Journal of Medical Virology 2445-53 (1988)

Use of Serotype-Specific Monoclonal Antibodies to Study the Epidemiology of Rotavirus Infection C.J. Birch, R.L. Heath, and I.D. Gust

Virology Department, Fairfield Hospital, Fairfield, Victoria, Australia

The development of an enzyme immunoassay (EIA) capable of serotyping human rotavirus (HRV) in faecal extracts has enabled us to retrospectively study the epidemiology of rotavirus infection in Melbourne. Of 552 stored specimens obtained from individuals with rotavirus-associated gastroenteritis between 1975 and 1986, the serotype could be determined in 62%. Infection was most prevalent in two groups, neonates and children aged between 12 and 24 months. In these groups infection was due to different serotypes, type 1 in older children and an untypable virus in infants. Serotype 1 strains were detected in greater numbers than the other serotypes and circulated in each year of the study. Serotype 2 rotaviruses were associated with a large epidemic in 1978, but have been detected only rarely since.

Key words: faecal EIA, epidemiology of rotavirus serotypes

INTRODUCTION

The existence of more than one serotype of HRV was first suggested in the late 1970s, when sequential infections in young children were documented [Gust et al, 1977; Fonteyne et al, 1978; Rodriguez et al, 19781. Four HRV serotypes have now been described, each of which has been demonstrated to cause disease or asymptomatic infection in infants and young children in many parts of the world [Wyatt et al, 1983; Birch et al, 1983; Beards and Flewett, 19841. Genetic approaches have demonstrated that serotype specificity is determined by two polypeptides, VP3 and VP7, located on the exterior of the virus [Greenberg et al, 1983; Offit and Blavat, 19861.

Most information on the prevalence of infection with the different serotypes has been obtained by neutralization of strains isolated in cell culture with serotype-specific polyclonal antiserum [Wyatt et al, 19831. This technique has been employed because of

Accepted for publication May 13, 1987.

Address reprint requests to Dr. C.J. Birch, Virology Department, Fairfield Hospital, Yarra Bend Road, Fairfield 3078, Victoria, Australia.

@ 1988 Alan R. Liss, Inc.

46 Birch et al.

TABLE I. Age Distribution of Patients and HRV Serotypes Detected in 552 Specimens Collected Between 1975 and 1986

Age group (months)

Serotype 0-3 4-6 7-12 13-24 25-36 3 7 4 0 61 + Total

1 9 12 26 51 21 25 15 159 2 3 2 8 23 15 20 11 82 3 43 1 7 23 9 5 3 60 4 1 1 0 I 1 1 2 13 0" 104 6 18 34 36 23 7 238

Total 160 22 59 134 65 74 38 552

"Untypable.

the difficulties encountered in serotyping HRVs in faecal extracts by methods such as enzyme immunoassay (EIA). Because rotavirus serotypes share a number of antigenic sites, it has proven difficult to distinguish them using polyclonal reagents. However, recently a number of monoclonal antibodies have been produced which react against various rotavirus polypeptides [Taniguchi et al, 1985; Coulson et al, 1985; Heath et al, 19861 and some of them have serotype specificity.

The aim of the study reported here was to develop an assay system capable of determining the serotype of rotaviruses in faecal extracts, thereby eliminating the need for virus isolation, and to use this assay to study the epidemiology of HRV infection in Melbourne. In particular, we wished to establish the number of serotypes which were circulating, any trends in the pattern of their circulation, and their distribution among children with diarrhoea.

MATERIALS AND METHODS Collection, Processing, and Storage of Specimens

A total of 552 faecal specimens, previously demonstrated to be positive for rotavirus by an EIA utilising polyclonal reagents (results not shown) were analysed during the study. These specimens had been collected between 1975 and 1986 and stored at -20 "C as clarified 20% (w/v) suspensions in Hanks' balanced salt solution [Birch et al, 19771. No specimens were available from 1976, a year in which there was a low incidence of rotavirus-associated gastroenteritis.

The majority of specimens were obtained from children with gastroenteritis who required admission to Fairfield Hospital or other hospitals in Melbourne; the remainder were from neonates who had developed symptoms while still in hospital. Only a single specimen from each patient was tested. Of the 552 specimens studied, 549 were obtained from individuals aged from 2 days to 12 years, and three were from adults. The age distribution of the patients from whom specimens were obtained is shown in Table I.

