JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1997, p. 486–488 0095-1137/97/$04.0010 Copyright q 1997, American Society for Microbiology
Vol. 35, No. 2
Epidemiology of Group C Rotavirus Infection in Western New York Women of Childbearing Age MARIE RIEPENHOFF-TALTY,1,2,3,4* KAREN MORSE,2 CHEN HUI WANG,4 CAREN SHAPIRO,5 JENNIFER ROBERTS,1,4 MARK WELTER,6 MICHELLE ALLEN,7 MARY JO EVANS,1 2 AND THOMAS D. FLANAGAN Departments of Pediatrics,1 Microbiology,2 and Clinical Laboratory Science,3 State University of New York at Buffalo, Children’s Hospital,4 and Division of Mathematics and Natural Sciences, D’Youville College,5 Buffalo, New York; Oragen, Inc., Urbandale, Iowa6; and Ambico, Inc., Dallas Center, Iowa7 Received 15 July 1996/Returned for modification 24 September 1996/Accepted 29 October 1996
Umbilical cord serum samples (380), an average of 10 per month for 3 years (1990 to 1992), were tested by indirect immunofluorescence assay for group C rotavirus immunoglobulin G. Thirty percent were positive, suggesting that approximately one-third of women of childbearing age in western New York have experienced group C rotavirus infection. obtained from our assays most likely reflect the epidemiology of group C rotavirus infection in women of childbearing age. Umbilical cord serum samples were available from stored sera remaining after routine testing at the time of delivery of 380 apparently healthy newborns born at Children’s Hospital, Buffalo, N.Y., during 1990, 1991, and 1992. All sera were stored at 2208C and were thawed once and then refrigerated during the period of the study. An average of 10 samples per month were chosen randomly for testing for each month during the years 1990 to 1992. The IIF assay was carried out with swine testicular cells inoculated with trypsin-activated porcine group C rotavirus (AmC-1 strain) (16, 28). Infected and control cells were harvested at 24 h, fixed in cold acetone, and frozen at 2208C until employed in the IIF assay. In the screening assay, umbilical cord sera at a dilution of 1:10 were added to the coverslips, incubated for 1 h, and washed, and a 1:30 dilution of either goat anti-human IgG or IgM conjugated with fluorescein isothiocyanate (Cappel Research Products, Durham, N.C.) was added. Following a 30-min incubation and washing, the stained coverslips were mounted on glass slides. The slides were read with a BX-40 microscope (Olympus America, Inc., Lake Success, N.Y.). In the blocking assay, group C rotavirus at twofold dilutions from 1:10 to 1:40 was added to the fixed infected-cell monolayers and incubated for 30 min at 378C. After incubation the IIF assay was carried out as described above. For the EIA, 96-well plates (Costar, Inc., Cambridge, Mass.) were coated with virus or cell antigen at 1 mg/ml. The plates were washed, and 100 ml of human serum (1:20) was added and incubated at 378C for 1 h. After washing, 100 ml of horseradish peroxidase-conjugated rabbit or goat anti-human IgG (1:2,000) was added and incubated for 30 min at 378C. Plates were washed and substrate was added. After the reaction was stopped, plates were read at 492 nm in an EIA reader. Thirty percent (114 of 380) of the umbilical cord sera were positive for group C rotavirus IgG by the IIF assay. The pattern of fluorescence was granular and intracytoplasmic. Specific fluorescence was blocked by the addition of virus. The specific blocking was demonstrated in a dose-dependent fashion, with low dilutions of virus blocking more completely. In Table 1 the results of the IIF assay are displayed with monthly positive totals for the 3 years of the study. The data are displayed graphically in Fig. 1. Note that a significantly larger percentage of antibody-positive sera were detected in 1991 than in either
