Journal of Medical Virology 35:20&211 (1991)

Direct Serotyping of Porcine Rotaviruses Using VP7-Specific Monoclonal Antibodies by an Enzyme Immunoassay

H.S. Nagesha and I.H. Holmes School of Microbiology, University of Melbourne, Parkville, Victoria, Australia

Employing a serotyping EIA test using MAbs both cell culture adapted and faecal porcine rotaviruses were classified into serotypes G3, G3/5, G4, and G5. The MAbs have confirmed and extended the serotyping results obtained using polyclonal antisera. These MAbs are therefore potential reagents for serotyping of porcine ro- taviruses. Using subgroup specific MAbs sero- types G3, G3/5, and G5 were found to contain subgroup I antigens while G4 rotaviruses con- tained either subgroup II or subgroup I antigens.

KEY WORDS: viral gastroenteritis, pig faeces, diarrhoea diagnosis

INTRODUCTION Rotaviruses are the major cause of gastroenteritis in

children and young animals and efforts are being made to develop effective vaccines [Bohl et al., 1984; Holmes, 1983; Johnson et al., 1989; Vesikari et al., 19861. For development of vaccines better understanding of the role of serotypes in the epidemiology of the rotavirus is important.

Rotaviruses are double-shelled, nonenveloped vi- ruses with 11 segmented double stranded RNA in their genome [Holmes, 19831. Two outer capsid proteins, VP7 (product of gene 7,8, or 9) and VP4 (product of gene 4) are known to provoke a neutralizing antibody response [Bastardo et al., 1981; Hoshino et al., 1985; Liu et al., 1988; Offit and Blavat, 19861. Serotypes of rotaviruses are distinguished on the basis of a 20-fold difference in reciprocal antibody neutralization titres [Beards et al., 1980; Hoshino et al., 19841. Recent studies with genetic reassortants have demonstrated that in some cases occurrence of cross-reactions between serotypes is due to sharing of VP4 among different serotypes [Hoshino et al., 19851. Therefore, i t has been accepted to define all rotaviruses in terms of both their neutralizing proteins. Serotype specificity carried on VP7 is desig- nated as G serotype specificity and that carried on VP4 as P serotype specificity [Beards and Brown, 1988; Nagesha et al., 19891. There are a t least 10 defined G serotypes of rotaviruses from man and animals [Albert

et al., 1987; Clark et al., 1987; Hoshino et al., 1984; Paul et al., 1988; Ruiz et al., 19881. A numbering system for P serotypes has been proposed [Estes and Cohen, 19891.

Conventional methods of serotyping by neutraliza- tion tests require much time and skilled personnel and also sometimes it is still difficult to adapt rotaviruses to cell culture. Therefore, the monoclonal antibodies (MAbs) produced against serotype specific antigens could conveniently be used in enzyme immunoassays (EIA) for direct serotyping of porcine rotaviruses as has been described for human rotaviruses [Coulson et al., 1987; Shaw et al., 1985; Taniguchi et al., 19871. How- ever, there are no reports in the literature describing MAb based direct serotyping EIAs for porcine rotavi- rus. Previously we have reported production of serotype specific MAbs against porcine rotaviruses [Nagesha et al., 19893. In this report the application of MAb based EIA for direct serotyping of porcine rotaviruses is described.

MATERIALS AND METHODS Viruses and Cells

Viruses CRW-8 (G3), MDR-13 (G3/5), BEN-144 (G4), and TFR-41 (G5) were isolated in our laboratory [Nage- sha and Holmes, 19881. The AT/76 strain of porcine rotavirus was kindly provided by J. Albert, Royal Children’s Hospital, Melbourne, Australia. To evaluate the serotyping EIA additional viruses were isolated from diarrhoeic faecal samples and serotyped following the method described previously [Nagesha and Holmes, 19881.

All viruses were propagated in MA-104 cells in the presence of porcine trypsin. Rotavirus-infected or mock-infected cells were processed as described previ- ously [Coulson et al., 19871 for EIA antigen and MA- 104 control antigen and used at 1:400.

Accepted for publication June 12, 1991. Address reprint requests to I. H. Holmes, School of Microbiol-

ogy, University of Melbourne, Parkville, Victoria 3052, Australia.

0 1991 WILEY-LISS, INC.

