the former, sera were conjugated with fluorescein isothiocyanate (British Drug Houses Ltd) and for the latter, fluorescent sheep antirabbit serum (Wellcome Reagents Ltd) was used. Nonspecific staining was masked by rhodamine counterstain (Difco Ltd) or by Evans blue. Sections were examined on a Zeiss Universal microscope with CS 200 W lamp, 2 KP 500 exciter filters and KP 50 and KP 44 barrier filters. The method of expression of the degree of staining is described in the footnotes to Table III. Organ cultures.-The method of Hoorne and Tyrrell (1969) as modified by Basarab and Smith (1970) was used. Brains were removed aseptically from mice and immersed in Hanks' salt solution. For each experiment pieces (1 mm3) cut with very sharp knives from each of 5 brains were placed on scratched grids in plastic Petri dishes (Esco AA), wvith 1-2 ml Eagle's medium containing 0.2% bovine serum albumin (Armour). They were infected with 0-2 ml of the appropriate virus dilution, which was spotted onto the tissue pieces. The dishes were incubated at 370 in a humid atmosphere in plastic boxes containing 5%/' carbon dioxide. After the stated time interval, infective virus produced was assayed after homogenizing the tissue pieces in their surrounding medium using a Griffiths tube (preliminary experiments showed this gave more reproducible titres than assaying the media alone). Each point shown in the Figures is the average virus yield from 4 dishes. Histological exa.n?nination of uninfected and infected organ cultures.-We are indebted to Dr I. Ziotnick of the Microbiological Research Establishment, Porton, Wriltshire for these examinations. RESULTS
Comparison of infectivity titres and virus
antigen estimations in mouse brains 4
days after inoculation with A7 and V13 Table I summarizes a comparison of infectivity and haemagglutination titres of homogenates of brains of mice 4 days after intraperitoneal inoculation of chick cell grown A7 or V13. The haemagglutination titres on the brain homogenates from the mice infected with V13 are what might be expected from previous work (see Materials and Methods) for 20% w/v suspensions of brains with infectivities of 1-3 x 108 pfu/g. However, the titres of brain homogenates from mice infected
366
C. G. WOODWARD AND H. SMITH
TABLE I.-Comparison of Infectivity and Haemagglutination Titres of Homogenates of the Brains of Mice 4 days after Intraperitoneal Inoculation with Avirulent (A7) or Virulent (VI 3) Strains of Semliki Forest VirUs Mouse brain homogenates*
Haemagglutination titres A
,
After adding non-immune
Infectivity pfu/ml (x 10-6)
Alone
After adding
antiseLrumt
sera
A
A7 V13 A7 V13 Uninfected A7 V13 Experiment 1-2 1 36 2 8 1 2 0- 3 40 2 8
PRODUCTION OF DEFECTIVE INTERFERING VIRUS IN THE BRAINS OF MICE BY AN AVIRULENT, IN CONTRAST WITH A VIRULENT, STRAIN OF SEMLIKI FOREST VIRUS C. G. WOODWARD A:m H. SMITH From the Department of Microbiology, Univerfity of Birmingham, Birmingham B15 2TT Received for publication March 25, 1975
Summary.-An avirulent strain (A7) of Semliki Forest virus formed nearly as much haemagglutinating and complement fixing antigen in the brains of adult mice as a virulent (V13) strain, yet the infectivities of the brain tissues were different by about 100-fold. It appeared therefore that defective virus particles were formed by A7 but these were not demonstrated by fluorescent antibody studies. In short-term organ cultures of adult mouse brain, A7 derived from mouse brain showed a typical interference pattern in inoculum infectivity response curves. Furthermore, when mixed suspensions of brain-grown V13 and A7 with equal infectivities were inoculated the inoculum infectivity response patterns showed significant depressions of the V13 response at higher inocula. Such interference was not detected if chick cell grown A7 and V13 were substituted for the mouse grown virus. The avirulence of A7 in adult mice and its rapid protective effect against lethal V13 infection could be due to the production of defective interfering virus particles in the brain.
