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Rubella Virus in the Cataractous Lens of Congenital Rubella Syndrome
RUBELLA VIRUS IN THE CATARACTOUS LENS OF CONGENITAL RUBELLA SYNDROME E D W A R D COTLIER, M.D.,
JEAN FOX,
AND M O R T O N S M I T H ,
B.S.,
M.D.
St. Louis, Missouri Congenital cataracts in infants born fol lowing maternal rubella infection was re ported by Gregg in 1941. 1 Since then, the occurrence of rubella during the first tri mester of pregnancy has been associated with a series of congenital malformations (rubella syndrome) in addition to ocular le sions, such as cataracts, pigmentary retinopathy and glaucoma. 2 Rubella virus has been recovered, follow ing rubella infections, from army recruits 3 and children. 10 I n addition, the virus has been found in nasopharynx, urine and feces of patients with congenital rubella syn dromes. 5 ' 6 Rubella virus isolated from the cataractous lenses of infants affected by maternal rubella is reported here. MATERIAL AND METHODS A.
C A S E REPORTS
Case 1. R. R. P., an eight-month-old white boy, was delivered normally following a pregnan cy complicated by German measles in the third week. The pregnancy lasted only seven months. Birth weight was five pounds, 13 ounces. The child was described as having cataracts at birth. He was examined at the age of two months for a heart murmur. Other than otitis media, the child has not had any other illnesses. Physical examination revealed a healthy child in no distress. The child had horizontal nystag mus. White, lenticular opacities were seen bilateral ly. The apex beat was at the fifth intercostal space and left midclavical line. The heart rate was 120/minute. Systolic murmur grade III/VI was heard over the entire precordium, loudest along the left sternal border at the third and fourth intercostal space. No thrill was palpable. Findings for the rest of the physical examination were negative. The clinical impression was: postFrom the Department of Ophthalmology, Wash ington University Medical School, and the Virology Section of Central Diagnostic Laboratories, Barnes Hospital Group. This study was supported in part by research grant B-621 from the National Institute of Neurological Diseases and Blindness, National Institutes of Health, Bethesda, Maryland. 233
rubella syndrome with (1) bilateral congenital cataracts, and (2) a ventricular septal defect. Additional investigations included: hemogramhemoglobin was 10.4 gm, hematocrit 33%, white blood cells and differential were normal. The urinalysis was normal. Long-bone X-ray films showed multiple growth arrest lines; otherwise normal long bones. A cardiac series showed slight cardiomegaly with left atrial and right ventricular enlargement and pulmonary overcirculation, compatible with left-to-right shunt at the ventricular level. An aberrant right subclavian ar tery was also seen. The EKG revealed right ven tricular enlargement. A needle aspiration of the cataract of the right eye was followed five weeks later by a similar procedure in the left eye. The postoperative course was uneventful. Case 2. W. J., an eight and one-half month-old Negro boy, had a history of maternal rubella during the second month of pregnancy. The preg nancy was also complicated by "low-iron anemia" and a vaginal infection. The child was full term, and weighed three pounds, nine ounces at birth. The delivery was uncomplicated. Physical exami nation at birth showed a mildly icteric baby with echymosis and petechiae over the entire body. A systolic murmur grade III/IV was heard over the entire precordium. Liver and spleen were pal pable. At birth, laboratory work revealed: hemo globin 11.7 gm and thrombocytopenia (platelets 10,000). Hemoglobin dropped to 9.7 gm during the second day. Blood type B ( + ), mother B ( + ) and Coombs (—). VDRL in mother and child were negative. Blood cultures were negative. On day 2 the child was transfused with 30 cc of whole blood. Ophthalmologic examination re vealed bilateral cataracts. Bone-marrow morphol ogy on day 3 was compatible with thrombocyto penia. The patient also developed "myotonic jerk ing of extremities" and a lumbar puncture showed xanthochromic fluid. The "seizures" dis appeared during the hospitalization. A small left upper lobe pneumonia developed during the hospi talization but cleared rapidly with antibiotics. Clinical impressions were: (1) postrubella syn drome with thrombocytopenic purpura. When admit ted for needle aspiration of congenital cataract, the eye examination revealed congenital nearly mature cataracts, nystagmus, esotropia and microcornea. The cataractous lens of one eye was aspi rated. The postoperative course was uneventful. P>. L E N S AND TISSUE MATERIAL
T h e lens material was obtained by needle
234
AMERICAN JOURNAL OF OPHTHALMOLOGY
A U G U S T , 1966
TABLE 1 SUMMARY OF VIRAL STUDIES IN CONGENITAL RUBELLA SYNDROMES
Specimen
1. 2. 3. 4. 5. 6. 7.