Porcine Rotavirus Serotypes

Antisera and Monoclonal Antibodies Production of antisera and neutralizing MAbs

against CRW-8, MDR-13, BEN-144 and TFR-41 has been described previously [Nagesha and Holmes, 1988; Nagesha et al., 19891. Rabbit anti-ST-3 and AT/76 were obtained from B.S. Coulson and J. Albert, Royal Chil- dren’s Hospital, Melbourne, Australia. MAbs specific for subgroup I and subgroup I1 antigens were kindly provided by H. Greenberg, Veterans Administration Medical Center, Palo Alto, California.

Faecal Samples Diarrhoeic faecal samples were obtained from differ-

ent piggeries in geographically separated areas in Australia. They include 1) Bunge Meat Industries Ltd., Corowa, New South Wales; 2) Melbourne Univer- sity Piggery, Mount Derrimut, Victoria; 3) Willow Grove Piggery, Petersville, Trafalgar, Victoria; 4) samples from different farms around Bendigo were obtained through the Regional Veterinary Laboratory, Bendigo, Victoria. These were collected from diar- rhoeic piglets aged one to four weeks and transported as soon as possible to the laboratory on ice. Approximately 10% (VN) of faecal suspension was made in Eagle minimal essential medium supplemented with 0.02 M N-2-hydroxyethylpiperazine-Nt-ethane sulfonic acid (HEPES) buffer and clarified at 1600 g for 10 minutes.

Enzyme Immunoassays (EIA) EIA for rotavirus detection. This was carried out

in polystyrene Nunc immunoplates following the pre- viously described method of Beards et al. [19841. Ap- proximately 10% (v/v) of clarified faecal suspension was used for detection of rotavirus group A antigens with hyperimmune NIC rotavirus antiserum (diluted 15000 in 0.1M carbonate-bicarbonate buffer) as the capture antibody. Bound antigen was detected with peroxidase conjugated rabbit immunoglobulins to rotavirus (Da- kopatts, Denmark) and 2,2l azinodi (3-ethyl) benzthi- azoline sulphonic acid as substrate. Results were read using a Titertek Multiskan spectrophotometer (Flow Laboratories, Inc., Irvine, Scotland) at 420nm. Absor- bance of ~ 0 . 1 was considered to indicate positive reactions. Positive samples were stored at 4°C for 6-24 months before being tested in serotyping EIA.

Subgrouping EIA. The MAb based EIA described previously by Greenberg et al. [19831 was used for subgrouping rotavirus strains.

This was used for evaluating MAbs by end point titrations and for direct serotyping of rotavirus. The method is essentially the same as that of Coulson et al. [19871 described for human rotavirus serotyping. Briefly, hyperimmune rabbit antisera to porcine rotaviruses CRW-8 (serotype G3), MDR-13 (serotype G3/5), TFR-41 (serotype G5), human rotavi- rus ST-3 (serotype G4), and preimmune rabbit sera were used as capture antibodies a t 15000 to 1:6000 dilutions (in PBS pH 7.2). Hyperimmune ST-3 serum was used as capture antibody for porcine G4 viruses

Serotyping EIA.

207

because it had high neutralizing activity against por- cine BEN-144 virus [G4, Nagesha et al., 19891. A neutralizing MAb reactive with the same virus sero- type as the corresponding coating serum was used as detector antibody. After reactions with conjugate (per- oxidase conjugated rabbit immunoglobulins to mouse immunoglobulin, Dakopatts) and substrate (3,3l,5,5l- tetramethyl benzidine) results were read using the EIA reader a t 450nm. The results were expressed as the mean OD 450 x1000. Values 3 100 in each EIA test were considered to show positive reactions. As negative controls MA-104 cell lysate or diarrhoeic faecal sam- ples negative for rotavirus group antigen by EIA or rotavirus ds RNA by polyacrylamide gel electrophore- sis were included [Nagesha and Holmes, 19881.

Immunofluorescence and neutralization tests. These tests were performed to determine the virus titres and serotype specificity of some cell culture adapted and faecal viruses [Thouless et al., 1977; Nagesha and Holmes, 19881. Faecal viruses or serum virus mixtures were centrifuged onto cell monolayers to determine virus infectivity or neutralization titres, respectively.

RESULTS In our previous study eight VP7-specific MAbs de-

rived against porcine rotaviruses were highly specific for viruses of the same serotype as the immunizing strain as determined by neutralization tests [Nagesha et al., 19891. Four of these were chosen and examined for their use for direct serotyping of porcine rotaviruses as described below.

