BACTERIOLOGICAL REVIEWSVol. 28, No. 4, p. 444-451 December, 1964Copyright © 1964 American Society for Microbiology

Printed in U.S.A.

EMERGING PERSPECTIVE OF RUBELLA1

FRANKLIN A. NEVA, CHARLES A. ALFORD, JR., AND THOMAS H. WELLERDepartment of Tropical Public Health, Harvard School of Public Health, Boston, Massachusetts

INTRODUCTION.................................................................................. 444ISOLATION AND PROPAGATION OF THE VIRUS............................................ 444CHARACTERISTICS OF RUBELLA VIRUS............................................ 445SEROLOGICAL STUDIES WITH RUBELLA VIRUS............................................ 447CLINICAL AND EPIDEMIOLOGICAL ASPECTS OF RUBELLA.......................................... 447MATERNAL RUBELLA AND CONGENITAL FETAL DAMAGE ......................................... 449LITERATURE CITED............................................ 449

INTRODUCTION

"There is probably no class of diseases in whichinaccurate diagnosis is more frequently made thanin those of the skin; nor is error more likely-thanin that subdivision of cutaneous complaints,which have been denominated Exanthemata, orRashes" (18).The above quotation, from Maton's paper

describing rubella as a clinical entity and de-livered before the Royal College of Physicians in1814, anticipated remarkably some of theproblems of the subsequent century and a halfwith respect to clinical differentiation of exan-them diseases. For many years, diagnosticdifficulties with respect to the various exanthe-mata served as little more than embarrassmentor intellectual discomfort to the physician. AfterGregg's discovery of the relationship betweenmaternal rubella and congenital defects (6), how-ever, the practical implications of rubella stimu-lated an increasing effort towards specificidentification of the viral exanthemata. Un-doubtedly, the sophistication of recent epi-demiological studies has contributed to a morerealistic estimate of the risks of maternal rubella.However, such information has raised as manyquestions as it has answered. As with every in-fectious disease, the availability of specificdiagnostic tests, an understanding of the mech-anism of pathogenesis, and the development ofprophylactic measures all depend upon specificcharacterization of the etiological agent.Although there has long been experimental1 A contribution to the Symposium on "Current

Progress in Virus Diseases" presented as part ofthe program for the Centennial of the Boston CityHospital, 1 June 1964, with Maxwell Finland serv-ing as Consultant Editor, and John H. Dingle andHerbert R. Morgan as moderators.

evidence indicating a viral etiology of rubella(8, 7, 2, 11), characterization of the causativeagent was accomplished only recently. Since thedual report of isolation of rubella virus in 1962 byParkman, Buescher, and Artenstein (24), andindependently by Weller and Neva (36), con-firmatory findings have quickly followed (35, 30,33, 19). Even though these data are rapidly beingextended by current studies, it may be propitious,at this time, to review some of the recently avail-able information on rubella. From the ground-work of clinical, laboratory, and epidemiologicalinvestigations, now based upon specific labora-tory methods, will the true perspective of rubellafinally emerge.

ISOLATION AND PROPAGATION OF THEVIRUS

The methods in current use for isolation andpropagation of rubella virus derive from the twobasically different techniques originally employedfor detecting the presence of the virus. One wasbased upon direct recognition of cytopathiceffects (CPE) produced in cultures of primaryhuman amnion (PHA) cells (36); the otherprocedure, also called the indirect or exclusiontechnique, utilized the finding that rubella-infected cultures of grivet monkey kidney cells(GMK) were resistant to challenge with ECHO-11 virus (24). Subsequently, modifications in bothtechniques have been introduced by otherworkers. McCarthy et al. found a special line ofrabbit kidney cells (RK13) to exhibit rubella-induced CPE (19). The interference procedure hasbeen found applicable to kidney-cell cultures fromother species of monkeys and cell lines thereof(35, 30), and other enteroviruses, such as Cox-sackie A-9, can be used as challenge virus (30, 33,19).