R. R. P. Cataract O.D. Urine Aqueous humor Conjunctival swab Cataract O.S. Conjunctival tissue Throat swab
W.J.
1. Cataract 2. Aqueous humor
Days in Days in 2nd Passage Inhibition Passage Inhibition Inhibition when of Echo when of Echo of Echo 11 Challenged 11 Challenged 11 (repeat) with with Echo 11 Echo 11
Amount Inocu lated per Tube (ml)
Cell Line Inoculated
0.2 0.2 0.1 0.1 0.2 0.1 0.2
LLCMK 2 LLCMK 2 LLCMK 2 LLCMK 2 LLCMK 2 LLCMK 2 LLCMK 2
12 13 12 12 12 12 12
No No No No No No Partial
12 12 11 11 11 11 11
Complete No No No No No Partial
Complete No No No No No
0.2 0.2
AGMK AGMK
12 12
No No
11 12
Complete No
Complete No
aspiration. A fixation suture was placed in the superior rectus, aqueous humor was as pirated, the anterior chamber was entered with a Ziegler needle-knife and the lens in cised. Then a 24-gauge needle connected to a syringe with two cc of saline was intro duced into the anterior chamber and the lens contents aspirated by slow to-and-fro move ments. The material for viral studies con sisted of the contents of the first syringe ob tained after the lens incisions. Conjunctival and throat swab and other biopsied tissues were immersed in one ml sterile saline. The amounts inoculated per tube are recorded in Table 1. C. VIRUS ISOLATION TECHNIQUES
The indirect interference method for virus isolation originally described by Parkman and co-workers3 was used. Tissue cul tures of African green monkey kidney cells (AGMK*) or continuous rhesus monkey kidney (LLCMK 2 f ) were maintained in 0.25% calf Eagles (Hanks base) containing 300 units of penicillin, 300 y.g streptomycin and 25 \hg mycostatin. Specimens were in oculated in 0.2-ml amounts into four culture tubes each. Inoculation was carried out short* Obtained from Baltimore Biological Labora tories, Baltimore, Maryland. t Obtained from Eli Lilly.
—
ly after collection. After 11 to 13 days' in cubation, cultures were challenged with 1,000 to 10,000 tissue culture infective doses (TCID 5 0 ) of Echo 11 virus. If interference was not evident on the first passage of the test material, second passages were under taken before the specimen was considered negative. Observations for cytopathologic effects were made every second day. Rubella antibody neutralization tests with immune rabbit serum confirmed the identity of the virus. Virus titrations are reported in a separate paper. RESULTS
As shown in Table 1, the cataractous lens (specimen 1)) from the right eye of R. R. P. (age eight months) contained rubella virus. However, urine, the lens material, aqueous humor, conjunctival swab and tissue were negative for virus five weeks later (speci mens 2, 3, 4, 5 and 6.) A throat swab at that time contained rubella virus (specimen 7). Lens material obtained from W. J. (aged eight and one-half months) was positive for rubella virus while no virus was recovered from aqueous humor of the same eye. COMMENTS
The presence of the rubella virus in the cataractous lens of maternal rubella children is not a totally unexpected finding, as the
VOL. 62, NO. 2
RUBELLA VIRUS IN CATARACTOUS LENS
virus has been recovered from adrenal glands, bone, brain, kidney, intestine, liver, muscle, spleen, nasopharynx, urine and other tissues of embryos and children with congenital rubella.5'8 Persistence of the virus has been found at birth and for sever al months thereafter.6 Rubella neutralizing antibodies present at birth persist for years. 