Binding Specificities of MAbs This was tested by end point titration of MAbs in

serotyping EIA. End point titre for each MAb was determined against the homologous rotavirus strain using antiserum to this as the capture antibody and the results are shown in Table I. All the MAbs were found to be specific to strain/serotype. The MAb C1/1 al- though it reacted to high titres with CRW-8 and AT/76 (both serotype G3) showed very low level reactivity with MDR-13 strain which usually reacts as serotype G3/5 [Nagesha et al., 19901. The EIA specificities of MAbs derived against the porcine MDR-13 (G3/5), BEN-144 (G4) and TFR-41 (G5) viruses were similar in that they all reacted with same serotypic strains as the immunizing antigens.

The MAbs C1/1 (G3 specific), E l (G3/5 specific), B2/4 (G4 specific), and T1/1 (G5 specific) were evaluated for their suitability in serotyping EIA by constructing antibody binding curves. The antibody binding curves for each MAb were constructed using positive-negative (P-N) values of each antibody dilution obtained in EIA titration (four-fold serial dilutions) and the results are shown in Figure 1. The MAbs El , B2/4 and T1/1 showed similar antibody binding curves with maximum P-N values over the full range of dilutions indicating the strong binding specificity and suitability of these MAbs

208 Nagesha and Holmes

TABLE I. End Point Titration of Monoclonal Antibodies Antibody titrea Virus

(serotype) c1/1 E l B2/4 T1/1 CRW-8 (G3) 604,000 3,200 200 140

BEN-144 (G4) 800 400 1,600,OO 400 MDR-13 (G3/5) 282 1,360,000 565 200

TFR-41 (G5) 400 282 565 100,000 numbers are the reciprocal antibody dilutions determined by end point titration in

enzyme immunoassays [Coulson et al., 1987; Kurstak, 19851. The homologous values are in boldface type.

1.4

1.2

1 .o UJ 2 0.8 - (0 > 0.6

2 0.4

0.2

- E l - 8214 - T111 Cell lysate

Y

o . o f * -r--.-r -. - . . - . . r *-..--i - -r=--*-r 4. ..*... l o 2 l o 3 lo4 1 o 5 l o 6 10’

Reciprocal antibody dilutions

Fig. 1. Antibody binding curves of MAbs C l i l , El, B214, and Tl i l titrated against porcine CRW-8, MDR-13, BEN-144, and TFR-41 viruses respectively. P-N values (positive-minus-negative) were obtained from results of titration (four-fold serial dilutions from 1:lOO) of each MAb following the method of Coulson et al. [1987]. MA-104 cell lysate served as negative control antigen. Actual (N 1 values are shown for cell lysate.

for use in a serotyping EIA. The MAb C1/1 showed shallow titration curve with approximately 3-fold lower P-N values compared to other three MAbs, however the end point titre of this MAb appeared to be similar to other three MAbs. The lowest dilution of the antibody at which the antigen was still saturated was considered optimum for use in serotyping EIA [Coulson et al., 19871. This was 1:20,000 for C1/1 and T l / l ; and 1:100,000 for E l and B2/4.

Serotyping EIA With Cell Culture Adapted and Faecal Viruses of Known Serotype Specificity To evaluate the specificity of MAb based serotyping

EIA, nine cell culture adapted viruses and ten faecal viruses of known serotype specificity were examined and the results are presented in Table 11. The serotype specificity of cell culture adapted and faecal viruses determined by MAbs corresponded to their serotype specificity obtained using polyclonal antisera. None of the MAbs reacted with the cell lysate or faecal control antigen. When three cell culture adapted viruses of unknown serotype specificity were reacted with four

MAbs, two viruses (CRW-6 and CRW-9) were recog- nised by C1/1 as G3 and one (MDR-24) by E l as G315. These results suggested that it is necessary to include two separate MAbs C1/1 and E l to identify virus strains belonging to serotype G3 and G3/5, respec- tively. None the less, to identify porcine TFR-41 like (G5) viruses MAb T1/1 appears to be quite sufficient. VP7 specific MAb B2/4 reacted only with BEN-144 strain and did not react with any of the virus strains belonging to other serotypes indicating that this MAb specifically recognizes the porcine BEN-144 like vi- ruses.

