38
tive,
or even beneficial, changes were more frequent among patients in this group. Fears that laparoscopic electrocoagulation leads to disrupted menstrual patterns are thus not warranted. Problems such as bowel burns and the prognosis for later re-anastomosis are of much greater concern, and it is for these reasons that minilaparotomy or the application of mechanical occlusion devices via laparoscopy should replace laparoscopic electrocoagulation in interval female sterilisation.
International Fertility Research Program, North Carolina 27709, U.S.A.
MARGARET F. McCANN ELTON KESSEL
DIFFERENT SEROTYPES OF HUMAN ROTAVIRUSES
SiR,-We have been detecting rotaviruses in stools with a sensitive micro complement-fixation (c.F.) technique for more than two years.’ This method is as sensitive and specific as electronmicroscopy (E.M.), and use of a simple absorption method has avoided difficulties with anticomplementary factors. The only problem is the need for a high-titre human serum, 128 on c.F.; convalescent calf serum is not sensitive enough for the detection of human reovirus-like particles (H.R.V.L.). We have tried to produce guineapig antiserum as an alternative, and this led us, quite by chance, to consider whether different serotypes OfH.R.V.L. might exist. In the first attempt to produce high-titre guineapig serum a guineapig was immunised according to the technique of McLure et a1.2 A fxcal suspension (about 30%, w/v) of stool no. 7 (table I) was twice centrifuged for 10 min, and the supernatant (crude H.R.v.L. antigen) was then sedimented through a 60/45/30% sucrose gradient at 100 000 g for 2t h. Complete rotavirus particles (purified H.R.V.L. antigen), banding at a density of 1.22, were then used as antigen for immunisation. 1. 2.
Fig. I-Immune E.M. : homologous antiserum. Negative stain of H.R.V.L. particles clumped together by homologous antiserum. (x 90 000.) "
Zissis, G., Lambert, J. P., Dekegel, D. J. clin. Path. (in the press). McLure, A. R., MacFarlane, D. E., Somerville, R. G. Arch. ges. Virusforsch.
1972, 37, 6.
TABLE !——C.F. REACTIVITY OF
12
CRUDE FACAL ROTAVIRUS
SUSPENSIONS OF HUMAN ORIGIN WITH THREE DIFFERENT IMMUNE SERA
Fig. 2-Immune E.M. : heterologous antiserum. Negative stain of H.R.V.L. particles after incubation gous antiserum. (x 30 000.)
with heterolo-
After 5 weeks the guineapig was bled by heart puncture. A chessboard titration was then performed with calf rotavirus
antigen against both a positive human serum and the guineapig serum (G.P. 1). These sera, used at four times their endpoint dilution, were then tested against 12 stools shown tct be rich in rotavirus particles by electronmicroscopy. G.P. 1 antireacted with 9 of the stools and the human serum with all 12 (table i). These results were surprising, especially since technical errors seemed to have been excluded and the titres obtained with the two sera against individual stools 1-9 were not significantly different from each other. We therefore tried again, this time using purified H.R.V.L. antigen extracted from stool no. 12. The procedures were the same as for G.P. 1. G.P. 2 detected as positive the 3 stools which had been negative with G.p.lbut gave negative results with the stools which had been positive with G.P. 1. Again the human serum reacted with all 12 crude antigen extracts. We then titrated the sera against purified H.R.v.L. antigen to see whether the purification procedure might have been responsible for the discrepancies. The results, however, showed a similar pattern (table 11) though the titres were lower. serum
(R)=residual red bloodcells (>95%
hxmolysis).