Viruses

The methods for isolation, propagation and purification of the strains of HRV used in these studies have been described elsewhere [Liu et al, 1984; Heath et al, 19861. Table I1 shows the designation and serotype of each strain used. SAI I was originally obtained from H. Malherbe, and Hu7 from J. Albert, Royal Children's Hospital, Melbourne. The other viruses, which are all of human origin, were isolated and characterised in this laboratory.

Rotavirus Serotypes in Faecal Extracts 47

TABLE 11. Characteristics of Viruses and Monoclonal Antibodies Used in EIA Serotvpine Experiments

Virus

FH4232 FH6228 FH 1064 Hu 7 SAI 1 FH6 139 FH4154 FH4453

Serotype Monoclonal designation

Dilutions used in test

Monoclonal Rabbit serum Guinea pig serum

5C8 2G10 5D2

2D7 5 F4 4G7

1 : 16,000 1: 8,000 1: 8,000

1 : 16,000 1: 4,000 1: 8,000

1 : 16,000 -

-

1 :32,0OO

1 : 16,000

1 :64,000

-

-

Polyclonal Antisera

Polyclonal serum against the rotaviruses shown in Table I1 were prepared in guinea pigs as previously described [Liu et al, 19841. Serotype-specific sera were produced in rabbits by subcutaneous and intramuscular inoculation with a purified virus preparation in Freund's incomplete adjuvant. Four doses were given to each animal over a 13-week period. The antisera produced were serotype-specific as determined by neutralization of homologous virus. They were not absorbed for use in the EIA. Table I1 shows the dilutions at which each of the sera were used in the assay.

Monoclonal Antibodies

The methods used for production of monoclonal antibodies and mouse ascitic fluid have been previously described [Heath et al, 19861, and the hybridoma designation and serotype specificity of each is shown in Table 11. The specificity of two of the monoclonal antibodies used (5C8 and 2D7) have been discussed in a previous publication [Heath et al, 19861. They detect serotype 1 (5C8) and 3 (2D7) strains as determined by monoclonal neutralization of rotavirus isolates and do not give nonspecific results with faecal extracts containing untypable strains. The same parameters apply to the additional monoclonal antibodies reported here; 2G 10 detects a population of serotype 1 strains not detected by 5C8 and 5F4 detects serotype 3 strains not detected by 2D7; 5D2, and 4G7 detect, respectively, known serotype 2 and 4 isolates.

If monoclonal antibodies with the same serotype specificity were used in the EIA serotyping test, they were pooled at their optimal dilutions. For example, monoclonal antibodies 5C8 and 2G 10, both made against serotype 1 strains, were pooled at dilutions of 1:16,000 and 1:8,000, respectively (see Table 11). The final panel consisted of 4 serotype-specific reagents, the serotype 1 and 3 components each being made up of pools of two monoclonal antibodies, and the serotype 2 and 4 components each being discrete serotype-specific monoclonal antibodies.

Serotyping EIA

A 96-well microtitre plate (Nunc Immunoplate 1, Denmark) was divided into six equal sections (1 6 wells per section), thereby enabling six strains of HRV to be serotyped per plate. One hundred pl of the optimal dilution in phosphate buffered saline (PBS, pH 7.2) of serotype-specific ascitic fluid (determined by chequerboard titration against homologous, cell culture-derived virus) was added to 4 wells in each section and the plate incubated at 4 "C overnight. The plate was then washed three times with distilled water,

48 Birch et al.

100 pl of 0.5% bovine serum albumin (BSA) in PBS (PBS-BSA) added to all wells, and the plate incubated for one hour at 37 "C. Following three washes with PBS, 50 pl of each faecal extract was added to all 16 wells of its designated section and the plate incubated at 4 "C overnight. After six washes with PBS, 50 p1 of each serotype-specific polyclonal rabbit serum (diluted 1 in 50 in PBS-BSA) was added to two wells per specimen in each of the specific monoclonal anti body-coated wells. That is, serotype- 1 -specific polyclonal serum was added to those wells that had been coated with serotype-1-specific monoclonal antibodies, serotype-2-specific polyclonal serum was added to those wells that had been coated with serotype-Zspecific monoclonal antibodies, etc. The same volume of preim- mune rabbit serum, diluted 1 in 50 in PBS-BSA, was added to the corresponding two wells of each extract and the plates incubated at 37°C for 1 hour. After six washes in PBS, 50 p1 of the optimal dilution of each polyclonal guinea pig serum (in PBS-BSA, see Table I1 for dilutions used) was added to each of the four wells corresponding to its serotype specificity, and the plate incubated at 37 "C for 1.5 hour. After six more washes in PBS, 50 pl of horseradish peroxidase-conjugated rabbit immunoglobulins to guinea pig immunoglobu- lins (Dako, Denmark), diluted 1 in 4,000 in PBS-BSA, was added and the plate incubated at 37 "C for 1 hour.