Serotyping EIA With Diarrhoeic Faecal Samples Application of EIA for serotyping viruses in diar-

rhoeic specimens from piglets was examined using 87 rotavirus samples positive by group antigen detection EIA. These samples originated from different piggeries in geographically separated areas in Australia. Ten specimens negative for rotavirus ds RNA and group antigen were used as negative controls. 32 out of 87 specimens were clearly serotyped by this method; 6

Porcine Rotavirus Serotypes 209

TABLE 11. Examination of the Specificity of Monoclonal Antibodies Using Viruses of Known Serotype Specificity

Monoclonal antibodya C1/1 E l B2/4 Tl/1 Serotype SubgroupC

Cell culture adapted viruses

(serotype) b CRW-6 353 12 1 10 G3 I CRW-8 (G3) 67 1 3 1 4 G3 I CRWB 112 5 19 9 G3 I AT/76 (G3) 300 2 1 9 G3 I MDR-13 (G3/5) 12 1244 7 9 G3/5 I MDR-24 12 827 1 12 G3/5 I BEN-144 (G4) 11 13 751 1 G4 I1 TFR-33 (G5) 1 4 11 103 G5 I TFR-41 (G5) 13.5 4 1 1357 G5 I BEN-128 (G5) 2 4 1 930 G5 I BEN-226 (G5) 9 6 1 270 G5 I

BEN-215 (G5) 12 8 2.5 345 G5 I BEN-226 (G5) 9 1 8 270 G5 I BEN-227 (G3) 1092 2.5 7 10 G3 I BEN-229 (G5) 12 7 29 163 G5 I BEN-260 (G5) 40 11 6.5 227 G5 I BEN-262 (G5) 38 12 19 254 G5 I BEN-263 (G5) 40 27 13 128 G5 I BEN-264 (G5) 30 40 63 240 G5 I TFR-74 (G5) 37 35 12 139 G5 I TFR-77 (G5) 6 1 10 117 G5 I Fecal control 1.2 0.8 1.2 0.8

Cell lysate 2.25 2.5 2.2 1.7 Faecal viruses (serotype)

aValues are the mean optical density 450 X 1000. Values 2100 were considered to show positive teactions. Significant values are in boldface type. The serotype specificity shown in parenthesis was obtained using reference antisera following

the method described previously (see Materials and Methods). ‘These viruses were subgrouped following the method of Greenberg et al. (1983). NT Not tested.

were typed as serotype G3, 5 as G3/5, 4 as G4, and 17 as G5 (Table 111). None of the control faecal specimens reacted with any antibodies. Non assignability of 55 samples into any serotypes will be discussed later.

Subgrouping EIA MAb based subgrouping EIA showed that majority of

the cell culture adapted as well as faecal viruses belonged to subgroup I and a small proportion of viruses belonged to subgroup I1 (Tables I1 and 111). Amongst four serotype G4 viruses two (BEN-144, BEN- 156) were subgroup 11, while the other two (CRW-61, CRW-61) were subgroup I.

DISCUSSION This study shows that VP7-specific MAbs could be

used for serotyping of porcine rotaviruses in diarrhoeic faecal samples by EIA which is simple and specific. This method is not only useful for typing the most common porcine rotavirus serotype G5 but also appears useful for typing viruses belonging to other serotypes including the G3, G4 type as well as the unusual G3/5 types. The majority of samples contained serotype G5 which shows that viruses belonging to this serotype are

the common pathogens in pigs as it has been reported previously [Bohl et al., 19841.

Faecal viruses belonging to each serotype were de- tected in samples which originated from two different places (Table 111). The serotype G5 viruses were de- tected in samples from Trafalgar and Bendigo, G3 from Corowa and Bendigo, G315 from Mount Derrimut and Corowa, and G4 from Corowa and Bendigo. This indi- cated that samples from Bendigo had G3, G4 and G5 serotypes, while those from Corowa had G3, G3/5 and G4 serotypes. However, samples from Trafalgar and Mount Derrimut contained only G5 and G3/5, respec- tively. The detection of a serotype G3/5 strain in a sample which originated from a diarrhoeic piglet from Corowa shows that this unusual serotype is not con- fined to one population [Nagesha et al., 19901.