TABLE 11-C.F. REACTIVITY OF TWO PURIFIED ROTAVIRUS SUSPENSIONS OF HUMAN ORIGIN WITH THREE DIFFERENT IMMUNE
SERA
39 The 12 stool samples had been chosen simply because they all showed a large amount of rotavirus by E.M. Stools 1-8, reacting with G.P. 1, came froma nursery where there had been an epidemic of gastroenteritis. The other 4 came from different general hospitals. The serological results and the origin of the stools thus seemed to favour the existence of at least two serotypes of human rotavirus. Experiments with immune E.M.3 suggest that human and calf rotaviruses share a common group antigen but show antigenic differences in their outer capsid layer. To see whether the type-specific antigens were located at the periphery of the virions we incubated 100 1 of purified H.R.V.L. antigen (types 1 and 2 from stools 7 and 12, respectively) with 100 pi of human serum, G.P. 1, and G.P. 2 (each at four times end-point dilution) at 37°C for 1 h. E.M. grids were then placed on one drop of the mixture, stained, and examined with a Philips 201 instrument. Human serum clumped both rotavirus types in enormous aggregates while the guineapig antisera only did so with the homologous antigen (fig. 1). With the heterologous antiserum the aggregates were absent or few and small (fig. 2). This slight degree of cross-reaction may also explain the small residual quantities of red blood-cells (R) observed in the c.F. tests (tables i and n). Our c.F. and immune E.M. results favour the existence of at least two human rotavirus types.4,s The type-specific receptors are located at the periphery of the virions although slight heterologous agglutinations seem to indicate that the different types might also share a minor surface antigen. It would be most interesting to see whether the neutralisation technique (indirect immunofluorescence) of Flewett et al.6 could confirm these results. Department of Microbiology, Free University of Brussels, B-1000 Brussels, Belgium
G. Zissis
J. P. LAMBERT
SEROTYPES OF HUMAN ROTAVIRUS a serum neutralisation test6 which between rotaviruses from different species. The distinguishes three human sera and human rotavirus strains used in that study were serologically the same. Rotavirus samples sent to us from various world-wide laboratories have now been tested with a Birmingham child’s convalescent serum and with sera raised in rabbits against a rotavirus extracted from a sample of human faeces from Birmingham and another from Vellore, India. The results (see table) show that there are at least two and probably three different serotypes in the samples we have examined. We have previously6 suggested that serum neutralising titres of 1/10 and 1/20 are probably not rotavirus specific. On that basis the convalescent child’s serum neutralised three of the six isolates. The rabbit anti-Birmingham rotavirus serum neutralised the same three sera to high titre and gave a lower titre with the remaining three. We have previously shown6 cross-reactions with a high titre serum, and in the table the ratios of titre of homologous to heterologous virus with each serum are 40:1and 32:1. The rabbit antiserum to the Indian rotavirus neutralised two isolates and a third isolate was neutralised by none of the sera. The Indian rotavirus and South African rotavirus 139 were not neutralised by antisera to calf, pig, and lamb rotavirus. All the viruses described above
SiR,-We have described
3. Flewett, T.
H., Bryden, A. S., Davies, H., Woode, G. N., Bndger, N. C. Lan1974, ii, 61. 4. Zissis, G., Fonteyne, J., Lambert, J. P., Butzler, J. P. Paper read at ScottishScandinavian Conference on Infectious Diseases, held in Uppsala, in June, cet,
1977. 5. Zissis, G.
Paper read at winter meeting of the British Society for the Study of Infection, held in Glasgow, in December, 1977. 6. Thouless, M E., Bryden, A. S., Flewett, T. H., Woode, G. N., Bridger, J. C., Snodgrass, D. R., Herring, J. A. Archs Virol. 1977, 53, 287.
NEUTRALISATION OF ISOLATES OF HUMAN ROTAVIRUS BY HUMAN CONVALESCENT SERUM AND RABBIT ANTISERA.
I
I
I
I
The numbers represent the reciprocal of the serum dilution 50% or greater reduction of fluorescent foci in LLC MK2 cells. *Doubling dilutions of this serum were made from 1/500.
causing a
isolated from children but we cannot be sure that the children with the "new" serotypes were not infected by an animal rotavirus to which we have no antiserum. We also screened a small number of Birmingham rotavirus isolates with the two rabbit sera. One was neutralised by the anti-Indian rotavirus serum and not by the anti-Birmingham serum. The Birmingham serotype is thus found in distant countries and the Indian serotype occurs in Birmingham. We propose, for convenience, to call the original Birmingham isolate serotype 1 and the Indian isolate serotype 2. Without an antiserum to the South Africa 139 isolate we cannot designate it. Information on the total number of serotypes of human rotavirus, which is currently unknown, is crucial for the production of a vaccine. were
We thank Prof. M. M. Mathan, India; Dr T. Johnsson and Prof. T. Wadstrom, Ethiopia; and Prof. O. W. Prozesky, South Africa, for the virus isolates. We also thank those in other countries who have sent us virus samples which will be needed for a comprehensive survey. We would be grateful for 10-20 ml samples of faeces containing rotavirus from any part of the world for antiserum preparation.