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lp) to the present time, one or another varia-tion of the interference method has been mostwidely used for isolation of rubella virus. Despitenonspecificity of the interference procedure,reports have not indicated this to be a seriousproblem in practice, but more extensive experi-ence is needed. The relatively short period of 10to 14 days before tentative results are obtainedconstitutes a distinct advantage of the exclusiontechnique. It is, of course, mandatory that theidentity of all presumptive rubella isolates ob-tained by the interference procedure be con-firmed by acceptable serological tests.The use of PHA cultures for isolation and

growth of rubella virus offers the advantage thatpresence of virus is indicated directly by thedistinctive cytopathology observable in bothfresh and stained preparations. Another advan-tage is that the presence of rubella virus in PHAcultures may also be detected by the interferencephenomenon upon addition of Sindbis virus (36).Thus, PHA cultures may be used in a combineddirect and indirect system for detection ofrubella virus, permitting parallel studies of thephenomena (22). The limitations of this methodinclude the necessity for cell cultures of goodquality that contain confluent monolayers of cells,and the requirement that an experienced observerbe available for the detection of the involved cells,which are not always abundant in infectedcultures. Use of chemically defined, instead ofbovine embryonic fluid, medium may result indiminished or suppressed CPE (36).

Reports, now available only in abstract form,covering studies by Giles et al. at WillowbrookState School and by Buescher et al. (4) at theWalter Reed Army Institute of Research indicatethat rubella virus is present in the blood or throatof patients as early as 6 or 7 days before ap-pearance of the rash, and disappears from theblood shortly after the exanthem, but maylersist in the pharynx as long as 7 days after on-set of rash. The virus has also been recoveredfrom the feces and may be present in the urine atthe time of the exanthem. However, virus is mosteasilv recovered from the throat, and this sourcenow appears preferable for the attempted isola-tion of the responsible agent.No published data are available assessing the

comparative effectiveness of different proceduresfor isolation of rubella virus from clinical speci-mens. Several methods, including the use of RK13cell line cultures, were successfully employed in

one study (19), but not in a comparative fashion.Preliminary results of Burnett and Alford in thislaboratory suggest that interference in GMK ismore rapid than either the indirect or directprocedures with PHA cultures, and may be moresensitive for the detection of minimal amounts ofrubella virus. Critical studies to select sensitivemethods for the detection of both wild and invitro-propagated strains of rubella virus willassume increasing importance in future workrelated to development of vaccines.

In evaluating the relative merits of differentprocedures for isolation and propagation ofrubella virus, it is important to recall that theindications for the use of a particular virus-hostcell system may be variable. Thus, the optimalsystem for isolation of virus from clinical speci-mens may not be the best method for the assayof neutralizing antibodies, production of antigen,study of viral kinetics, or demonstration of cyto-pathology.

CHARACTERISTICS OF RUBELLA VIRUSOn the basis of available centrifugation, filtra-

tion, and electron microscopic studies, it wouldappear that infectious rubella virus particles arerelatively large, in the range of 100 to 300 mu(24, 36, 23, 25), although one report indicates thatsome infectious particles may be less than 100 m/uin size (19). In the presence of 2% serum, theinactivation rate of rubella at 37 C was found tobe 0.3 to 0.4 log ID50 per hour (25), but in othermedia with higher protein content virus in-fectivity was not completely destroyed after 1hr at 56 C (36). Some loss in infectivity occurs onstorage for several weeks at 4 and -20 C (36),but the virus is stable for many months at -60 C(25). Rubella virus is capable of replication in thepresence of 5-iodo-2'-deoxyuridine, and infec-tious virus is destroyed on exposure to ether, chlo-roform, and sodium deoxycholate.The most distinctive cellular alterations at-

tributable to rubella virus are those occurring ininfected PHA cultures (36). At any particulartime, relatively few of the cells in an infectedculture exhibit changes in either fresh or stainedpreparations. Affected cells appear enlarged orrounded in the fresh culture, and will oftenexhibit amoeboid pseudopods whose cytoplasmiccontents are transparent and may show thepresence of round inclusions. When stained, theinvolved cells show various abnormalities, in-cluding disappearance of the nuclear membrane,

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prominent clumping of nuclear chromatin, andthe presence of round or irregular cytoplasmic,eosinophilic inclusions. The progression ofrubella CPE in PHA cultures is slow, but thecytopathic process can be enhanced by continuedin vitro passage of the virus. After 15 to 20 serialtransfers, we generally find that rubella strains

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produce 50'S or more destruction of the cell sheetwithin 3 weeks after inoculation (Fig. 1).

