Br. J. exp. Path. (1976) 57, 521
IMMUNIZATION OF MICE AGAINST COXSACK1EVIRUS B3 AND PREVENTION OF FOETAL GROWTH RETARDATION A. B. G. LANSDOWN AND J. D. BROWN From the Clinical Research Centre, Watford Road, Harrow, Middlesex HAL 3 UJ Received for publication April 20, 1976
Summary.-Coxsackievirus B3 infection in pregnant mice leads to a severe pancreatitis with a retardation of foetal growth and increased wastage. The present study demonstrates that animals may be immunized actively or passively against this infection to allow foetal development to proceed normally. Active immunization was achieved by injecting a low dose of live virus into 4-weekold animals. These mice were then mated at 10 weeks and given a high dose of virus on the eighth day of pregnancy. Examination at 18 days' gestation revealed that foetal growth was not significantly different from the controls injected with heat-killed virus, and pathological changes in the mothers were not seen. Animals were passively immunized against Coxsackievirus B3 in pregnancy by injecting serum from immunized animals 1 day before the high dose of live virus was given. This procedure also protected against the effects of the virus and litter sizes and foetal weights were normal.
COXSACKIEVIRUS B infections in human pregnancy are frequently inapparent and diagnosed retrospectively by serological analysis. However, as a result of epidemiological studies these viruses have been implicated in the production of congenital heart disease, abortions, stillbirths and low birth weight (Kilbrick and Benirischke, 1958; Czeizel, 1967; Brown and Karunas, 1972). Experimental studies in pregnant mice, characterized by an acute inflammation and necrosis of the exocrine pancreatic tissue, have also resulted in retarded foetal growth and plasma protein development (Coid and Ramsden, 1973; Lansdown and Coid, 1974; Coid, Ramsden and Healy, 1974). This impaired foetal development is attributed largely to the maternal pancreatic insufficiency which was first identified 2 days after injection of the virus (Lansdown and Ellaby, 1974). Subsequent work demonstrated that if the pregnant mice were fed diets containing hydrolysed protein to overcome the maternal pancreatic defect, then foetal growth did not differ significantly from
normal (Lansdown, Coid and Ramsden, 1975; Lansdown, 1975). Previous observations that neonatal mice are noticeably more susceptible to Coxsackievirus B infections than adults, suggested that antibody production has an important role in controlling the infection (Rager-Zisman and Allison, 1973a). The evidence presented was that if antibody production was suppressed by cyclophosphamide, infections which are often mild or inapparent under normal conditions prove to be lethal (Rager-Zisman and Allison, 1973b). The present studies were carried out to examine the possibility that antibodies to Coxsackievirus B3 formed in an animal as a result of a previous mild infection, or passively transferred in serunm from an infected animal, may protect pregnant mice from the pancreatotropic effects of a subsequent infection to allow normal foetal growth. MATERIALS AND METHODS
Virus Stock. The Coxsackievirus B3 used in these studies was from a stock strain maintained
522
A. B. G. LANSDOWN AND J. D. BROWN
at the Public Health Laboratory Service at Colindale, London. It was derived from the original Nancy Strain, passaged through suckling mice, isolated in primary rhesus monkey kidney cells and subsequently passed 3 times through vervet monkey kidney cells (VERO). The undiluted virus suspension was shown to have a tissue culture infective dose of 106 85/ml
(TCID50).
