Yasuko MATSUNAGA, Toshihiko MATSUKURA1, Shudo YAMAZAKI, Motoyasu SUGASE2 and Rikuichi IZUMI

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1 Japan. J. Med. Sci. Biol., 40, ,1987. Short Communication HYDROPS FETALIS CAUSED BY INTRAUTERINE HUMAN PARVOVIRUS INFECTION Yasuko MATSUNAGA, Toshihiko MATSUKURA1, Shudo YAMAZAKI, Motoyasu SUGASE2 and Rikuichi IZUMI Central Virus Diagnostic Laboratory and (Department of Enteroviruses, National Institute of Health, 4-7-1, Gakuen, Musashimurayama-shi, Tokyo , 2Nagano Red Cross Hospital Wakasato, Nagano 380, and 3Department of Obsterics and Gynecology, Faculty o f Medicine, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama930-1 (Received January 12, Accepted March 2, 1988) SUMMARY: During a large outbreak of erythema infectiosum in 1987 in Toyama prefecture, Japan, a 32-year-old woman acquired a mild rash on her arms and legs at 18 weeks of gestation. At 26 weeks and 4 days of gestation, the fetus died by hydrops fetalis and pregnancy was terminated. Histological studies of the fetus revealed degeneration of erythroblastic cells in the liver and bone marrow. Extensive extramedullary hematopoiesis and hemosiderin deposits were observed in the liver. Antibody response to human parvovirus B19 virus was demonstrated in maternal sera by ELISA. Furthermore, dot hybridization with the molecularly cloned DNA probe revealed the presence of human parvovirus DNA sequence in the fetal liver, spleen, lung, kidney and placenta. This report describes the first case in Japan of hydrops fetalis caused by human parvovirus B19 infection. 165

2 Human parvovirus (B19), first found in sera of healthy blood donors by Cossart et al. (1), is now recognized as an agent causing aplastic crisis in inherited hemolytic anemia like sickle cell disease (2), hereditary spherocytosis (3) and Thalassaemia (4), and also causing erythema infectiosum (5) and arthropathy (6). Among them erythema infectiosum (fifth disease) is a mild, acute exanthematous disease which commonly affects children. When a pregnant woman is contracted, however, her fetus may be affected more severely. There have been several reports on intrauterine human parvovirus infection resulting in spontaneous abortion or fetal death of hydrops fetalis (7-10). Since we had a large outbreak of erythema infectiosum in Japan in involving approximately 3% of the population under 15 years of age, we have tried to obtain the evidence showing that hydrops fetalis was indeed induced by intrauterine parvovirus infection. Here we describe the first case in Japan confirmed to have been caused by human parvovirus B19. A 32-year-old pregnant woman (multiparous, blood group B, and Rh positive) acquired a mild rash on her arms and legs at 18 weeks of gestation (March, 1987). At about the same time, her five-year-old daughter contracted erythema infectiosum. At 26th week of gestation, ultrasonic scanning showed features of hydrops fetalis. Fetal death was observed four days later and pregnancy was terminated (May 5th, 1987). A 1,372-g female fetus with generalized edema was delivered. Ascites (150 ml) and bilateral pleural effusion (left 4 ml and right 5 ml) were found. The placenta and fetal liver weighed 635 g and 59 g, respectively. There was no other anomalies in the fetus. Histological studies revealed degeneration of erythro-blastic cells (ballooned nucleus, peripheral chromatin condensation and central eosinophilic changes) in the liver and bone marrow. Extensive extramedullary hematopoiesis and hemosiderin deposits were demonstrated in the liver. There was no serological evidence of recent infection with Toxoplasma, rubella virus, cytomegalovirus or herpes simplex virus. The indirect coombs test was negative. Maternal sera were tested for parvovirus antibodies by ELISA. Specific IgG antibody was assayed by modified sandwich ELISA using 96-well vinyl assay plates (foster, MA, USA) coated with anti B19 monoclonal antibody (VRL/B19/11) supplied by Dr. B. J. Cohen, Central Public Health Laboratory, London (11). The B19 antigen partially purified by pelleting from the Nakatani (B19) antigen (12)-positive plasma by ultracentrifugation (100,000 ~g, 16 hr), then serum test samples diluted to 1:200 or more, and finally peroxidaseconjugated, goat anti-human IgG (ć-chain specific) (Tago Inc., Burlingame, CA, 166