Finally, the plates were washed six times with PBS and the substrate (0.1 mgjml 3,3,5,5-tetramethylbenzidine in sodium acetate buffer, pH 5.8) added. After 15 minutes incubation at room temperature, the absorbance of each well was measured at 650 nm. Zero was taken as the absorbance of wells incubated with substrate alone. Specimens were considered positive for a particular serotype if the two wells incubated with normal rabbit serum gave a reading greater than 0.100 and the corresponding two wells incubated with the serotype-specific rabbit serum were reduced in absorbance value by at least 50%.

RESULTS EIA Serotype Determinations.

Serotypes detected. In practice the EIA produced several possible outcomes. In some known-positive extracts there was no visible reaction in any wells, or when reaction did occur it was in one or more specifically coated wells but without blocking by any one of the polyclonal rabbit sera. Either of these reactions resulted in the virus being classified as untypable. However, the majority of extracts produced either a visible reaction in more than one set of specifically coated wells that was then reduced by 50% or more by only one polyclonal rabbit serum, or a visible reaction in only one set of specifically coated wells that was then reduced by at least 50% by the relevant polyclonal reagent. These last two reactions satisfied our definition of a confirmed serotyping result.

Of the 552 specimens examined, the rotavirus serotype was established in 345 (62.5%). Of this group, 159 (28.8%) were serotype 1, 82 (14.8%) were serotype 2, 91 (16.5%) were serotype 3, and 13 (2.4%) were serotype 4. The rotaviruses in the remaining 207 specimens (37.5% of the total) could not be serotyped using the existing monoclonal reagents.

In some cases it was possible to assess the specificity of these results by comparing them to the serotype result obtained when virus isolation from the faecal extract had been successful. Of the 552 specimens tested, rotaviruses had previously been isolated and serotyped by polyclonal neutralization from 45. Concordant EIA serotyping results on the faecal extracts and neutralization results on the isolates were obtained on 26 of these 45 specimens, while the viruses in the remaining 19 extracts were untypable by EIA (see

Rotavirus Serotypes in Faecal Extracts 49

TABLE 111. Comparison of Faecal ELA Serotyping With Virus Isolation and Identification by Neutralization

Outcome of serotyping test

Faecal virus serotyped, isolate serotyped” Faecal virus not serotyped, isolate serotyped” Faecal virus not serotyped, isolate not serotyped’

“Isolate serotyped using monoclonal and plyclonal antisera. bNumber in brackets refers to the serotype as identified by polyclonal neutralization. “Isolate not serotyped by monoclonal antibody neutralization. Serotype established only by plyclonal neutraliza- tion.

Number of specimens and serotype

26 [21(l),b4(3), 1(4)1 10 [6(1), 4(4)1 9 M3)1

Table 111). There were no specimens that gave contradictory EIA and polyclonal serotyping results.

As an additional indicator of the specificity of the EIA, we made use of the generally accepted observation that serotype 2 strains possess a short RNA electrophoretic profile. When sufficient volume of specimen was available to enable extraction and detection of viral RNA after electrophoresis and staining, each of the specimens identified as containing serotype 2 virus by EIA were found to contain rotaviruses with short RNA electrophoretic patterns (results not shown).