Animals.-The mice used were of the " TO" outbred strain of the original Swiss stock (Theiler). Virgin females weighing between 20 and 25 g were used in these experiments. Experimental.-In an initial series of experiments, groups of 20 4-week-old mice were injected i.m. with 0-1 ml of a diluted suspension of Coxsackievirus B3 containing 103 85/ml TCID50 which did not cause overt illness or produce severe pancreatic damage. Control mice were injected with an equivalent dose of virus suspension that had been inactivated by heating. Six weeks following treatment, tt and control mice were mated with proven males and on Day 8 of pregnancy were injected with 0 3 ml of undiluted virus suspension. Groups of 10 mice not previously treated were injected with a similar dose of live virus or with heatinactivated virus suspension as a further set of controls. Animals were killed on Day 18 of pregnancy and the number of live foetuses and resorption sites enumerated. Foetuses and placentae were weighed and representative sections of maternal liver and pancreas fixed in phosphate-buffered formalin for histological
10 4-week-old mice were injected with 0-1 ml of diluted Coxsackievirus B3 (TCID50 = 103 85/ml) or with heat-inactivated virus. These animals were killed after 6 weeks and 1 ml of blood obtained from each by cardiac puncture. The serum was separated by centrifugation and stored at - 4C. Groups of 10 pregnant mice were injected i.m. with 0-2 ml of this serum on Day 7 of pregnancy and on the following day received an injection of 0-3 ml of undiluted virus suspension. As in the first part of the work, these animals were killed on the 18th day of pregnancy and the foetuses and placentae examined for signs of reduced growth and maternal organs for pathological changes. RESULTS
The table shows that the foetuses from pregnant mice pretreated with a dilute suspension of live virus, and later injected with a higher titre of live virus in the course of their gestation, did not differ significantly in weight from the control group injected only in pregnancy with inactivated virus. However, in those mice pretreated with diluted inactivated virus and subsequently injected with live virus on Day 8 of pregnancy, the foetal weights were significantly lower, with an increased number of resorption sites (P< 0-001). A similar retarded growth examination. In a second series of experiments, groups of with increased resorption rate was also TABLE.-The Influence of Pretreatment of Mice with Live Coxsackievirus B3 or Coxsackievirus B3 Antiserum on Viral Infections Occurring in Pregnancy
Total TotalI no. Foetal Placental No. Maternal no. of of of livce weight weight weight (g) Treatment animals foetusees resorptions (g) (g) Injected dil. live Coxsackievirus 16 50-006±0-957 169 8 1-200±0-010 0-102±0-002 B3 at 4 weeks and challenged with live virus in pregnancy 50 Injected heat-killed virus at 4 8 37-420±2- 107 31 0-862±0-039 0 082±0-004 weeks and challenged with live virus in pregnancy Live Coxsackievirus B3 injected 66 10 28-194±2-035 20 0-803±0-027 0-091±0-003 in pregnancy only Heat-killed virus given in 82 9 48-975±3-415 7 1-252±0-012 0-119±0-003 pregnancy only Injected with serum from 0 1-277±0-014 0-121±0-002 8 43-743±2-482 69 animal given live Coxsackievirus B3 and challenged with live virus in pregnancy 5 32-625±3-371 18 Injected with serum from 13 0-987±0-051 0-093±0-004 animal given heat-killed virus and challenged with live virus in pregnancy + s.e. mean
IMMUNIZATION OF MICE AGAINST COXSACKIEVIRUS B3
seen in those mice injected once only, in pregnancy, with live Coxsackievirus. Histological observations of maternal tissues revealed severe pancreatitis and hepatocellular vacuolation due to labile protein loss in those mice pretreated with heat-inactivated virus and given injections of live virus in pregnancy. These types of pathological changes were also identified in those mice injected once only with live virus. When animals had received pretreatment with live virus followed in pregnancy with a further dose of live virus (higher titre), or were treated only once with inactivated virus, normal pancreatic exocrine tissue was present and the liver profile was consistent with the stage in pregnancy. The Table also shows that the foetuses from mice injected with serum from animals given injections of live virus and then injected with live virus on Day 8 of pregnancy did not differ in weight from the control group given heat-inactivated virus only. Also, the mothers did not exhibit pancreatic or hepatic changes. The foetuses obtained from mothers injected with the serum taken from mice treated with inactivated virus which were later given live virus on Day 8 of pregnancy were significantly smaller than the control, (P
IMMUNIZATION OF MICE AGAINST COXSACK1EVIRUS B3 AND PREVENTION OF FOETAL GROWTH RETARDATION A. B. G. LANSDOWN AND J. D. BROWN From the Clinical Research Centre, Watford Road, Harrow, Middlesex HAL 3 UJ Received for publication April 20, 1976
Summary.-Coxsackievirus B3 infection in pregnant mice leads to a severe pancreatitis with a retardation of foetal growth and increased wastage. The present study demonstrates that animals may be immunized actively or passively against this infection to allow foetal development to proceed normally. Active immunization was achieved by injecting a low dose of live virus into 4-weekold animals. These mice were then mated at 10 weeks and given a high dose of virus on the eighth day of pregnancy. Examination at 18 days' gestation revealed that foetal growth was not significantly different from the controls injected with heat-killed virus, and pathological changes in the mothers were not seen. Animals were passively immunized against Coxsackievirus B3 in pregnancy by injecting serum from immunized animals 1 day before the high dose of live virus was given. This procedure also protected against the effects of the virus and litter sizes and foetal weights were normal.