3 Table I. Human parvovirus antibody titers in maternal sera *Reciprocal of the highest serum dilution giving positive r eaction in ELISA. USA) were added to the plates in sequence. The substrate was o- phenylenediamine (0.4 mg/ml) and hydrogen peroxide (0.015%) in citrate phosphate buffer. IgM antibody was assayed by capture ELISA modified from the method of Anderson et al (13). Serum obtained four days after delivery contained both IgM and IgG antibodies, but one month later, the IgM antibody diminished (Table I). The results showed that she had recently been infected with human parvovirus. Then, we tried to detect viral DNA sequence in fetal tissues by dot hybridization with the molecularly colned B19 virus DNA as a probe (14). Formalin fixed tissues were rinsed with PBS and treated overnight with proteinase K (Sigma, St. Louis, USA) at 37 C, then extracted with phenol. DNA was precipitated from aqueous phase with ethanol at -80 C. A human parvovirus B19-cloned DNA in the vector pgem-1 was supplied by Dr. J. P. Clewley, Central Public Health Laboratory, London, and propagated in E. coli HB101 in the presence of ampicillin. A 5.2-kb B19 DNA was excised from the plasmid DNA by EcoRI digestion and recovered by preparative gel electrophoresis as mentioned by Clewley (personal communication) and labelled with 32P by the nick translation method. Dot hybridization was performed according to the method of Shimizu et al (15). Presence of human parvovirus DNA sequence was demonstrated in the fetal liver, spleen, lung, and kidney (Fig. 1). A small amount of viral DNA was detected also in the placenta after prolonged exposure of the autoradiogram (data not shown). These results show that the fetus had been infected with human parvovirus, resulting in hydrops fetalis. The risk of occurrence of hydrops fetalis after maternal human parvovirus infection has been reported mostly in the United Kingdom. Here described is the 167

4 Fig. 1. Detections of the B19 sequence in fetal tissues. Approximately 0.5 Đg aliquots of DNA extracted from fetal tissues and denatured were spotted on nitrocellulose membranes and hybridized with the 32Plabelled B19 DNA probe, and autoradiographed. A: tissues from the affected fetus, B: Tissues from normal fetus. Liver (1,6), spleen (2,7), kidney (3,8), placenta (4,9), lung (5,10). C: Positive controls, B19 DNA 10 pg (11), 2 pg (12), 1 pg (13). first case in Japan proved virologically and serologically. As one of the potential agents causing fetal death and spontaneous abortion, human parvovirus should be taken into consideration, especially when erythema infectiosum is prevalent. The dot hybridization technique using molecularly cloned DNA as a probe, detecting 1-2 pg of viral DNA (Fig. 1), was found to be a useful method to demonstrate human parvovirus in tissues. 168

5 ACKNOWLEDGEMENTS We thank Dr. J. P. Clewley and Dr. B. J. Cohen for supplying the B19 DNA clone and monoclonal antibody. We also thank Dr. K. Fukada, Fukuoka Red Cross Blood Center for providing the Nakatani antigen (B19)-positive plasma. The authors are grateful to Dr. Y. Moritsugu, Department of Enteroviruses, National Institute of Health, Japan, for his valuable advice in preparing the ELISA assay system. REFERENCES 1. Cossart, Y. E., Field, A. M., Cant, B, and Widows, D. (1975): Lancet, i, Pattison, J. R., Jones, S. E., Hodgson, J., Davis, L. R., White, J. M., Stroud, C. E, and Murtaza, L. (1981): Lancet, i, Kelleher, J. F., Luban, N. L. C., Mortimer, P. P. and Kamimura, T. (1983): J. Pediatr.,102, Rao, K. R. P., Patel, A. R., Anderson, M. J., Hodgson, J., Jones, S. E. and Pattison, J. R. (1983): Ann. Int. Med., 98, Anderson, M. J., Jones, S. E., Fisher-Hoch, S. P., Lewis, S., Hall, S. M., Bartlett, C. L. R., Cohen, B. J., Mortimer, P. P. and Pereira, M. S. (1983): Lancet, i, White, D. G., Woolf, A. D., Mortimer, P. P., Cohen, B. J., Blake, D. R. and Bacon, B. A. (1985): Lancet, i, Brown, T., Anand, A., Richite, L. D., Clewley, J. P. and Reid, T. M. S. (1984): Lancet, ii, Knott, P. D., Welply, G. A. C and Anderson, M. J. (1984): Brit. Med. J., 289, Bond, P. R., Caul, E. O., Usher, J., Cohen, B. J., Clewley, J. P. and Field, A. M. (1986): Lancet, i, Anand, A., Gray, E. S., Brown, T., Clewley, J. P. and Cohen, B. J. (1987): New Engl. J. Med., 316, Cohen, B. J., Mortimer, P. P, and Pereira, M. S. (1983): J. Hyg. Camb., 91, Okochi, K., Mori, R., Miyazaki, M., Cohen, B. J. and Mortimer, P. P. (1984): Lancet, i, Anderson, L. J., Tsou, C., Parker, R. A., Chorba, T. L., Wulff, H., Tattersall, P. and Mortimer, P. P. (1986): J. Clin. Microbiol., 24, Clewley, J. P. (1985): J. Med. Virol., 15, Shimizu, Y., Ida, S., Matsukura, T. and Yuasa, T. (1984): Microbiol. Immunol., 28,