Rubella virus can also multiply in cultures ofbovine embryonic tissues (22), as well as in thosederived from various human and simian sources,and the special cell line of rabbit kidney alreadymentioned. With most systems, maximal yields of

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FIG. 1. Cytopathic effect of rubella virus in human amnion cultures. (A) Appearance of normal amnioncell culture as viewed microscopically under low magnification (33X). (B) Rubella-infected culture withestimated 20% destruction of cells on 14th day after inoculation (55th culture passage, Bell strain; 33X).(C) Rubella infected culture with 80%l; cell destruction on 28th day after inoculation (53rd culture passage,

Bell strain; 3SX). (D) Single affected cell with adjacent uninvolved cells on 10th day after inoculation (54thculture passage, Bell strain; 132X). (E) Scattered infected cells showing amoeboid distortion on 10th dayafter inoculation (54th culture passage, Bell strain; 132X). (F) Infected cell with large eosinophilic cyto-plasmic inclusion and basophilic aggregation of nuclear chromatin, as well as portions of two normal cells(4th culture passage, 35th day after inoculation, RW strain; hematoxylin and eosin stain, 3,500X). Re-produced by permission with addenda from F. A. Neva and T. H. Weller (21).

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infectious virus are relatively low, usually in therange of 104 to 105 IDw per ml. As yet, no comple-ment-fixing, hemagglutinating, or hemadsorbingactivity has been demonstrable in rubella-infected cultures. Representative growth curvesof rubella virus as obtained by us in human andbovine cell cultures are depicted in Fig. 2.The exact nature of the interference phe-

nomenon resulting from growth of rubella virusunder different in vitro conditions is not yet clear.Parkman et al. reported evidence that in GMKcultures the interference between rubella andECHO-11 virus is not mediated by interferon(25); others (28) have suggested that interferon

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does play a role in this system. The interferingactivity for Sindbis virus in the fluids of rubella-infected PHA cultures can readily be dissociatedfrom infectious rubella virus, and it also exhibitsother properties compatible with interferon (22).

SEROLOGICAL STUDIES WITH RUBELLA VIRUS

Serological procedures for measurement of anti-body to German measles virus, and for identifica-tion of the agent, are presently limited to neu-tralization tests. These are applications of thetwo basic methods for isolation of virus, namely,inhibition of rubella CPE, or inhibition of therubella interference phenomenon. Technicalproblems associated with both types of tests arerelated to factors such as (i) absence of rapid andobvious rubella CPE, (ii) incomplete neutraliza-tion of virus by antibody, (iii) critical adjust-ment of rubella virus dose, and (iv) difficulties instandardization of the interference neutraliza-

tion procedure. These problems have beendiscussed in detail elsewhere (22, 26).Although simplified procedures are needed for

routine diagnostic purposes, the methods incurrent use for assay of neutralizing antibody torubella have yielded considerable information.Patients with German measles exhibit increasesin serum-neutralizing antibody after their illness.The titers of antibody found in individuals whohave presumably experienced rubella in the pastsuggest that substantial levels of antibody aremaintained for prolonged periods. The sameinference can be drawn from the fact thatneutralizing antibody is readily demonstrable bythe interference test in specimens of pooled 'y-globulin (31, 26, 22).The first laboratory evidence indicating a

considerable degree of antigenic homogeneity ofrubella virus strains was the demonstration ofantibody responses to recent virus isolates inpaired serum samples collected from rubella pa-tients during previous years (36, 21). Similarconclusions regarding the absence of majorantigenic differences among rubella strains havebeen obtained from cross-neutralization tests withantisera prepared in rabbits (25).

CLINICAL AND EPIDEMIOLOGICAL ASPECTS OFRUBELLA

Since the diagnosis of rubella hitherto has beenbased upon clinical and epidemiological criteria,the advent of laboratory methods for specificdiagnosis of rubella infection may be expected todefine the clinical spectrum of the disease. Thereis little, however, at present that can be added toclinical rubella that was not covered in theexcellent review of the subject by Wesselhoeft in1947 (38). Some features of the disease, such asthe transitory polyarthritis, have received in-creased attention recently. The first patient fromwhom we isolated rubella virus (36) would hardlyqualify as a typical example of rubella; he hadfever and severe joint symptoms lasting a week,associated with an atypical rash most prominenton the palms of the hands and soles of the feet.Thrombocytopenia accompanying rubella may bedeserving of special comment; platelets are oftendecreased even though bleeding or purpura is notclinically manifest (1, 34), and a number of casesof neonatal purpura following maternal rubellahave been reported (3). A point of clinical interestin two adult males with laboratory verified

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rubella and thrombocytopenia encountered by uswas that the appearance of purpura on the lowerlegs 3 or 4 days after onset of rash was interpretedas recurrence of exanthem by the patients. Themore unusual complications, such as relapses ofthe disease and rubella encephalitis, can now beinvestigated in the laboratory; search for virusin the spinal fluid in the latter instance, forexample, would be of interest.