Forty-three out of 87 samples with mean OD 450

value of 0.999 by group antigen detection EIA could not be serotyped. All these samples remained untypable even after concentration of faecal extracts (data not shown). The OD value of these 43 samples did not show any observable difference when compared to that of 32 serotypable specimens which had the mean OD 450

value of 0.834. Moreover, these 43 samples did not

Nagesha and Holmes

contain any infectious particles while the 32 typable samples contained infectious particles (Table 111). Therefore, it is likely that the outer capsid in the untypable faecal samples had disintegrated or been degraded. Shaw et al. [19851 and Coulson et al. [19871 in their studies on human rotavirus serotyping re- ported that a proportion of samples remained un- typable; the former group postulated that ETA serotyp- ing is likely to be less sensitive than group antigen detection EIA by virtue of the greater amount of group antigen per virion, while the latter group suggested that the outer capsid degradation is the major factor in the occurrence of false-negative serotyping results. In the present study, the fact that 43 untypable samples did not contain any infectious particles suggests that the occurrence of false-negative serotyping results is frequently due to the degradation or loss of the outer capsid which carries the neutralizing antigens VP7 and VP4. It is possible that the long time storage of faecal samples of PBS at 4°C might have resulted in loss of outer capsid, however this awaits further study by electronmicroscopy.

Another potential problem with MAb based serotyp- ing EIA is the presence of untypable viruses in samples containing infectious particles. In the present study twelve such untypable viruses were found and the serotyping results did not show any significant differ- ence even after treatment of these samples with fluo- rocarbon, which rules out the possibility of any lipid- like materials interfering with the serotyping reactions (data not shown). Of these 12 untypable samples seven did not react with any serotyping MAbs, while the other five reacted with two or more MAbs. We intend to adapt some of these viruses to cell culture and it is hoped that VP7 sequence analysis of such viruses will soon lead to an explanation for the non assignability of viruses to any serotypes.

The MAb based subgrouping EIA shows that Austra- lian porcine rotaviruses belong to subgroup I or I1 (Tables I1 and 111) and this observation is consistent with the results obtained from the previous studies in the USA with porcine viruses [Greenberg et al., 1983; Hoshino et al., 19841. The Australian porcine rotavirus serotypes G3, G3l5, and G5 were found to contain subgroup I specific antigens while serotype G4 strains contained either subgroup I1 or I antigens (Tables I1 and 111). Subgrouping and serotyping results reveal that the antigenic nature of porcine rotaviruses is rather complex. Porcine BEN-144 and BEN-156 (both G4) carry subgroup I1 antigen in the inner capsid while porcine CRW-61 and CRW-68 (both G4) carry subgroup I antigen. Similarly it has been shown that the Amer- ican porcine isolates SB-2 and Gottfried share the same G4 serotype specificity but belong to different sub- groups: SB-2 is a subgroup I virus and Gottfried is a subgroup I1 virus [Hoshino et al., 19841.

ACKNOWLEDGMENTS We thank Paula Kelly for excellent technical assis-

tance and the National Health and Medical Research Council of Australia for financial support.

Porcine Rotavirus Serotypes 211

REFERENCES Albert MJ, Unicomb LE, Tzipori SR, Bishop RF (1987): Isolation and

serotyping of animal rotaviruses and antigenic comparison with human rotaviruses. Archives of Virology 93:123-130.

Bastardo JW, McKimm-Breschkin JL, Sonza S, Mercer LD, Holmes IH (1981): Preparation and characterization of antisera to electro- phoretically purified S A l l virus polypeptides. Infection and Im- munity 34541447.

Beards GM, Pilfold JN, Thouless ME, Flewett TH (1980): Rotavirus serotypes by serum neutralization. Journal of Medical Virology 5231-237.

Beards GM, Campbell AD, Cottrell NR, Peiris JSM, Rees N, Sanders RC, Shirley JA, Wood HC, Flewett TH (1984): Enzyme-linked immunosorbent assays based on polyclonal and monoclonal anti- bodies for rotavirus detection. Journal of Clinical Microbiology

Beards GM, Brown DWG (1988): The antigenic diversity of rotavi- ruses: significance to epidemiology and vaccine strategies. Euro- pean Journal of Epidemiology 4:l-11.

Bohl EH, Theil KW, Saif LJ (1984): Isolation and serotyping of porcine rotaviruses and antigenic comparison with other rotaviruses. Journal of Clinical Microbiology 19:105-111.

Clark HF, Hoshino Y, Bell M, Groff J , Hess G, Bachman P, Offit PA (1987): Rotavirus isolate W161 representing a presumptive new human serotype. Journal of Clinical Microbiology 25:1757-1762.

Coulson BS, Unicomb LE, Pitson GA, Bishop RF (1987): Simple and specific enzyme immunoassay using monoclonal antibodies for serotyping human rotaviruses. Journal of Clinical Microbiology 25509-515.

