rotavirus shedding by newborn children

Journal of Medical Virology 14: 127-136 (1984)

Rotavirus Shedding by Newborn Children Irene Perez-Schael, Georgette Daoud, Laura White, Gidalia Urbina, Naimeh Daoud, Mireya Perez, and Jorge Flores

lnstituto Nacional de Dermatologia (I. P.-S., L. W , G. U.), lnstituto Nacional de Nutricion (I. P.-S.), Hospital "Miguel Perez Carretio" (G. D., N. D.), Hospital de Nitios J. M. De Los Rios (M. P.), Caracas, Venezuela, and Laboratory of Infectious Diseases, National Institutes of Health, Bethesda, Maryland (J. F.)

We studied the shedding of rotavirus by newborn children in the nurseries of a large maternity hospital in Caracas, Venezuela, throughout the year 1982. Sixty- two (57%) of 108 children examined shed the virus within the first few days of life. Four (6%) of the 62 children who shed rotavirus had diarrhea but only one of them required oral rehydration therapy. The rotavirus specimens were identified as subgroup 2 in an ELISA subgrouping assay that employs monoclonal antibodies. Analysis of the RNA extracted from 52 of the samples by electrophoresis revealed a similar migration pattern in all the specimens; their identity was confirmed by crosshybridization analyses which revealed a strong degree of genomic homology among the strains studied.

Key words: rotavirus, neonatal infections, molecular epidemiology

INTRODUCTION

The shedding of rotavirus by newborn children initially documented in England in 1975 [Chrystie et al, 19751 has since been documented in several studies from different regions of the world [Cameron et al, 1975; Madeley and Cosgrove, 1975; Murphy et al, 1977; Rocci et al, 1981; Rodriguez et al, 1982; Soenarto et al, 1981; Van Renterghem et al, 19801. Rotavirus shedding may occur in neonates as early as in the first day after birth [Murphy et al, 19771. Although rotavirus gastroenteritis during the neonatal period has been reported [Bishop et al, 1979; Cameron et al, 1975; Murphy et al, 1977; Rocci et al, 19811, most of the studies point to the common asymptomatic character of the infection. These observations have been looked upon with optimism since they may provide important clues on the possibility of adminis- tering rotavirus vaccines to newborn children when they become available. It is not known whether the infection occurring at this early age is attenuated because of host factors related to the status of maturation of the small intestine in the neonate, or by passive protection afforded by breast milk or transplacental antibody, or perhaps

Accepted for publication January 16, 1984.

Address reprint requests to Jorge Flores, Laboratory of Infectious Diseases, National Institutes of Health, Bethesda, Maryland, 20205.

0 1984 Alan R. Liss, Inc.

128 Perez-Schael et al

because of the attenuation of rotavirus strains harbored by newborns. It is of interest that neonatal rotavirus infections have recently been shown to protect against subse- quent rotaviral illness [Bishop et al, 19831.

The present study was planned to investigate the presence of rotavirus in the stools of newborn children throughout a 1-year period of surveillance in a large maternity hospital in order to establish the rates of infection and the association of the virus with diarrheal illness. An attempt was made to characterize the rotavirus strains identified by means of electrophoretic and hybridization analyses.

MATERIALS AND METHODS

The Maternity Hospital “Concepcion Palacios” in Caracas, Venezuela, is probably one of the busiest in the world (about 100 children are born there daily). It serves the communities of lower socioeconomic resources in the center and west of the city. A total of 108 neonates were studied ranging in age from 1 to 15 days after birth. From 4 to 23 children were examined each month during the year 1982, and 2 to 7 stool samples obtained in consecutive days were studied from each child. The clinical condition of the children was documented at the time the samples were obtained and from the discharge records. Information was also obtained on whether or not the children were breastfed during their stay in the Hospital.