Sensitivity of the EIA. In assessing the sensitivity of the test, it is necessary to assume that the 209 specimens containing untypable rotaviruses fall into two main groups, one comprising specimens with insufficient particles for detection by EIA serotyping, and another containing variant rotaviruses not reacting with the monoclonal antibodies used in the panel. (A third group may contain variants without outer shell.) The number of specimens in the first group determines the sensitivity of the assay. To estimate this, EIA serotyping was carried out on the cell culture supernates of the 45 specimens from which virus had been isolated and the results compared with those obtained by direct serotyping of the faecal virus. In 26 of these specimens (58%) the direct assay provided the same serotyping result as the monoclonal neutralization result on the cell culture-adapted virus (see Table 111). However, in ten specimens (22%) the serotype of the virus could be determined by testing the supernate but not by testing the faecal extract (see Table III), suggesting that in this group the inability to assign serotype was owing to lack of sensitivity rather than being due to the presence of variants which were not reactive with the monoclonal antibodies used in the assay. In this group of ten specimens, amplification of the number of virus particles had enabled serotyping to be carried out. The remaining nine specimens (20%) contained rotaviruses that were not typable in either the direct EIA system, or by monoclonal neutralization. These appear to be variants, and all were serotype 3 as determined by polyclonal neutralization.

Temporal Distribution of Rotavirus Serotypes Figure 1 shows the distribution of serotypes (and untypable strains) during the

period 1975 to 1986. In 1977,1979,1981,1983,1984, and 1985,onlya limited numberof specimens were available, reflecting the minor nature of these outbreaks, and during these years no single serotype predominated. However, in 1975, 1978, 1980, 1982, and 1986, substantial epidemics occurred. In these years a single serotype predominated while other serotypes caused only sporadic infections or were not detected. For example, the 1975 epidemic was largely associated with serotype 3, while in 1978 serotype 2 predominated. The epidemics of 1980, 1982, and 1986 were associated with serotype 1. Following the

50 Birch et al.

Year

Temporal distribution of rotavirus serotypes in Melbourne Fig. 1.

large epidemic associated with serotype 2 rotavirus in 1978, only a few strains were isolated in 1979, and in the subsequent seven years of the study there has been no evidence of this serotype circulating in Melbourne.

Distribution of Rotavirus Serotypes by Age

Rotaviruses were detected with the highest frequency in two separate age groups, infants aged up to three months, and children from 12 to 24 months of age (see Table I). There was a marked difference in serotype distribution within these two groups, with serotype 1 predominating in the older children, and an as yet undetermined serotype almost exclusively infecting the neonates. Of the 143 specimens obtained from infants aged 14 days or less, only 21 (14.6%) were associated with an identifiable rotavirus serotype.

Serotype 2 strains were mostly associated with infection in older children (see Table I ) . During 1978, when this serotype caused a significant epidemic, only 5 of 54 strains identified (9.3%) were associated with infection in children under twelve months of age, and only one of these was a neonate. A similar pattern of age distribution was also evident in those years when serotype 2 virus circulated at nonepidemic levels.

Serotype 3 rotaviruses were relatively evenly spread throughout each age group (see Table I), although they were detected more frequently than the other serotypes in children under 3 months of age. Serotype 4 strains were detected infrequently during the 10-year study period, with the majority (7 of 13) producing infection in children aged twelve to 24 months.

Rotavirus Serotypes in Faecal Extracts 51

DISCUSSION

Monoclonal antibodies have been previously used in EIAs to detect HRV in faecal extracts [Cukor et al, 1984; Beards et al, 1984b], and have recently been used for direct serotyping of these viruses [Heath et al, 1986; Coulson et al, 19871. Our results demonstrate that while monoclonal antibodies can be used to directly serotype HRV in many faecal extracts, the configuration of the EIA required to achieve optimal results is complicated and requires the use of specific blocking antibodies immediately after the antigen incubation step. Although several different configurations for coating and detector antibodies were tried (results not shown), optimal results were ultimately obtained when the monoclonal antibodies were used as coating reagents and polyclonal antibodies were used as detector reagents.