Regional Virus Laboratory, East Birmingham Hospital, Birmingham B9 5ST
M. E. THOULESS A. S. BRYDEN T. H. FLEWETT
ANTI-ROTAVIRUS ANTIBODY IN HUMAN COLOSTRUM
SiR,-During investigation of the epidemiology of human rotavirus infection in Costa Rica,’ the anti-human rotavirus capacity of colostrum was studied. We were looking for an explanation for the relatively low incidence of acute diarrhoea! disease among exclusively breast-fed infants. Colostrum was obtained, 1-76 h post partum, from thirty-three women aged 16-27 y from San Jose, the capital of Costa Rica, a city of about half a million inhabitants. Colostrum was used as a blocking agent in the enzymelinked immunosorbent assay (ELISA).2 A "double sandwich" variation of the assay (developed by Dr R. H. Yolken, National Institute of Health, Bethesda, Maryland) permitted a visual reading of the colour. Polyvinyl microtitre plates (Cooke) are precoated with goat anti-rotavirus antibody. Stool suspensions are added in duplicate wells and incubated for 1 h at 37 °C. Guineapig anti-rotavirus serum is added to each well and allowed to react for 1 h at 37° C. A further incubation of 1 h at 370C with goat anti-guineapig antibody labelled with alkaline phosphatase (Sigma) completes the double sandwich. All steps are followed by three washings with phosphate-buffered saline containing 0.5 ml of ’Tween 20’ per litre of solution. Addition of p-nitrophenyl phosphate substrate after a 30 min 1. Mata, L., Lizano, C., Hernández, F., Mohs, E., Herrero, L., Peñaranda, M. E., Gamboa, F., Le6n, J. Med. Hosp. Inf. (Méx.), 1977, 5, 955. 2. Yolken, R. H., Kim, H. W., Clem, T., Wyatt, R. G., Kalica, A. R., Chanock, R. M., Kapikian, A. Z. Lancet, 1977, ii, 163.
tive,
or even beneficial, changes were more frequent among patients in this group. Fears that laparoscopic electrocoagulation leads to disrupted menstrual patterns are thus not warranted. Problems such as bowel burns and the prognosis for later re-anastomosis are of much greater concern, and it is for these reasons that minilaparotomy or the application of mechanical occlusion devices via laparoscopy should replace laparoscopic electrocoagulation in interval female sterilisation.
International Fertility Research Program, North Carolina 27709, U.S.A.
MARGARET F. McCANN ELTON KESSEL
DIFFERENT SEROTYPES OF HUMAN ROTAVIRUSES
SiR,-We have been detecting rotaviruses in stools with a sensitive micro complement-fixation (c.F.) technique for more than two years.’ This method is as sensitive and specific as electronmicroscopy (E.M.), and use of a simple absorption method has avoided difficulties with anticomplementary factors. The only problem is the need for a high-titre human serum, 128 on c.F.; convalescent calf serum is not sensitive enough for the detection of human reovirus-like particles (H.R.V.L.). We have tried to produce guineapig antiserum as an alternative, and this led us, quite by chance, to consider whether different serotypes OfH.R.V.L. might exist. In the first attempt to produce high-titre guineapig serum a guineapig was immunised according to the technique of McLure et a1.2 A fxcal suspension (about 30%, w/v) of stool no. 7 (table I) was twice centrifuged for 10 min, and the supernatant (crude H.R.v.L. antigen) was then sedimented through a 60/45/30% sucrose gradient at 100 000 g for 2t h. Complete rotavirus particles (purified H.R.V.L. antigen), banding at a density of 1.22, were then used as antigen for immunisation. 1. 2.