COXSACKIEVIRUS B infections in human pregnancy are frequently inapparent and diagnosed retrospectively by serological analysis. However, as a result of epidemiological studies these viruses have been implicated in the production of congenital heart disease, abortions, stillbirths and low birth weight (Kilbrick and Benirischke, 1958; Czeizel, 1967; Brown and Karunas, 1972). Experimental studies in pregnant mice, characterized by an acute inflammation and necrosis of the exocrine pancreatic tissue, have also resulted in retarded foetal growth and plasma protein development (Coid and Ramsden, 1973; Lansdown and Coid, 1974; Coid, Ramsden and Healy, 1974). This impaired foetal development is attributed largely to the maternal pancreatic insufficiency which was first identified 2 days after injection of the virus (Lansdown and Ellaby, 1974). Subsequent work demonstrated that if the pregnant mice were fed diets containing hydrolysed protein to overcome the maternal pancreatic defect, then foetal growth did not differ significantly from
normal (Lansdown, Coid and Ramsden, 1975; Lansdown, 1975). Previous observations that neonatal mice are noticeably more susceptible to Coxsackievirus B infections than adults, suggested that antibody production has an important role in controlling the infection (Rager-Zisman and Allison, 1973a). The evidence presented was that if antibody production was suppressed by cyclophosphamide, infections which are often mild or inapparent under normal conditions prove to be lethal (Rager-Zisman and Allison, 1973b). The present studies were carried out to examine the possibility that antibodies to Coxsackievirus B3 formed in an animal as a result of a previous mild infection, or passively transferred in serunm from an infected animal, may protect pregnant mice from the pancreatotropic effects of a subsequent infection to allow normal foetal growth. MATERIALS AND METHODS
Virus Stock. The Coxsackievirus B3 used in these studies was from a stock strain maintained
522
A. B. G. LANSDOWN AND J. D. BROWN
at the Public Health Laboratory Service at Colindale, London. It was derived from the original Nancy Strain, passaged through suckling mice, isolated in primary rhesus monkey kidney cells and subsequently passed 3 times through vervet monkey kidney cells (VERO). The undiluted virus suspension was shown to have a tissue culture infective dose of 106 85/ml
(TCID50).