7 However, virus was not recovered from the nasopharynx, urine or serum of patients with congenital rubella syndromes at the ages of five to 22 years.4 The latter findings indicate a limited persistence of virus in congenital rubella. Of interest is the recov ery of the virus from the first but not the second cataractous lens in Case 1 (R. R. P.) aspirated five weeks after the first. The persistence of virus in the lens may be re lated to the period of time following intrauterine infection. The reasons for the formation of these congenital rubella cataracts are unknown. The pathology of embryonic rubella cata racts was studied by Cordes and Barber.8 The lens is surrounded by blood vessels during the first trimester of development. At that stage, the rubella virus has easy ac cess to the lens structures. Electron micro scopic studies of embryonic rabbit lenses have revealed a wealth of "free" RNA particles and endoplasmic reticulum.9 Possi ble interactions of viral DNA and RNA and lens nucleotides or competition for enzyme systems needed by both lens and virus may result in the formation of such cataracts. It is worthwhile to point out that cataracts were not found in 58% of cases of congenital rubella with thrombocytopenic purpura. 5 A high and persistent maternal and fetal viremia may be needed for cataract formation in congenital rubella. SUMMARY
Rubella virus was recovered from cata ractous lenses of two patients with congenital rubella syndromes. In one patient, rubella virus was found in the lens at the age of eight months. Virus was not recovered in the contralateral lens aspirated five weeks
235
later. In the second case, rubella virus was recovered from the lens but not from the aqueous humor of an eight and one-half month-old child with congenital rubella syn drome and associated thrombocytopenic pur pura. The rubella virus may persist in the crystalline lens as well as in other tissues following birth. 660 South Euclid Avenue (63110). ADDENDUM
Rubella virus has been identified in lens and/or aqueous humor of three additional patients with congenital rubella syndromes, aged five weeks to 10 and one-half months. The virus was also recovered from the lens of an infant with congenital cataracts without maternal history of rubella or rubella ex posure. Since this paper was accepted for publication Sir Norman Gregg has called our attention to similar viral isolations, using another technique,* from cataracts of three congenital rubella patients by R. R. Reid, A. M. Murphy, A. M. Gillespie, D. C. Dorman, M. A. Menser, R. Hertzberg and J. D. Harley in Australia. REFERENCES
1. Gregg, N. M.: Congenital cataracts follow ing German measles in the mother. Tr. Ophth. Soc. Australia, 3:35, 1941. 2. Krugman, S.: Rubella: New light on an old disease. J. Pediat. 67:159, 1965. 3. Parkman, P. D., Buescher, E. L., and Artenstein, M. S.: Recovery of rubella virus from Army recruits. Proc. Soc. Exper. Biol. Med. 111:225, 1962. 4. Sever, J. L., and Gilles, M.: Limited persis tence of virus in congenital rubella. Am. J. Dis. Child. 110:452, 1965. 5. Cooper, L. Z., Green, R. H., Krugman, S., Giles, J. P., and Mirick, G. S.: Neonatal throm bocytopenic purpura and other manifestations of rubella contracted in utero. Am. J. Dis. Child. 110:416, 1965. 6. Alford, C. A., Jr., Neva, F. A., and Weller, T. H.: Virologic and serologic studies on human products of conception after maternal rubella. New England J. Med. 211:1275, 1964. 7. Platkin, S. A., Dudgeon, J. A., and Ransay, A. M.: Laboratory studies on rubella and rubella syndrome. Brit. Med. J. 2:1296, 1963. 8. Cordes, F. C, and Barber, A. N.: Changes in lens of embryo after rubella. Arch. Ophth. 36:135,1946. 9. DuPree, A.: Unpublished observations. 10. Green, R. H., Balsamo, M. R., Giles, J. P., Krugman, S., and Mirick, G. S.: Studies on the natural history and prevention of rubella. Am. J. Dis. Child. 110:348, 1965. * Cytopathologic effect of rubella virus on the RKu rabbit kidney cell line.