Fig. I-Immune E.M. : homologous antiserum. Negative stain of H.R.V.L. particles clumped together by homologous antiserum. (x 90 000.) "
Zissis, G., Lambert, J. P., Dekegel, D. J. clin. Path. (in the press). McLure, A. R., MacFarlane, D. E., Somerville, R. G. Arch. ges. Virusforsch.
1972, 37, 6.
TABLE !——C.F. REACTIVITY OF
12
CRUDE FACAL ROTAVIRUS
SUSPENSIONS OF HUMAN ORIGIN WITH THREE DIFFERENT IMMUNE SERA
Fig. 2-Immune E.M. : heterologous antiserum. Negative stain of H.R.V.L. particles after incubation gous antiserum. (x 30 000.)
with heterolo-
After 5 weeks the guineapig was bled by heart puncture. A chessboard titration was then performed with calf rotavirus
antigen against both a positive human serum and the guineapig serum (G.P. 1). These sera, used at four times their endpoint dilution, were then tested against 12 stools shown tct be rich in rotavirus particles by electronmicroscopy. G.P. 1 antireacted with 9 of the stools and the human serum with all 12 (table i). These results were surprising, especially since technical errors seemed to have been excluded and the titres obtained with the two sera against individual stools 1-9 were not significantly different from each other. We therefore tried again, this time using purified H.R.V.L. antigen extracted from stool no. 12. The procedures were the same as for G.P. 1. G.P. 2 detected as positive the 3 stools which had been negative with G.p.lbut gave negative results with the stools which had been positive with G.P. 1. Again the human serum reacted with all 12 crude antigen extracts. We then titrated the sera against purified H.R.v.L. antigen to see whether the purification procedure might have been responsible for the discrepancies. The results, however, showed a similar pattern (table 11) though the titres were lower. serum
(R)=residual red bloodcells (>95%
hxmolysis).
TABLE 11-C.F. REACTIVITY OF TWO PURIFIED ROTAVIRUS SUSPENSIONS OF HUMAN ORIGIN WITH THREE DIFFERENT IMMUNE
SERA
39 The 12 stool samples had been chosen simply because they all showed a large amount of rotavirus by E.M. Stools 1-8, reacting with G.P. 1, came froma nursery where there had been an epidemic of gastroenteritis. The other 4 came from different general hospitals. The serological results and the origin of the stools thus seemed to favour the existence of at least two serotypes of human rotavirus. Experiments with immune E.M.3 suggest that human and calf rotaviruses share a common group antigen but show antigenic differences in their outer capsid layer. To see whether the type-specific antigens were located at the periphery of the virions we incubated 100 1 of purified H.R.V.L. antigen (types 1 and 2 from stools 7 and 12, respectively) with 100 pi of human serum, G.P. 1, and G.P. 2 (each at four times end-point dilution) at 37°C for 1 h. E.M. grids were then placed on one drop of the mixture, stained, and examined with a Philips 201 instrument. Human serum clumped both rotavirus types in enormous aggregates while the guineapig antisera only did so with the homologous antigen (fig. 1). With the heterologous antiserum the aggregates were absent or few and small (fig. 2). This slight degree of cross-reaction may also explain the small residual quantities of red blood-cells (R) observed in the c.F. tests (tables i and n). Our c.F. and immune E.M. results favour the existence of at least two human rotavirus types.4,s The type-specific receptors are located at the periphery of the virions although slight heterologous agglutinations seem to indicate that the different types might also share a minor surface antigen. It would be most interesting to see whether the neutralisation technique (indirect immunofluorescence) of Flewett et al.6 could confirm these results. Department of Microbiology, Free University of Brussels, B-1000 Brussels, Belgium
G. Zissis
J. P. LAMBERT
SEROTYPES OF HUMAN ROTAVIRUS a serum neutralisation test6 which between rotaviruses from different species. The distinguishes three human sera and human rotavirus strains used in that study were serologically the same. Rotavirus samples sent to us from various world-wide laboratories have now been tested with a Birmingham child’s convalescent serum and with sera raised in rabbits against a rotavirus extracted from a sample of human faeces from Birmingham and another from Vellore, India. The results (see table) show that there are at least two and probably three different serotypes in the samples we have examined. We have previously6 suggested that serum neutralising titres of 1/10 and 1/20 are probably not rotavirus specific. On that basis the convalescent child’s serum neutralised three of the six isolates. The rabbit anti-Birmingham rotavirus serum neutralised the same three sera to high titre and gave a lower titre with the remaining three. We have previously shown6 cross-reactions with a high titre serum, and in the table the ratios of titre of homologous to heterologous virus with each serum are 40:1and 32:1. The rabbit antiserum to the Indian rotavirus neutralised two isolates and a third isolate was neutralised by none of the sera. The Indian rotavirus and South African rotavirus 139 were not neutralised by antisera to calf, pig, and lamb rotavirus. All the viruses described above