Animals.-The mice used were of the " TO" outbred strain of the original Swiss stock (Theiler). Virgin females weighing between 20 and 25 g were used in these experiments. Experimental.-In an initial series of experiments, groups of 20 4-week-old mice were injected i.m. with 0-1 ml of a diluted suspension of Coxsackievirus B3 containing 103 85/ml TCID50 which did not cause overt illness or produce severe pancreatic damage. Control mice were injected with an equivalent dose of virus suspension that had been inactivated by heating. Six weeks following treatment, tt and control mice were mated with proven males and on Day 8 of pregnancy were injected with 0 3 ml of undiluted virus suspension. Groups of 10 mice not previously treated were injected with a similar dose of live virus or with heatinactivated virus suspension as a further set of controls. Animals were killed on Day 18 of pregnancy and the number of live foetuses and resorption sites enumerated. Foetuses and placentae were weighed and representative sections of maternal liver and pancreas fixed in phosphate-buffered formalin for histological
10 4-week-old mice were injected with 0-1 ml of diluted Coxsackievirus B3 (TCID50 = 103 85/ml) or with heat-inactivated virus. These animals were killed after 6 weeks and 1 ml of blood obtained from each by cardiac puncture. The serum was separated by centrifugation and stored at - 4C. Groups of 10 pregnant mice were injected i.m. with 0-2 ml of this serum on Day 7 of pregnancy and on the following day received an injection of 0-3 ml of undiluted virus suspension. As in the first part of the work, these animals were killed on the 18th day of pregnancy and the foetuses and placentae examined for signs of reduced growth and maternal organs for pathological changes. RESULTS
The table shows that the foetuses from pregnant mice pretreated with a dilute suspension of live virus, and later injected with a higher titre of live virus in the course of their gestation, did not differ significantly in weight from the control group injected only in pregnancy with inactivated virus. However, in those mice pretreated with diluted inactivated virus and subsequently injected with live virus on Day 8 of pregnancy, the foetal weights were significantly lower, with an increased number of resorption sites (P< 0-001). A similar retarded growth examination. In a second series of experiments, groups of with increased resorption rate was also TABLE.-The Influence of Pretreatment of Mice with Live Coxsackievirus B3 or Coxsackievirus B3 Antiserum on Viral Infections Occurring in Pregnancy
Total TotalI no. Foetal Placental No. Maternal no. of of of livce weight weight weight (g) Treatment animals foetusees resorptions (g) (g) Injected dil. live Coxsackievirus 16 50-006±0-957 169 8 1-200±0-010 0-102±0-002 B3 at 4 weeks and challenged with live virus in pregnancy 50 Injected heat-killed virus at 4 8 37-420±2- 107 31 0-862±0-039 0 082±0-004 weeks and challenged with live virus in pregnancy Live Coxsackievirus B3 injected 66 10 28-194±2-035 20 0-803±0-027 0-091±0-003 in pregnancy only Heat-killed virus given in 82 9 48-975±3-415 7 1-252±0-012 0-119±0-003 pregnancy only Injected with serum from 0 1-277±0-014 0-121±0-002 8 43-743±2-482 69 animal given live Coxsackievirus B3 and challenged with live virus in pregnancy 5 32-625±3-371 18 Injected with serum from 13 0-987±0-051 0-093±0-004 animal given heat-killed virus and challenged with live virus in pregnancy + s.e. mean
IMMUNIZATION OF MICE AGAINST COXSACKIEVIRUS B3
seen in those mice injected once only, in pregnancy, with live Coxsackievirus. Histological observations of maternal tissues revealed severe pancreatitis and hepatocellular vacuolation due to labile protein loss in those mice pretreated with heat-inactivated virus and given injections of live virus in pregnancy. These types of pathological changes were also identified in those mice injected once only with live virus. When animals had received pretreatment with live virus followed in pregnancy with a further dose of live virus (higher titre), or were treated only once with inactivated virus, normal pancreatic exocrine tissue was present and the liver profile was consistent with the stage in pregnancy. The Table also shows that the foetuses from mice injected with serum from animals given injections of live virus and then injected with live virus on Day 8 of pregnancy did not differ in weight from the control group given heat-inactivated virus only. Also, the mothers did not exhibit pancreatic or hepatic changes. The foetuses obtained from mothers injected with the serum taken from mice treated with inactivated virus which were later given live virus on Day 8 of pregnancy were significantly smaller than the control, (P