We were not able to determine the serotype of many (37.9%) rotaviruses in our EIA, and although some of these could represent new serotypes, we believe there are several alternative explanations. The efficiency of the test used is directly related to the number of structurally intact virions present in the faecal extract. The rotavirus polypeptides associated with serotype specificity are situated on the outer shell of the virion [Offit and Blavat, 19861 and may be lost following virus shedding from infected cells along the small intestine, or during specimen processing. Although not all the reasons for breakdown of virus particles are understood, some factors influencing the number of complete virions are pH, and the levels of enzymes such as trypsin in the gut. As well, the number of complete virus particles in faecal extracts also appears to be type specific; we have observed that serotype 4 strains appear to readily lose their outer shell, even when adapted to growth in cell culture. It was thought that prolonged storage of faecal extracts at -20 "C might also contribute to loss of outer shell. However, the proportion of specimens on which we obtained a definitive serotyping did not decrease with length of storage. For whatever reasons loss of outer shell occurs, the end result is that incomplete particles will not be bound by the monoclonal antibodies coating the solid phase and will therefore remain undetected (and therefore unserotyped). Although we did not attempt to concentrate faecal extracts in order to increase the number of complete particles in the specimen, another study has demonstrated that a threefold concentration of the extract significantly improves the sensitivity of direct EIA serotyping [Coulson et al, 19871.

The second reason for our inability to serotype all strains may be the considerable antigenic variation which occurs even within serotypes [Heath et al, 19861. This phenomenon has increased the difficulties involved in producing uncomplicated yet specific EIA serotyping tests. For example, we found that some monoclonal antibodies that neutralized individual rotavirus serotypes were not useful for detecting all viruses of the same serotype in faecal extracts (unpublished observations). Accordingly, we have used pools of monoclonal antibodies raised against serotype 1 and 3 strains as solid-phase coating reagents in our EIA system.

Finally, the sensitivity of the assay may be influenced by interfering factors in the faecal extract, the most likely of these being coproantibodies. Even though one of the monoclonal antibodies used in the EIA was made to a neonatal serotype 3 rotavirus, strains associated with neonatal infections constituted a large proportion of our untypable viruses. Antibodies to rotaviruses, acquired both across the placenta and via maternal colostrum and milk, have been reported in the faeces of infants with rotavirus-associated gastroenter- itis [McLean and Holmes, 1980; Stals et al, 19841, and it is possible that these may interfere with assays that utilise reagents specific for an epitope that determines serotype,

52 Birch et al.

perhaps by binding directly to that epitope and blocking subsequent binding of the virus to the monoclonal antibody on the solid-phase.

The ability to serotype HRV directly in faecal extracts has enabled us to carry out a retrospective epidemiological study of rotavirus infections in children living in Melbourne, using extracts stored since 1975 as source material. The results obtained show that the pattern of circulation of HRV serotypes, particularly in epidemic years, appears to favour the predominance of a single serotype, with minor input from the others. In 1986, only serotype 1 strains circulated. We were able to detect the circulation of each of the recognized serotypes in only one year, 1978, during which there was a large epidemic associated with serotype 2 virus.

Of interest was the abrupt disappearance from circulation of serotype 2 strains. This serotype was seen in 1979 after causing a large epidemic in 1978, but has not been detected since. Assuming that there has not been a major alteration in the ability of this serotype to cause symptomatic infection, thereby enabling it to circulate asymptomatically in susceptibles, it would seem that few children aged under seven years of age who live in Melbourne will possess antibodies to it. In addition, rotaviruses with short electrophero- types, which to date have all been serotype 2 strains, are reported to have circulated in 1983 and 1984 in Hong Kong, a city often visited by Australian tourists [Tam et al, 19861. Under these circumstances, it does not seem unreasonable to predict an epidemic due to this serotype within the next few years, possibly introduced into the susceptible population by adults who have visited this region.

Analysis of the distribution of HRV serotypes by age of infection revealed two major peaks, in the neonatal period, and between 12 and 24 months of age, each associated with antigenically distinct viruses. Untypable viruses were primarily associated with infection in neonates, while serotype 1 strains were associated with infection above three months of age. Rotavirus strains specific to neonates have been previously reported [Rodger et al, 1981; Bishop et al, 19831, and in our sample of 552 patients, 155 (28%) were in the first month of life. Other studies have demonstrated the Occurrence of endemic, rotavirus- associated diarrhoea in the nurseries of several major Melbourne hospitals [Cameron et al, 1978; Rodger et al, 19811, and in one of these studies [Rodger et al, 19811 the circulating strain appeared to be unique to that hospital. It may therefore be necessary to produce monoclonal antibodies to a number of neonatal strains in order to improve the serotyping efficiency of the EIA for this group.

ACKNOWLEDGMENTS

This study was supported by a grant from the National Health and Medical Research Counci 1.

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