Rotavirus Identification

The initial detection of rotavirus was carried out by a confirmatory ELISA assay [Kapikian et al, 19791. Microtiter plates were precoated with serum obtained before or after parenteral immunization of a goat with rotavirus antigen; 5% stool suspensions were incubated in duplicate wells precoated with either of the two sera. Antirotavirus guinea pig serum was used as the detecting antibody and a phosphatase- labeled anti-guinea pig antiserum as the conjugate. OD readings from reactions in the wells coated with immune serum which were at least twofold greater than those obtained in the preserum coated wells were regarded as positive for rotavirus. In addition to the ELISA for detection, a subgrouping ELISA was performed on 38 of the specimens. Such testing employs monoclonal antibodies which were generously provided to us by Dr. Harry Greenberg (LID, National Institutes of Health, Bethesda, MD) [Greenberg et al, 19831. Microtiter plate wells were precoated with hyperim- mune goat serum; hybridoma fluids specific for subgroup 1 (hybridoma 255/60) or subgroup 2 (hybridoma 639/1) rotavirus were used as second antibodies and an anti- mouse IgG antibody labeled with alkaline phosphatase was used as the conjugate.

Electrophoretic Analysis of Rotavirus RNA

From 15 to 20 ml of 5 % stool suspensions in PBS was used to prepare rotavirus RNA from the positive samples. The suspensions were first treated with trichloro- trifluoro-ethane (Genetron 113- ICI, Allied Chemical Corp., Morristown, NJ) and then pelleted through a 30% sucrose cushion for 2 hr in a Beckman SW 27 rotor at 25,000 rpm. One third of the material pelleted was extracted with 50 mM Tris, 100 mM NaCl, 1 mM EDTA, and 0.2% sodium dodecyl sulfate (SDS) at 4°C for 30 min and then with an equal volume of phenol. The aqueous phase was then precipitated overnight at -20°C by adding 3 vol of ethanol. Electrophoresis of the RNAs extracted in this way was carried out in nondenaturing polyacrylamide Laemmli gels

Rotavirus Shedding by Newborn Infant 129

as previously described [Flores et al, 1982bl. The RNA bands were visualized and photographed under UV light after staining the gels with ethidium bromide.

Hybridization Analysis In some instances and depending on the amount of virus available to study,

transcription probes were prepared from single capsid rotavirus particles purified by cesium chloride gradient centrifugation from the specimens and from two laboratory strains (Wa and DS-1). In vitro transcription was performed as described previously [Flores et al, 1982bl in the presence of 32P-labeled guanosine triphosphate. Hybridi- zation of genomic RNA obtained from the virus particles to labeled single stranded RNA obtained by in vitro transcription was performed as previously described [Flores et al, 1982al. The resulting hybrid RNA molecules were electrophoresed in polyacrylamide gels as described above, dried, and autoradiographed.

RESULTS

Sixty two of the 108 (57.4%) newborn children studied shed rotavirus as detected by the confirmatory ELISA test. The presence of the virus had also been confirmed in 34 of the specimens by recognition of rotavirus RNA in the samples when assayed by dot hybridization [Flores et al, 1983al. Rotaviruses were identified throughout the entire year of the study. The days in which rotaviruses were first identified in the stools are represented in Table I. In some cases it was not possible to obtain samples from the first 2 or 3 days after birth. Some stool samples obtained the actual day of birth yielded negative results.

The mean birth weight was 3.25 k 0.35 (SE) kg for the children who shed rotaviruses versus 3.1 k 0.17 who did not shed them; 15 of 25 (60%) low birth weight children studied shed the virus.

Only 8 of the children studied were not breastfed prior to obtaining the stool samples. Four of them were positive for rotavirus. Nine of the 108 children had diarrhea; 4 of them shed rotavirus whereas Campylobacter fetus was detected in 1 of the 5 who were rotavirus negative. In 1 of the children with diarrhea in whom rotavirus was detected, a temporal relationship between the presence of diarrhea and shedding of rotavirus was not present (virus shedding stopped 5 days before the diarrhea started).

TABLE I. Age at Which Rotaviruses Were Initially Detected

Rotavirus oositive Datients Days after Cumulative birth No. Dercentaee

1 6 10 2 8 22 3 14 44 4 12 63 5 9 78 6 2 81 7 6 90 > 7 5 100

130 Perez-Schael et a1

In only one of the four cases of diarrhea among the children who shed rotavirus was rehydration treatment necessary; this patient was successfully treated by means of oral rehydration; this patient had recurrent severe nonrotavirus diarrhea at 5 weeks of age when the diagnosis of pyloric stenosis was made; the condition was surgically corrected. However, new episodes of diarrhea developed at 3, 5,7, 10, and 13 months of age (all rotavirus negative) of mild to severe degree; the patient has been intolerant to regular formula and aside from the diarrheal episodes has also presented two episodes of bronchiolitis which required hospitalization, two of pharyngitis and one of oral candidiasis.