In contrast to measles, the clinical diagnosis ofrubella is often impossible due to the lack ofcharacteristic signs or symptoms, and variation inextent and nature of the rubella rash. It isunderstandable that many of the entities on theexpanding list of enterovirus exanthems (13)may clinically be labeled as rubella. This was ap-parent in Massachusetts, during an epidemic of"Boston Exanthem" or ECHO-16 disease in1951 (20), when a record number of "rubella"cases were notified for the month of August, dur-ing an otherwise low-incidence rubella year.Additional evidence of misdiagnosis of rubella canbe derived from an analysis of annually andmonthly reported rubella in Massachusetts from1940 to the present time. (We are indebted toNicholas J. Fiumara, Director of Division ofCommunicable Diseases, Massachusetts Depart-ment of Public Health, for making the monthlyand annual tabulations available to us. Therecent experience with rubella in Massachusetts issummarized in Fig. 3; by the end of September,the reported number of cases for 1964 was inexcess of 36,000.) Customarily, as noted by In-galls et al. (9), there is a seasonal peak of rubelladuring the months of March through June in thenorthern hemisphere. If the monthly distributionof annual cases in Massachusetts during years ofhigh incidence is compared with the plot for yearsof low rubella incidence, an interesting divergencein patterns results (Fig. 4). A higher proportion ofthe total cases of "rubella" was reported fromAugust to January during the 8 years of lowestannual incidence than during the correspondingperiod for the 6 years of highest incidence. Al-though one cannot exclude the possibility thatrubella occurring during the summer and fall maybe an inherent epidemiological characteristic ofthe disease during low periods of endemicity,such a distribution of cases would more likely beexpected for years of high rubella incidence.These data incriminate enterovirus exanthemsand other viral exanthems as a source of mis-

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diagnosed rubella; they may also help explainSiegel and Greenberg's finding that the risk ofcongenital rubella was very low during non-epidemic years (32).

Interesting results on the occurrence of sub-clinical rubella, heretofore an epidemiologicalhiatus in our understanding of the disease, have

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recently come from laboratory-supported studiesof rubella in military recruits by Buescher andassociates (4). These workers found that 10 to15% of young adult males from the eastern sea-board entering military service were susceptibleto rubella. Then, by carefully following events intwo training groups, the ratio of subclinicalinfection to overt disease was estimated to be6.5:1. Under these circumstances, virtually allsusceptibles became infected. However, theepidemiological pattern of rubella in a civiliansetting remains to be determined, for rubella inmilitary recruits may differ from that occurring incivilian populations in a manner somewhatanalogous to adenovirus disease. If studies incivilian populations yield results comparable tothe situation in military recruits, it would haveimportant implications regarding the problem ofmaternal rubella.

MATERNAL RUBELLA AND CONGENITALFETAL DAMAGE

Despite the relative abundance of informationconcerning the risk of fetal damage followingmaternal rubella, re-evaluation of the problem isindicated under circumstances whereby thediagnosis of rubella can be verified by laboratorystudy. Carefully designed and well-controlledprospective investigations (17, 16, and as re-viewed in 5) have yielded variable estimates ofrisk of major congenital defects after maternalrubella during the first trimester of pregnancy.Campbell (5) places the overall risk of abortionand of malformation at 30 to 70% for exposuresoccurring during the first 4 weeks of pregnancy,declining to 10 to 25% during the fourth 4 weeksof gestation. Such estimates are complicated bythe fact that long term follow-up is necessary, ascertain defects require the passage of severalyears before becoming clinically apparent (15,10). Some experiences of high risk maternalrubella have been attributed to unusually severeepidemics (14, 12). In view of the seasonalpattern and possible misdiagnosis of rubella,referred to earlier, the data from individualepidemics may be more accurate than those fromlarger, long-term investigations. The possibilitythat the virulence of rubella virus for the fetusvaries from year to year cannot be excluded, butit seems more likely that errors in diagnosis haveconstituted the major difficulty in accurately

assessing the risk of congenital defects subse-quent to maternal rubella.