IN MIcE after intraperitoneal inoculation an avirulent (A7) strain of Semliki Forest virus replicated in muscle and produced a viraemia as well as, if not better than, a virulent (V13) strain. In the brain, however, V13 was detectable about 12 h earlier than A7, increased to a high infectivity, and killed the mice in 4-5 days. A7 was detectable at about 48 h but the infectivity remained at levels approximately 100-fold lower than V13 for 4-5 days and then receded (Pusztai, Gould and Smith, 1971; Smillie, Pusztai and Smith, 1973). A similar pattern of infection was also obtained when the 2 strains were inoculated directly into the brain (Pusztai et al., 1971). One explanation for the above result was that A7 either induced stronger host defence mechanisms than V13 or was more prone to destruction by them. However, defence mechanisms differentially induced or resisted by V13 or A7, which might explain the different patterns of infection in mice were not detected in virus-free extracts of muscle, blood and spleen from 25
mice infected with either strain using inhibition tests in vitro and in vivo (Smillie et al., 1973). Interferon production in mouse brains was poor and greater with V13 (Bradish and Alner, 1972), nor could other differentially induced defence mechanisms be detected in brain homogenates. Infections of either strain in mouse brain organ culture (see below) were slightly inhibited by ultraviolet light irradiated homogenates of mouse brains from A7 and V13 infections, but only to the same extent as by homogenates from uninfected brains (Woodward and Smith, unpublished observations). The specific immune defence mechanisms probably have little bearing on the differences between A7 and V13 in the brain since these differences occur within 24-48 h in this immunologically privileged site. This was supported by observations on X-irradiated mice. The pattern of A7 infection was similar to that in normal mice and the extent of the brain infection did not resemble the fatal infection of V13 in normal animals
364
C. G. WOODWARD AND H. SMITH
(Smillie et al., 1973). The lack of influence of antibody production on the differential behaviour of the strains was also indicated by a recent study (Allner et al., 1974) showing the potentiation of A7 infection by near-toxic levels of aurothiomalate which left antibody production unaffected but may have depressed phagocytosis of the virus by macrophages. Another possible explanation for the difference in behaviour of A7 and V13 in the brain is that A7 produces defective interfering particles in this site. Many viruses produce defective particles that interfere with virus infections in animals as well as in tissue culture (Huang, 1973; Holland and Doyle, 1973; Doyle and Holland, 1973). The production of defective interfering particles by A7 might also explain the extremely rapid (within 12-24 h) and therefore probably nonspecific protection that an injection of A7 can provide against an otherwise lethal inoculation of V13 (Pusztai et al., 1971; Bradish et al., 1972); such rapidly arising nonspecific protection has also been noticed between arboviruses of different antigenic groups, including Semliki Forest virus, although it was evoked by formalin treated virus and may have been associated with an interferon-like activity in the serum (Casals, Buckley and Barry, 1973). Recently, defective interfering particles were detected in murine cells infected with high multiplicities of Semliki Forest virus (Levin, Ramseur and Grimley, 1973) but not in chick embryo cells. This paper describes the estimation of virus antigens in mouse brains infected with A7 and V13 by haemagglutination, complement fixation and fluorescent antibody techniques, to see if the amount of such antigens in the A7 brains approached that in V13 brains despite the 100-fold differences in infectivity. Then separate mouse brain organ cultures were infected with different concentrations of mouse brain grown A7 and V13 and with equal mixtures of the 2 strains to see if evidence of interference by A7 defective particles could be obtained in A7 titrations alone
and when mixed with V13. A preliminary report of some of this work has been published (Woodward and Smith, 1974). MATERIALS AND METHODS
Mice.-Porton white mice were used, usually aged 25-30 days. Viru8.-Semliki Forest virus (SFV) strains A7 (avirulent) and V13 (virulent) were those described as A7.C1 and V13.C1 by Bradish et al. (1971) or A7 and V13 by Pusztai et al. (1971). Chick cell grown virus was obtained from chick embryo cells as described by Pusztai et al. (1971). Brain grown virus was produced by inoculating 104 pfu of the chick cell grown virus intraperitoneally into mice and removing the brains 4 days later. After homogenization in Parker's medium 199 containing 5% v/v calf sertum and centrifugation (2000g, 15min, room temperature) the virus was stored at _70°. Infectivity assay.-This was done by plaque counts on chick embryo cells as described by Bradish et al. (1971). Samptel of peritoneal mu8cle, brain and blood from infected mice.-These were obtained and processed for virus assay as described by Pusztai et al. (1971). Haemagglutination as8ay.-Brains and occasionally other tissues taken from groups (5-10) of mice were homogenized and extracted by the sucrose/acetone method of Clarke and Casals (1958). The haemagglutinin was titrated against goose or day-old-chick red blood cells (Tissue Culture Services Ltd) using the method of Clarke and Casals (1958) in Perspex WHO plates with 0-2 ml red blood cells and 0-2 ml antigen; the end point was taken as the last well to give complete agglutination and replicate assays agreed within one two-fold dilution. The pH optimum for assay of antigen from either chick cells or mouse brain was shown to be pH 6-2. To check the validity of the assay a suspension of chick cell grown virus (V13 or A7) was assayed and titres of 1/256-1/512 were obtained for an infectivity of 1 x 109 pfu/ml; this agreed with the figures of uthers for SFV (Cameron and Bradish, 1972, 1973; personal communication from C. J. Bradish). Then suspensions of the chick cell grown virus (V13 and A7) and virus (V13) harvested from the brains of suckling mice and having known infectivities and haemagglutination titres were added to normal adult mouse brain and assayed after homogenization and extraction as described above. In 5 tests, a four-fold reduction in haemagglutination titre was observed after extraction and assay. Thus, suspensions of chick cell grown virus and suckling mouse brain grown virus each with an infectivity of 1 x 109 pfu/ml and haemagglutination titres of 1/256-1/512 and 1/1024