More than 200 samples obtained from newborn children from other hospitals in the metropolitan area of Caracas were also examined. Only 2 of those samples were positive for rotavirus. The results are shown in Table 11. The newborn child from the “ Algodonal” Hospital who shed rotavirus presented diarrhea; he was a 26-day-old premature baby who was not being breastfed at the time of the illness; the one child who shed virus from “Los Teques” Hospital was 25 days old and was asymptomatic.

The ELISA subgrouping test was performed in 36 of the positive samples studied; 3 1 of them reacted more strongly or exclusively with subgroup 2 hybridoma, whereas they reacted poorly or not at all with subgroup 1 hybridoma (the OD ratios between subgroup 2 and subgroup 1 reactivity were 2.3 or higher). The remaining 5 samples did not react with either monoclonal antibody presumably due to low titers of antigen (as observed from the ELISA confirmatory assay).

RNA Analysis Enough material for electrophoretical analysis of viral RNA was available in 52

of 62 positive samples examined. All of them had a similar and characteristic migration pattern which can be seen in Figure 1 in which 10 of the samples examined are compared with RNAs from the strains Wa and DS-1. Of the samples from other hospitals, only the one from the “Los Teques” Hospital had enough material for electrophoretic analysis. The migration pattern of its RNA segments was different from the electrophoretype commonly found at the “Concepcion Palacios” Maternity (not shown).

Crosshybridization analyses were carried out by denaturing the genomic double stranded RNA of some of the rotavirus specimens and hybridizing it to labeled single stranded RNA prepared by in vitro transcription from specimens obtained at other times during the study. Forty such crosshybridization reactions were performed. In every case, complete identity was observed between any pair of specimens studied; that is, heterologous reactions yielded exactly the same hybridization pattern as did

TABLE 11. Search for Rotavirus in Other Maternity Hospitals of Caracas and Adjacent Cities

Number Rotavirus Maternity Hospital examined positive

(A) Private 7 0 (B) Social Security Hospital 6 0 (C) Algodonal Hospital 33 1 (D) Maternidad Santa Ana 45 0 (E) Petare Hospital 24 0 (F) Los Teques Hospital 107 1

Total 222 2


Fig. 1. RNA electrophoretical patterns of 12 rotavirus specimens obtained from newborn children at the maternity “Concepcion Palacios” at various times during the year of study (1982) from January (sample 12) through October (sample 162). Wa and DS-1 human rotavirus RNAs were included as controls. Gel electrophoresis was performed on 7.5% gels at 15-mA constant current.

homologous reactions in which the labeled probes and the genomic RNA originated from the same virus. An example of such hybridization experiments is found in Figure 2A. It should be noted that the quality of the transcripts obtained in vitro from the maternity specimens and used as probes was not uniform and in many cases less abundant full-sized copies were obtained from the larger genes (segments 14) ; this was probably due to the scarcity of the material and the difficulties in purifying the virus from the stools; however, in every case homologous hybridizations were performed as controls.


Fig. 2. (A) Autoradiography of the RNA hybridization patterns obtained by denaturing the genomic RNAs of three of the maternity rotaviruses and hybridizing them to a single stranded RNA probe prepared by in vitro transcription from one of the strains (M-37). Lane A represents a homologous hybridization of RNAs from strain M-37; lanes B and C represent hybridization of two other maternity strains to a probe prepared from virus M-37. (B) Autoradiography of the RNA crosshybridization patterns obtained by hybridizing genomic RNAs from one of the maternity strains to single stranded RNA probes prepared by in vitro transcription from the prototype rotaviruses Wa (lane B) and DS-I (lane C). Lanes A and D represent hybridizations of genomic RNAs from Wa and DS-1, respectively, to their homologous probes.