JEAN FOX,
AND M O R T O N S M I T H ,
B.S.,
M.D.
St. Louis, Missouri Congenital cataracts in infants born fol lowing maternal rubella infection was re ported by Gregg in 1941. 1 Since then, the occurrence of rubella during the first tri mester of pregnancy has been associated with a series of congenital malformations (rubella syndrome) in addition to ocular le sions, such as cataracts, pigmentary retinopathy and glaucoma. 2 Rubella virus has been recovered, follow ing rubella infections, from army recruits 3 and children. 10 I n addition, the virus has been found in nasopharynx, urine and feces of patients with congenital rubella syn dromes. 5 ' 6 Rubella virus isolated from the cataractous lenses of infants affected by maternal rubella is reported here. MATERIAL AND METHODS A.
C A S E REPORTS
Case 1. R. R. P., an eight-month-old white boy, was delivered normally following a pregnan cy complicated by German measles in the third week. The pregnancy lasted only seven months. Birth weight was five pounds, 13 ounces. The child was described as having cataracts at birth. He was examined at the age of two months for a heart murmur. Other than otitis media, the child has not had any other illnesses. Physical examination revealed a healthy child in no distress. The child had horizontal nystag mus. White, lenticular opacities were seen bilateral ly. The apex beat was at the fifth intercostal space and left midclavical line. The heart rate was 120/minute. Systolic murmur grade III/VI was heard over the entire precordium, loudest along the left sternal border at the third and fourth intercostal space. No thrill was palpable. Findings for the rest of the physical examination were negative. The clinical impression was: postFrom the Department of Ophthalmology, Wash ington University Medical School, and the Virology Section of Central Diagnostic Laboratories, Barnes Hospital Group. This study was supported in part by research grant B-621 from the National Institute of Neurological Diseases and Blindness, National Institutes of Health, Bethesda, Maryland. 233
rubella syndrome with (1) bilateral congenital cataracts, and (2) a ventricular septal defect. Additional investigations included: hemogramhemoglobin was 10.4 gm, hematocrit 33%, white blood cells and differential were normal. The urinalysis was normal. Long-bone X-ray films showed multiple growth arrest lines; otherwise normal long bones. A cardiac series showed slight cardiomegaly with left atrial and right ventricular enlargement and pulmonary overcirculation, compatible with left-to-right shunt at the ventricular level. An aberrant right subclavian ar tery was also seen. The EKG revealed right ven tricular enlargement. A needle aspiration of the cataract of the right eye was followed five weeks later by a similar procedure in the left eye. The postoperative course was uneventful. Case 2. W. J., an eight and one-half month-old Negro boy, had a history of maternal rubella during the second month of pregnancy. The preg nancy was also complicated by "low-iron anemia" and a vaginal infection. The child was full term, and weighed three pounds, nine ounces at birth. The delivery was uncomplicated. Physical exami nation at birth showed a mildly icteric baby with echymosis and petechiae over the entire body. A systolic murmur grade III/IV was heard over the entire precordium. Liver and spleen were pal pable. At birth, laboratory work revealed: hemo globin 11.7 gm and thrombocytopenia (platelets 10,000). Hemoglobin dropped to 9.7 gm during the second day. Blood type B ( + ), mother B ( + ) and Coombs (—). VDRL in mother and child were negative. Blood cultures were negative. On day 2 the child was transfused with 30 cc of whole blood. Ophthalmologic examination re vealed bilateral cataracts. Bone-marrow morphol ogy on day 3 was compatible with thrombocyto penia. The patient also developed "myotonic jerk ing of extremities" and a lumbar puncture showed xanthochromic fluid. The "seizures" dis appeared during the hospitalization. A small left upper lobe pneumonia developed during the hospi talization but cleared rapidly with antibiotics. Clinical impressions were: (1) postrubella syn drome with thrombocytopenic purpura. When admit ted for needle aspiration of congenital cataract, the eye examination revealed congenital nearly mature cataracts, nystagmus, esotropia and microcornea. The cataractous lens of one eye was aspi rated. The postoperative course was uneventful. P>. L E N S AND TISSUE MATERIAL
T h e lens material was obtained by needle
234
AMERICAN JOURNAL OF OPHTHALMOLOGY
A U G U S T , 1966
TABLE 1 SUMMARY OF VIRAL STUDIES IN CONGENITAL RUBELLA SYNDROMES
Specimen
1. 2. 3. 4. 5. 6. 7.