Rotaviruses are double-stranded RNA viruses belonging to the reovirus family. They cause diarrhea in human and animal infants worldwide (10, 11, 14). To date there have been seven serogroups (A to G) identified, of which group A is the most widely known and studied. All of the serogroups are morphologically identical to group A but are antigenically distinct (1–3, 7–9, 17, 20). Group B rotaviruses have been found associated with adult diarrhea, primarily in China (5, 6). Group C rotaviruses were first detected in 1980 in piglets (21). Since that time studies showing high antibody seroprevalence in herds have suggested that group C rotaviruses may be important pathogens in pigs (1, 2, 21, 22, 24). Group C rotaviruses also play a role in human diarrhea. These viruses have been detected in sporadic outbreaks of diarrhea in children and adults in many geographical areas including Asia, Australia, Europe, Central America, South America, and North America (2, 4, 7–10, 12, 13, 15, 17, 18, 20, 22, 27). Recently, we reported the detection of group C rotavirus RNA in liver samples from children with extrahepatic biliary atresia (19). Diagnostic assays for group C rotaviruses are not routinely available, and the methods used for detection have been electron microscopy and polyacrylamide gel electrophoresis (2, 22). However, with the adaptation of a porcine group C rotavirus to tissue culture (23, 28), some assays have been developed to detect virus and virus-specific antibody. The indirect immunofluorescence (IIF) assay, immune electron microscopy, indirect hemagglutination, the neutralization test, and the blocking enzyme immunoassay (EIA) have been used in seroepidemiological studies done on the prevalence of group C rotavirus antibody in humans (2, 7, 12, 16, 23–26). These studies have found between 3 and 40% of the sera from adults and children tested to be positive. Generally, group C rotavirus antibodies were not detected in children under 3 years of age, in contrast to group A rotavirus, to which the majority of children have antibodies by age 3. The primary focus of the present studies was to analyze umbilical cord blood sera for the presence of group C rotavirus-specific immunoglobulin G (IgG). Since maternal IgG crosses the placenta, the results
* Corresponding author. Mailing address: Division of Infectious Diseases, Department of Pediatrics, Children’s Hospital, 219 Bryant St., Buffalo, NY 14222. Phone: (716) 878-7084. Fax: (716) 888-3804. E-mail:
[email protected]. 486
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NOTES
TABLE 1. Monthly total of seropositive subjects No. (%) positive ina: Mo 1990
Jan. Feb. Mar. Apr. May June July Aug. Sept. Oct. Nov. Dec. Total
b
7 5 2 4 5 3 2 0 1 6 5d 2 42 (30)
1991 c
3 3 5 7 4 5 3 3 5 4 3 3
48 (41)
1992
2 3 4 3 2 1 4 1 1 1 2 1 25 (21)
a
Ten tested per month unless otherwise indicated. Twenty tested. Eight tested. d Twenty-two tested. b c
1990 or 1992 (P , 0.002 and P , 0.05, respectively; Fisher’s exact test). In addition, proportionally more antibody-positive sera were detected from January through June (67 of 188) than for July through December (47 of 192) for the combined 3 years (P , 0.02; Fisher’s exact test). Following the testing for group C rotavirus IgG, the samples which showed the brightest fluorescence in the IIF screening assay were tested for the presence of virus-specific IgM. None of 10 samples tested were positive for group C rotavirus IgM. The EIA was much less sensitive than the IIF assay. Only 58 of 343 samples were positive by the EIA, compared with 112 positive by the IIF assay (P , 0.001; Fisher’s exact test). Three EIA-positive samples were negative by the IIF assay. It is difficult to reconcile
FIG. 1. Number of seropositive subjects, based on 10 samples per month.