To obtain information on the genetic relatedness among the maternity strains and other rotavirus strains, double stranded RNA from three of the newborn rotavirus specimens from the Concepcion Palacios Maternity was denatured and hybridized to probes prepared from the Wa and DS-1 human rotavirus prototype strains. An example is shown in Figure 2B. When the maternity strains were hybridized to Wa probe, only two hybrid bands were resolved in the gel; they did not comigrate with any of the genes of the Wa or the maternity strain. Similarly, when the maternity strains were hybridized to DS-1 probe, only one hybrid band was apparent which did not comigrate with any of the segments of either the DS-1 virus or the maternity strain. In other experiments, double stranded RNAs from the animal rotavirus strains originally isolated from monkeys (Rh-2), dogs (CU-l), cats (feline), and calves (B-


682) [Flores et al, 1983bl were denatured and hybridized to a 32P probe prepared from one of the maternity specimens. None of the genes of any of those strains formed hybrids with the maternity strain that could be resolved in the electrophoretic system employed (results not shown).

DISCUSSION

Infectious diseases and prominently diarrhea during the neonatal period are commonly associated with high rates of mortality and with predisposition to severe nutritional retardation and increased susceptibility to further infectious processes. Rotavirus infections are commonly observed during this period of life. Several groups have presented evidence on the etiological role of rotavirus as a diarrhea-causing agent in this particular age group; however, more commonly, the infections observed in neonates are asymptomatic or mild. In the present study we found that a large proportion (57 %) of neonates at the Maternity Hospital “Concepcion Palacios” in Caracas were infected with rotavirus. The virus was found in all the nursery wards investigated with the paradoxic exception of the infectious cases nursery (although only 11 cases were examined in this area). Forty two percent of the positive cases were found in children as early as the day after birth, although in some children virus shedding was observed for the first time only in the sixth or seventh day after birth. No significant differences were observed in the birth weight of infected versus noninfected children; also the occurrence of rotavirus in children of low birth weight (less than 2.5 kg) was not different from that in children with normal range birth weight.

Nine of the 108 children studied at this hospital had diarrhea and in 4 of them rotavirus was identified in the stools. The significance of this observation is difficult to assess since the stools of healthy newborn babies are frequently of loose consist- ency. Only 1 of the children who shed rotavirus had a severe case of diarrhea necessitating treatment with oral rehydration therapy. Several studies point to the frequently asymptomatic character of rotavirus infection in newborn babies. Thus, in the study of Chrystie et a1 [ 19781 only 8 % of 189 newborn children who shed rotavirus had diarrhea, and none of them required fluid replacement therapy. Similarly, only 28 % of the children in which Murphy et a1 [ 19771 detected rotavirus had diarrhea and only 21 % of premature children shedding the virus had diarrhea in the study of Van Renterghem et a1 [ 19801. On the other hand, severe rotavirus gastroenteritis has been documented affecting full-term [Bishop et al, 19791 or premature [Rocci et a1 19811 babies.

Nevertheless, the finding of rotavirus in newborns is not universal. Published reports from England [Appleton et al, 19781 and the United States [Steinhoff and Gerber, 19781 have failed to detect the virus or have found it only in rare instances [Santosham et al, 19821 despite the examination of large numbers of samples. Besides the Concepcion Palacios Maternity, we only found two positive specimens after examining 222 stool samples from five other nurseries in Caracas and one in Los Teques (a neighboring city). Two of those nurseries were in private hospitals whereas the other four served people from areas with a socioeconomic status similar to that of the people served by the Concepcion Palacios Maternity Hospital.

In our initial attempts to type the maternity strains a subgroup test was performed which utilizes monoclonal antibodies that recognize antigenic specificities in

134 Perez-Schael et al

the protein encoded by the sixth gene (the 42,000 dalton major inner capsid protein) [Greenberg et al, 19831. All the samples positive in this assay reacted as subgroup 2, of which the human cultivatable rotavirus “Wa” is a prototype. The genomic RNAs extracted from the rotavirus in the stools were analyzed by polyacrylamide gel electrophoresis. All the samples from which enough RNA could be extracted and independent of the time they were obtained presented an identical electrophoretic migration pattern; the only exception observed was that of the single positive sample obtained in “Los Teques” Hospital. Not enough RNA for analysis was obtained from the only positive sample from the “Algodonal” Hospital. This observation is very similar to that of Rodger et a1 [ 19811 who analyzed 72 samples from newborn children finding only two electrophoretic patterns with small differences between them at a time when 17 other electrophoretypes could be identified among strains circulating in the community.