PRODUCTION OF DEFECTIVE INTERFERING VIRUS IN THE BRAINS OF MICE 365
respectively showed titres of 1/64-1/128 and 1/256 respectively after mixing and extraction from adult mouse brain tissue. These figures are in good agreement with those obtained for the brains of adult mice infected with V13 (Table I); 20% w/v suspensions of adult mouse brains infected with V13 with infectivities of about 5 x 107 pfu/ml had haemagglutination titres of 1/4-1/8, that is 1/80-1/160 for an infectivity of 1 x 109 pfu/ml. To check the specificity of the haemagglutinin in the brains, haemagglutination inhibition tests were made with a rabbit antiserum raised against live A7. This had a titre of 1/1024 when measured against amounts of A7 or V13 suspensions having haemagglutination titres of 1/8. In the inhibition tests it was used at a final dilution of 1/100 with nonimmune rabbit serum (1/100) as control. Conmplement fixation. Chick cell grown virus was assayed directly but brain grown virus was extracted as for the haemagglutination tests. The assay was carried out with 01 ml volumes of reagents (complement and horse haemolytic serum from Wellcome Reagents Ltd, sheep blood from Tissuie Culture Services Ltd) in Perspex WA,'HO plates and the end point was taken as the dillution showing 50% haemolysis. In replicate assays end points varied by one two-fold dilution. The rabbit antiserum against live A7 was treated for anticomplementary activity (Lennette and Schmidt, 1964) and adsorbed 3 times with homogenates of normal adult mouse brains before use. The dilutions of antiserum and complement used with the mouse brain grown viruses were determined by initial titrations w ith chick cell grown SFV (A7 and V13). In these titrations complement fixing titres of 1/80-1/160 were obtained for A7 and V 13 corresponding to infectivities of 1 x 109 pfu/ml. This is in reasonable agreement with the figure obtained for mouse brains infected with V13 (Table II); 20% w/v suspensions with infectivities of about 5 x 107 pful/ml had titres of 1/16, that is 1/320 for an infectivity of 1 x 109 pfu/ml. It should be noted that suspensions of uninfected brains showed significant complement fixing titres although they were always less than suspensions of infected brains (see Table II). Attempts to reduce the effects of this nonspecific complement fixing activity by titrating the complement in the presence of uninfected brain antigens were not successful. Fluorescent antibody studies.-Brains were removed from control mice or infected (104 pfu, intraperitoneally, A7 or V13, 4 days previously) mice, frozen immediately in solid carbon dioxide and sectioned on a cryostat. The specific antiserum was prepared in rabbits against A7 and non-immune rabbit serum was the control. Sera were adsorbed (2 x) with homogenates of
normal mouse brain or whole mice. Direct and indirect staining was used (Nairn, 1969). For the former, sera were conjugated with fluorescein isothiocyanate (British Drug Houses Ltd) and for the latter, fluorescent sheep antirabbit serum (Wellcome Reagents Ltd) was used. Nonspecific staining was masked by rhodamine counterstain (Difco Ltd) or by Evans blue. Sections were examined on a Zeiss Universal microscope with CS 200 W lamp, 2 KP 500 exciter filters and KP 50 and KP 44 barrier filters. The method of expression of the degree of staining is described in the footnotes to Table III. Organ cultures.-The method of Hoorne and Tyrrell (1969) as modified by Basarab and Smith (1970) was used. Brains were removed aseptically from mice and immersed in Hanks' salt solution. For each experiment pieces (1 mm3) cut with very sharp knives from each of 5 brains were placed on scratched grids in plastic Petri dishes (Esco AA), wvith 1-2 ml Eagle's medium containing 0.2% bovine serum albumin (Armour). They were infected with 0-2 ml of the appropriate virus dilution, which was spotted onto the tissue pieces. The dishes were incubated at 370 in a humid atmosphere in plastic boxes containing 5%/' carbon dioxide. After the stated time interval, infective virus produced was assayed after homogenizing the tissue pieces in their surrounding medium using a Griffiths tube (preliminary experiments showed this gave more reproducible titres than assaying the media alone). Each point shown in the Figures is the average virus yield from 4 dishes. Histological exa.n?nination of uninfected and infected organ cultures.-We are indebted to Dr I. Ziotnick of the Microbiological Research Establishment, Porton, Wriltshire for these examinations. RESULTS
Comparison of infectivity titres and virus
antigen estimations in mouse brains 4
days after inoculation with A7 and V13 Table I summarizes a comparison of infectivity and haemagglutination titres of homogenates of brains of mice 4 days after intraperitoneal inoculation of chick cell grown A7 or V13. The haemagglutination titres on the brain homogenates from the mice infected with V13 are what might be expected from previous work (see Materials and Methods) for 20% w/v suspensions of brains with infectivities of 1-3 x 108 pfu/g. However, the titres of brain homogenates from mice infected
366
C. G. WOODWARD AND H. SMITH
TABLE I.-Comparison of Infectivity and Haemagglutination Titres of Homogenates of the Brains of Mice 4 days after Intraperitoneal Inoculation with Avirulent (A7) or Virulent (VI 3) Strains of Semliki Forest VirUs Mouse brain homogenates*
Haemagglutination titres A
,
After adding non-immune
Infectivity pfu/ml (x 10-6)
Alone
After adding
antiseLrumt
sera
A
A7 V13 A7 V13 Uninfected A7 V13 Experiment 1-2 1 36 2 8 1 2 0- 3 40 2 8