Recent laboratory studies have provided anadditional dimension, one with practical applica-tions, to the problem of congenital rubella in-fection. Two reports (27, 37) indicate that a stateof immunological tolerance is not induced in thefetus exposed to rubella virus, and that elevatedlevels of neutralizing antibody can persist foryears in the child with congenital rubella syn-drome (37). This finding may be very helpful inthe retrospective diagnosis of congenital damageconsequent to maternal rubella. Recovery ofrubella virus from a human fetus with presumedcongenital infection has also been accomplished(29). In our laboratory, rubella virus has beenisolated from 24 fetal or placental specimens ob-tained from 51 women within 49 days aftermaternal infection. In three instances, further-more, the virus has been demonstrated post-natally in the pharynx or urine of infants showingstigmata of congenital rubella syndrome. Furtherdefinitive investigations of the phenomenon notedby Gregg nearly 25 years ago (6), and which havesince directed most of the interest in rubella, maynow be expected.

ACKNOWLEDGMENTS

This investigation was supported in part byPublic Health Service research grant AI-01023from the National Institute of Allergy and Infec-tious Diseases, and by grants from Parke, Davis& Co. and United Cerebral Palsy Research andEducational Foundation, Inc.

LITERATURE CITED

1. ACKROYD, J. F. 1949. Three cases of thrombo-cytopenic purpura occurring after rubella.Quart. J. Med. N.S. 18:299-318.

2. ANDERSON, S. G. 1949. Experimental rubellain human volunteers. J. Immunol. 62:29-40.

3. BERGE, T., F. BRUNNHAGE, AND L. R. NILS-SON, 1963. Congenital hypoplastic thrombo-cytopenia in rubella embryopathy. ActaPediat. 52:349-352.

4. BUESCHER, E. L., P. D. PARKMAN, H. L.WEISBERGER, M. S. ARTENSTEIN, F. C.CADIGAN, AND R. E. NITZ. 1964. Studies ofrubella. Epidemiology in military recruits.J. Immunol., in press.

5. CAMPBELL, M. 1961. Place of maternal rubellain the aetiology of congenital heart disease.Brit. Med. J. 1:691-696.

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6. GREGG, N. M. 1941. Congenital cataract fol-lowing German measles in the mother.Trans. Ophthalmol. Soc. Australia (BMA)3:35-46.

7. HABEL, K. 1942. Transmission of rubella toMacacus mulatta monkeys. Public HealthRept. 57:1126-1139.

8. HIRO, Y., AND S. TASAKA. 1938. Die Rbtelnsind eine Viruskrankheit. Monatsschr.Kinderheilk. 76:328-332.

9. INGALLS, T. H., F. L. BABBOTT, JR., K. W.HAMPSON, AND J. E. GORDON. 1960. Rubella:its epidemiology and tetralogy. Am. J.Med. Sci. 239:363-383.

10. JACKSON, A. D. M., AND L. FISCH. 1958. Deaf-ness following maternal rubella. Results ofa prospective investigation. Lancet 2:1241-1244.

11. KRUGMAN, S., R. WARD, K. G. JACOBS, ANDM. LAZAR. 1953. Studies on rubella im-munization. 1. Demonstration of rubellawithout rash. J. Am. Med. Assoc. 151:285-288.

12. LAMY, M., AND M. E. SEROR. 1956. Resultatsd'une enqu6te sur les embryopathies d'ori-gine rubeolique. Bull. Acad. Natl. Med.(Paris). 140:196-203.

13. LERNER, A. M., J. 0. KLEIN, J. D. CHERRY,AND M. FINLAND. 1963. Medical progress.New viral exanthems. New Engl. J. Med.269:678-685, 736-740.

14. LIGGINS, G. C., AND L. I. PHILLIPS. 1963.Rubella embryopathy. An interim reporton a New Zealand epidemic. Brit. Med. J.1:711-713.

15. LOCK, F. R., H. B. GATLING, AND H. B. WELLS.1961. Difficulties in the diagnosis of congeni-tal abnormalities. Experience in a study ofthe effect of rubella in pregnancy. J. Am.Med. Assoc. 178:711-714.

16. LUNDSTROM, R. 1962. Rubella during preg-nancy. A follow-up study of children bornafter an epidemic of rubella in Sweden, 1951,with additional investigations on prophylax-sis and treatment of maternal rubella. ActaPediat. Suppl. 133:1-110.

17. MANSON, M. M., W. P. D. LOGAN, AND R. M.Loy. 1960. Rubella and other virus infec-tions during pregnancy. Reports on PublicHealth and Medical Subjects. No. 101,Ministry of Health, London, Her Majesty'sStationery Office. 1-101.