Since comigration of equivalent RNA segments does not rule out the possibility that nucleotide sequence differences may exist among them [Flores et al, 1982b] we attempted to obtain more information on the actual degree of homology among the maternity strains by performing crosshybridization studies in which the denatured genomic RNAs from some of the specimens were hybridized to 32P ssRNA probes produced by in vitro transcription from purified rotavirus from the specimens from which more material was available. In such hybridizations the pattern obtained in each case was identical to that obtained in homologous hybridizations (in which the genomic RNA from the viruses from which the probes were obtained were hybridized to probes from themselves). Some specimens were hybridized to rotaviruses obtained at different times of the year. In six of the crosses, enough material was available to analyze the suceptibility of the hybrids formed to S-1 nuclease which selectively digests single stranded nucleic acids or single stranded regions of partially homologous hybrids. The hybrids were in every case resistant to S-1 nuclease, which suggests the existence of a strong degree of homology among the strains studied.

These observations on the genomic uniformity among the maternity strains contrast with the high degree of genetic variation among strains circulating in the community [Rodger et al, 1981; Flores et al, 1982bl. The nosocomial spread of the infection explains this phenomenon and apparently under the conditions of transmis- sion in the nursery, rotavirus-or at least this strain-seems to exhibit a greater genetic stability than strains circulating outside the nursery. An indirect implication of this observation is that the genetic heterogeneity described by many authors may not be the result of accumulation of successive mutations as much as the possible expression of reassortment of genes among different rotavirus strains.

Several potential explanations may be appropriate for the unusually low fre- quency of diarrhea observed in rotavirus positive neonates: (1) special characteristics related to the maturation of the gastrointestinal tract may make the newborn less susceptible to rotavirus diarrhea; this does not explain the fact that rotavirus diarrhea of varying degrees of severity has been observed in neonates in other studies. (2) Positive protection may be afforded by breast milk or transplacentally acquired antibody; although breastfeeding has been associated with the prevention of rotavirus infection in the studies of Banatvala et a1 119781, Totterdell et a1 [1976, 19801, and McLean and Holmes [ 19811, this does not explain the frequently asymptomatic character of the infection that occurs in nonbreastfed babies [Chrystie et al, 19781. (3) Another possibility to explain the occurrence of asymptomatic rotavirus infections in


neonates is that the particular rotavirus strains that they harbor may be attenuated. The observation of Rodger et al [1981] that the electrophoretic pattern of the RNAs extracted from rotavirus from a series of newborn children was rather uniform at a time when rotaviruses with 17 other electrophoretic patterns were circulating among ill children supports this possibility. The results from the present study also support this possibility showing not only a uniformity in the subgrouping antigenicity and a constancy in the RNA as studied by electrophoresis and crosshybridization experiments, but also a distinctness from two prototype rotaviruses (Wa and DS-1) with which most rotaviruses isolated from ill children in Caracas share a strong degree of homology [Flores et al, 1982b, 1983b, manuscript in preparation].

Whichever explanation is correct for the commonly observed asymptomatic rotavirus infections in neonates it is clear that the observation touches on important issues relevant to the development of strategies for production and administration of rotavirus vaccines. First, the knowledge that newborn children will not necessarily develop illness when administered a rotavirus vaccine is valuable since such a vaccine will be preferably given in early life. Second, the strains isolated from asymptomatic newborn children might be attenuated or at least behave as if attenuated. Finally, at least some rotavirus strains seem to be, as shown in this study, genetically stable as they are transmitted from child to child.

In a recent report, Bishop et a1 [1983] followed for up to 3 years a group of children who shed rotavirus during the neonatal period and a control group. The children who were infected during the neonatal period suffered less frequent and less severe episodes of diarrhea than those in the control group. These results demonstrate the potential efficacy that early vaccination (possibly with these particular strains) may have in preventing rotavirus diarrhea.

ACKNOWLEDGMENTS

The authors wish to express their thanks to Dr. A. Z. Kapikian for valuable suggestions in the preparation of this manuscript; to Mrs. Mitzi Sereno for her valuable technical assistance, and to Dr. J. Esparza, from the “Instituto Venezolano de Investigaciones Cientificas” (IVIC), who supplied us with the specimens from the hospital in “Los Teques.” The portion of this work performed in Venezuela was supported by CONICIT (Consejo Venezolano de Investeigaciones Cientificas y Tec- nologicas), Project S1-1197.

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