R. R. P. Cataract O.D. Urine Aqueous humor Conjunctival swab Cataract O.S. Conjunctival tissue Throat swab
W.J.
1. Cataract 2. Aqueous humor
Days in Days in 2nd Passage Inhibition Passage Inhibition Inhibition when of Echo when of Echo of Echo 11 Challenged 11 Challenged 11 (repeat) with with Echo 11 Echo 11
Amount Inocu lated per Tube (ml)
Cell Line Inoculated
0.2 0.2 0.1 0.1 0.2 0.1 0.2
LLCMK 2 LLCMK 2 LLCMK 2 LLCMK 2 LLCMK 2 LLCMK 2 LLCMK 2
12 13 12 12 12 12 12
No No No No No No Partial
12 12 11 11 11 11 11
Complete No No No No No Partial
Complete No No No No No
0.2 0.2
AGMK AGMK
12 12
No No
11 12
Complete No
Complete No
aspiration. A fixation suture was placed in the superior rectus, aqueous humor was as pirated, the anterior chamber was entered with a Ziegler needle-knife and the lens in cised. Then a 24-gauge needle connected to a syringe with two cc of saline was intro duced into the anterior chamber and the lens contents aspirated by slow to-and-fro move ments. The material for viral studies con sisted of the contents of the first syringe ob tained after the lens incisions. Conjunctival and throat swab and other biopsied tissues were immersed in one ml sterile saline. The amounts inoculated per tube are recorded in Table 1. C. VIRUS ISOLATION TECHNIQUES
The indirect interference method for virus isolation originally described by Parkman and co-workers3 was used. Tissue cul tures of African green monkey kidney cells (AGMK*) or continuous rhesus monkey kidney (LLCMK 2 f ) were maintained in 0.25% calf Eagles (Hanks base) containing 300 units of penicillin, 300 y.g streptomycin and 25 \hg mycostatin. Specimens were in oculated in 0.2-ml amounts into four culture tubes each. Inoculation was carried out short* Obtained from Baltimore Biological Labora tories, Baltimore, Maryland. t Obtained from Eli Lilly.
—
ly after collection. After 11 to 13 days' in cubation, cultures were challenged with 1,000 to 10,000 tissue culture infective doses (TCID 5 0 ) of Echo 11 virus. If interference was not evident on the first passage of the test material, second passages were under taken before the specimen was considered negative. Observations for cytopathologic effects were made every second day. Rubella antibody neutralization tests with immune rabbit serum confirmed the identity of the virus. Virus titrations are reported in a separate paper. RESULTS
As shown in Table 1, the cataractous lens (specimen 1)) from the right eye of R. R. P. (age eight months) contained rubella virus. However, urine, the lens material, aqueous humor, conjunctival swab and tissue were negative for virus five weeks later (speci mens 2, 3, 4, 5 and 6.) A throat swab at that time contained rubella virus (specimen 7). Lens material obtained from W. J. (aged eight and one-half months) was positive for rubella virus while no virus was recovered from aqueous humor of the same eye. COMMENTS
The presence of the rubella virus in the cataractous lens of maternal rubella children is not a totally unexpected finding, as the
VOL. 62, NO. 2
RUBELLA VIRUS IN CATARACTOUS LENS
virus has been recovered from adrenal glands, bone, brain, kidney, intestine, liver, muscle, spleen, nasopharynx, urine and other tissues of embryos and children with congenital rubella.5'8 Persistence of the virus has been found at birth and for sever al months thereafter.6 Rubella neutralizing antibodies present at birth persist for years. 