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the differences in the results of the two assays, which both employed swine testicular cells infected with the Ambico strain of porcine group C rotavirus. The apparently greater sensitivity of the IIF assay could be related to the greater avidity of the antibody for the infected whole cells as a substrate than for the lysate, which was used for the EIA. The data presented here represent the first report of a seroepidemiological study of the incidence of group C rotavirus infection in a population in the United States. Group C rotavirus has recently been reported to be present in about 1.5% of infants with diarrhea in a study of fecal samples from nearly 1,100 infants in Rhode Island (13). Umbilical cord sera were chosen for this study because of our recent findings of the presence of group C rotavirus in neonates with extrahepatic biliary atresia (19). While we were interested in acquiring information about the incidence of group C rotavirus infection in the United States, we were also interested in investigating the immune status of pregnant women because of the potential for susceptible pregnant women to contribute to infection in newborns. The present studies add confirmatory data that group C rotaviruses circulate in the United States and also that approximately one-third of young adults may have experienced infection at some time. More studies are needed to help us gain an understanding of this virus and its epidemiology. REFERENCES 1. Bridger, J. C. 1983. Detection by electron microscopy of caliciviruses, astroviruses and rotavirus-like particles associated with diarrhea in young pigs. Vet. Rec. 107:532–533. 2. Bridger, J. C., S. Pedley, and M. A. McCrae. 1986. Group C rotaviruses in humans. J. Clin. Microbiol. 23:760–763. 3. Bridger, J. C. 1987. Novel rotaviruses in animals and man. Ciba Found. Symp. 128:5–23. 4. Caul, E. O., C. R. Ashler, J. M. Darville, and J. C. Bridger. 1990. Group C rotaviruses associated with fatal enteritis in a family outbreak. J. Med. Virol. 30:201–205. 5. Chen, G. M., T. Hung, J. C. Bridger, and M. A. McCrae. 1985. Chinese adult rotavirus is a group B rotavirus. Lancet ii:1123–1124. 6. Den, S. S., X. Q. Liong, and W. C. Chang. 1984. Waterborne outbreak of rotavirus diarrhoea in adults in China caused by a novel rotavirus. Lancet i:1139–1142. 7. Dimitrov, D. H., M. K. Estes, S. M. Rangelova, L. M. Shindarov, J. L. Melnick, and D. Y. Graham. 1983. Detection of antigenically distinct rotaviruses from infants. Infect. Immun. 41:523–526. 8. Eiden, J., S. Vonderfecht, K. W. Theil, A. Torres-Medina, and R. H. Yolken. 1986. Genetic and antigenic relatedness of human and animal strains of antigenically distinct rotaviruses. J. Infect. Dis. 154:972–982. 9. Espejo, R. T., F. Puerto, C. Soler, and N. Gonza ´lez. 1984. Characterization of a human pararotavirus. Infect. Immun. 44:112–116. 10. Flores, J., O. Nakagomi, T. Nakagomi, R. Glass, M. Gorziglia, J. Askaa, Y. Hoshino, I. Perez-Schael, and A. Z. Kapikian. 1986. The role of rotaviruses in pediatric diarrhea. Pediatr. Infect. Dis. J. 5:553–562. 11. Holmes, I. H. 1983. Rotaviruses, p. 359–423. In W. K. Joklik (ed.), The Reoviridae. Plenum Publishing Corp., New York, N.Y. 12. Ishimaru, Y., H. Nakano, M. Oseto, Y. Yamashita, N. Kobayashi, and S. Urasawa. 1990. Group C rotavirus infection and infiltration. Acta Paediatr. Jpn. 32:523–529. 13. Jiang, B., P. H. Dennehy, S. Spangenberger, J. R. Gentsch, and R. I. Glass. 1995. First detection of group C rotavirus in fecal specimens of children with diarrhea in the United States. J. Infect. Dis. 172:45–50. 14. Kapikian, A. Z., and R. M. Chanock. 1985. Rotaviruses, p. 863–906. In B. N. Field (ed.), Virology. Raven Press, New York, N.Y. 15. Matsumoto, K., M. Hatano, K. Kobayashi, A. Hasegawa, S. Yamazaki, S. Nakata, S. Chiba, and Y. Kimura. 1989. An outbreak of gastroenteritis associated with acute rotaviral infection in schoolchildren. J. Infect. Dis. 160:611–615. 16. Nilsson, M., and L. Svensson. 1993. Antibody prevalence and specificity against group C rotavirus, p. 303. In Program and abstracts of the IXth International Congress of Virology. American Society of Virology, Edinburgh, Scotland. 17. Pedley, S., J. C. Bridger, J. F. Brown, and M. A. McCrae. 1983. Molecular characterization of rotavirus with distinct group antigens. J. Gen. Virol. 64: 2093–2101. 18. Penaranda, M. E., W. D. Cubitt, P. Sinarachatanat, D. N. Taylor, S. Likanonsakul, L. J. Saif, and R. I. Glass. 1989. Group C rotavirus infections in
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