18. MATON, W. G. 1815. Some account of a rash,liable to be mistaken for scarlatina. Med.Trans. Roy. Coll. Phys. 5:149-165.

19. MCCARTHY, K., C. H. TAYLOR-RoBINSON,AND S. E. PILLINGER. 1963. Isolation of ru-

bella virus from cases in Britain. Lancet2:593-598.

20. NEVA, F. A., R. F. FEEMSTER, AND I. J. GOR-BACH. 1954. Clinical and epidemiologicalfeatures of an unusual epidemic exanthem.J. Am. Med. Assoc. 155:544-548.

21. NEVA, F. A., AND T. H. WELLER. 1964. Ad-vances in our knowledge of rubella, p. 90-95. In Industry and tropical health. Indus-trial Council for Tropical Health, HarvardSchool of Public Health, Boston.

22. NEVA, F. A., AND T. H. WELLER. 1964. Rubellainterferon and factors influencing the in-direct neutralization test for rubella anti-body. J. Immunol., in press.

23. NORRBY, E., R. MAGNUSSON, B. FRIDING, ANDS. GARD. A note on the morphology of ru-bella virus. Arch. Ges. Virusforsch. 13:421-424.

24. PARKMAN, P. D., E. L. BUESCHER, AND M. S.ARTENSTEIN. 1962. Recovery of rubella virusfrom army recruits. Proc. Soc. Exptl. Biol.Med. 111:225-230.

25. PARKMAN, P. D., E. L. BUESCHER, M. S.ARTENSTEIN, J. M. MCCOWN, AND F. K.MUNDON. 1964. Studies of rubella. I. Proper-ties of the virus. J. Immunol., in press.

26. PARKMAN, P. D., J. M. MCCOWN, F. K. MUR-DON, A. DRUZD, AND E. L. BUESCHER. 1964.Studies of rubella. II. Neutralization of thevirus by immune sera. J. Immunol., inpress.

27. PLOTKIN, S. A., J. A. DUDGEON, AND A. M.RAMSAY. 1963. Laboratory studies on rubellaand the rubella syndrome. Brit. Med. J.2:1296-1299.

28. ROzEE, K. R., K. F. GIVAN, F. W. DOANE,AND A. J. RHODES. 1963. A plaque methodfor rubella virus assay. Can. Med. Assoc.J. 89:314-315.

29. SELZER, G 1963. Virus isolation, inclusion-bodies, and chromosomes in a rubella in-fected human embryo. Lancet 2:336-337.

30. SEVER, J. L., G. M. SCHIFF, AND R. G. TRAUB.1962. Rubella virus. J. Am. Med. Assoc.182:663-671.

31. SCHIFF, G. M., J. L. SEVER, AND R. J. HUEB-NER. 1963. Rubella virus: neutralizing anti-body in commercial gamma globulin. Science142:58-60.

32. SIEGEL, M., AND M. GREENBERG. 1960. Fetaldeath, malformation, and prematurity aftermaternal rubella. New Engl. J. Med. 262:389-393.

33. SIGURDARDOTTIR, B., K. F. GIVAN, K. R. Ro-ZEE, AND A. J. RHODES. 1963. Association of

virus with cases of rubella studied in

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Toronto; propagation of the agent andtransmission to monkeys. Can Med. Assoc.J. 88:128-132.

34. STEEN, E., AND K. H. TORP. 1956. Encephalitisand thrombocytopenic purpura afterrubella. Arch. Disease Childhood 31:470-473.

35. VERONELLI, J. A., H. F. MAASSAB, AND A. V.HENNESSY. Isolation in tissue culture of an

interfering agent from patients with rubella.Proc. Soc. Exptl. Biol. Med. 111:472-476.

36. WELLER, T. H., AND F. A. NEVA. 1962. Propa-

/TIVE OF RUBELLA 451

gation in tissue culture of cytopathic agentsfrom patients with rubella-like illness. Proc.Soc. Exptl. Biol. Med. 111:215-225.

37. WELLER, T. H., C. A. ALFORD, JR., AND F. A.NEVA. 1964. Retrospective diagnosis byserologic means of congenitally acquiredrubella infection. New Engl. J. Med. 270:1039-1041.

38. WESSELHOEFT, C. 1947. Rubella (Germanmeasles). New Engl. J. Med. 236:943-950,978-988.

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