7 However, virus was not recovered from the nasopharynx, urine or serum of patients with congenital rubella syndromes at the ages of five to 22 years.4 The latter findings indicate a limited persistence of virus in congenital rubella. Of interest is the recov ery of the virus from the first but not the second cataractous lens in Case 1 (R. R. P.) aspirated five weeks after the first. The persistence of virus in the lens may be re lated to the period of time following intrauterine infection. The reasons for the formation of these congenital rubella cataracts are unknown. The pathology of embryonic rubella cata racts was studied by Cordes and Barber.8 The lens is surrounded by blood vessels during the first trimester of development. At that stage, the rubella virus has easy ac cess to the lens structures. Electron micro scopic studies of embryonic rabbit lenses have revealed a wealth of "free" RNA particles and endoplasmic reticulum.9 Possi ble interactions of viral DNA and RNA and lens nucleotides or competition for enzyme systems needed by both lens and virus may result in the formation of such cataracts. It is worthwhile to point out that cataracts were not found in 58% of cases of congenital rubella with thrombocytopenic purpura. 5 A high and persistent maternal and fetal viremia may be needed for cataract formation in congenital rubella. SUMMARY
Rubella virus was recovered from cata ractous lenses of two patients with congenital rubella syndromes. In one patient, rubella virus was found in the lens at the age of eight months. Virus was not recovered in the contralateral lens aspirated five weeks
235
later. In the second case, rubella virus was recovered from the lens but not from the aqueous humor of an eight and one-half month-old child with congenital rubella syn drome and associated thrombocytopenic pur pura. The rubella virus may persist in the crystalline lens as well as in other tissues following birth. 660 South Euclid Avenue (63110). ADDENDUM
Rubella virus has been identified in lens and/or aqueous humor of three additional patients with congenital rubella syndromes, aged five weeks to 10 and one-half months. The virus was also recovered from the lens of an infant with congenital cataracts without maternal history of rubella or rubella ex posure. Since this paper was accepted for publication Sir Norman Gregg has called our attention to similar viral isolations, using another technique,* from cataracts of three congenital rubella patients by R. R. Reid, A. M. Murphy, A. M. Gillespie, D. C. Dorman, M. A. Menser, R. Hertzberg and J. D. Harley in Australia. REFERENCES
1. Gregg, N. M.: Congenital cataracts follow ing German measles in the mother. Tr. Ophth. Soc. Australia, 3:35, 1941. 2. Krugman, S.: Rubella: New light on an old disease. J. Pediat. 67:159, 1965. 3. Parkman, P. D., Buescher, E. L., and Artenstein, M. S.: Recovery of rubella virus from Army recruits. Proc. Soc. Exper. Biol. Med. 111:225, 1962. 4. Sever, J. L., and Gilles, M.: Limited persis tence of virus in congenital rubella. Am. J. Dis. Child. 110:452, 1965. 5. Cooper, L. Z., Green, R. H., Krugman, S., Giles, J. P., and Mirick, G. S.: Neonatal throm bocytopenic purpura and other manifestations of rubella contracted in utero. Am. J. Dis. Child. 110:416, 1965. 6. Alford, C. A., Jr., Neva, F. A., and Weller, T. H.: Virologic and serologic studies on human products of conception after maternal rubella. New England J. Med. 211:1275, 1964. 7. Platkin, S. A., Dudgeon, J. A., and Ransay, A. M.: Laboratory studies on rubella and rubella syndrome. Brit. Med. J. 2:1296, 1963. 8. Cordes, F. C, and Barber, A. N.: Changes in lens of embryo after rubella. Arch. Ophth. 36:135,1946. 9. DuPree, A.: Unpublished observations. 10. Green, R. H., Balsamo, M. R., Giles, J. P., Krugman, S., and Mirick, G. S.: Studies on the natural history and prevention of rubella. Am. J. Dis. Child. 110:348, 1965. * Cytopathologic effect of rubella virus on the RKu rabbit kidney cell line.