Estes MK, Cohen J (1989): Rotavirus gene structure and function. Microbiological Reviews 53:410-449.

Greenberg H, McAuliffe V, Valdesuso J , Wyatt R, Flores J , Kalica A, Hoshino Y, Singh N (1983): Serological analysis of the subgroup protein of rotavirus, using monoclonal antibodies. Infection and Immunity 39:91-99.

Holmes IH (1983): Rotaviruses. In Joklik W (eds): “The Reoviridae.” New York: Plenum Publishing Corporation, pp 359-423.

Hoshino Y, Wyatt RG, Greenberg HB, Flores J, Kapikian AZ (1984): Serotypic similarity and diversity of rotaviruses of mammalian and avian origin as studied by plaque-reduction neutralization. Journal of Infectious Diseases 149:694-702.

Hoshino Y, Sereno MM, Midthun K, Flores J, Kapikian AZ, Chanock RM (1985): Independent segregation of two antigenic specificities (VP3 and VP7) involved in neutralization of rotavirus infectivity. Proceedings of the National Academy of Sciences USA 828701- 8704.

Johnson MA, Misra RM, Lardelli M, Messina M, Ephraums C, Reeves PR, Bolcevic Z, Noel JS, Hum CP, Mai HV, Dyall-Smith ML,

i9:248-254.

Holmes IH (1989): Synthesis in Escherichia coli of the major glycoprotein of human rotavirus: analysis of the antigenic regions. Gene 84:73-81.

Kurstak K (1985): Progress in enzyme immunoassays: production of reagents, experimental design, and interpretation. Bulletin of World Health Organization 63:793-811.

Liu M, Offit PA, Estes MK (1988): Identification of the simian rotavirus S A l l genome segment 3 product. Virology 163:26-32.

Nagesha HS, Holmes IH (1988): New porcine rotavirus serotype antigenically related to human rotavirus serotype. 3 Journal of Clinical Microbiology 26:171-174.

Nagesha HS, Brown LE, Holmes IH (1989): Neutralizing monoclonal antibodies against three serotypes of porcine rotaviruses. Journal of Virology 63:3545-3549.

Nagesha HS, Huang J , Hum CP, Holmes IH (1990): A porcine rotavirus strain with dual VP7 serotype specificity. Virology 175:319-322.

Offt PA, Blavat G (1986): Identification of two rotavirus genes determining neutralization specificities. Journal of Virology 57:376-378.

Paul PS, Lyoo YS, Andrews J J , Hill HT (1988): Isolation of two new serotypes of porcine rotavirus from pigs with diarrhoea. Archives of Virology 100:139-143.

Ruiz AM, Lopez IV, Lopez S, Espejo RT, Arias CF (1988): Molecular and antigenic analysis of porcine rotavirus YM, a possible new rotavirus serotype. Journal of Virology 62:43314336.

Shaw RD, Stoner-Ma DL, Estes MK, Greenberg HB (1985): Specific enzyme-linked immunoassay for rotavirus serotypes 1 and 3. Journal of Clinical Microbiology 22:286-291.

Steel RB, Torres-Medina A (1984): Effects of environmental and dietary factors on human rotavirus infection in gnotobiotic piglets. Infection and Immunity 43:906911.

Taniguchi K, Urasawa T, Morita Y, Greenberg HB, Urasawa S (1987): Direct serotyping of human rotavirus in stools by an enzyme- linked immunosorbent assay using serotypes 1-, 2-, 3-, and 4- specific monoclonal antibodies to VP7. Journal of Infectious Dis- eases 155:1159-1166.

Thouless ME, Bryden AS, Flewett TH, Woode GN, Bridger JC, Snodgrass DR, Herring JA (1977): Serological relationships be- tween rotaviruses from different species as studied by complement fixation and neutralization. Archives of Virology 53:287-294.

Torres-Medina A, Wyatt RG, Mebus CA, Underdahl NR, Kapikian AZ (1976): Diarrhoea caused in gnotobiotic piglets by the reovirus-like agent of human infantile gastroenteritis. Journal of Infectious Diseases 133:22-27.

Vesikari T, Kapikian AZ, Delem A, Zissis G (1986): A comparative trial of Rhesus monkey (RRV-1) and bovine (RIT 4237) oral rotavirus vaccine in young children. Journal of Infectious Diseases 153:832-839.