SiR,-We have described
3. Flewett, T.
H., Bryden, A. S., Davies, H., Woode, G. N., Bndger, N. C. Lan1974, ii, 61. 4. Zissis, G., Fonteyne, J., Lambert, J. P., Butzler, J. P. Paper read at ScottishScandinavian Conference on Infectious Diseases, held in Uppsala, in June, cet,
1977. 5. Zissis, G.
Paper read at winter meeting of the British Society for the Study of Infection, held in Glasgow, in December, 1977. 6. Thouless, M E., Bryden, A. S., Flewett, T. H., Woode, G. N., Bridger, J. C., Snodgrass, D. R., Herring, J. A. Archs Virol. 1977, 53, 287.
NEUTRALISATION OF ISOLATES OF HUMAN ROTAVIRUS BY HUMAN CONVALESCENT SERUM AND RABBIT ANTISERA.
I
I
I
I
The numbers represent the reciprocal of the serum dilution 50% or greater reduction of fluorescent foci in LLC MK2 cells. *Doubling dilutions of this serum were made from 1/500.
causing a
isolated from children but we cannot be sure that the children with the "new" serotypes were not infected by an animal rotavirus to which we have no antiserum. We also screened a small number of Birmingham rotavirus isolates with the two rabbit sera. One was neutralised by the anti-Indian rotavirus serum and not by the anti-Birmingham serum. The Birmingham serotype is thus found in distant countries and the Indian serotype occurs in Birmingham. We propose, for convenience, to call the original Birmingham isolate serotype 1 and the Indian isolate serotype 2. Without an antiserum to the South Africa 139 isolate we cannot designate it. Information on the total number of serotypes of human rotavirus, which is currently unknown, is crucial for the production of a vaccine. were
We thank Prof. M. M. Mathan, India; Dr T. Johnsson and Prof. T. Wadstrom, Ethiopia; and Prof. O. W. Prozesky, South Africa, for the virus isolates. We also thank those in other countries who have sent us virus samples which will be needed for a comprehensive survey. We would be grateful for 10-20 ml samples of faeces containing rotavirus from any part of the world for antiserum preparation.
Regional Virus Laboratory, East Birmingham Hospital, Birmingham B9 5ST
M. E. THOULESS A. S. BRYDEN T. H. FLEWETT
ANTI-ROTAVIRUS ANTIBODY IN HUMAN COLOSTRUM
SiR,-During investigation of the epidemiology of human rotavirus infection in Costa Rica,’ the anti-human rotavirus capacity of colostrum was studied. We were looking for an explanation for the relatively low incidence of acute diarrhoea! disease among exclusively breast-fed infants. Colostrum was obtained, 1-76 h post partum, from thirty-three women aged 16-27 y from San Jose, the capital of Costa Rica, a city of about half a million inhabitants. Colostrum was used as a blocking agent in the enzymelinked immunosorbent assay (ELISA).2 A "double sandwich" variation of the assay (developed by Dr R. H. Yolken, National Institute of Health, Bethesda, Maryland) permitted a visual reading of the colour. Polyvinyl microtitre plates (Cooke) are precoated with goat anti-rotavirus antibody. Stool suspensions are added in duplicate wells and incubated for 1 h at 37 °C. Guineapig anti-rotavirus serum is added to each well and allowed to react for 1 h at 37° C. A further incubation of 1 h at 370C with goat anti-guineapig antibody labelled with alkaline phosphatase (Sigma) completes the double sandwich. All steps are followed by three washings with phosphate-buffered saline containing 0.5 ml of ’Tween 20’ per litre of solution. Addition of p-nitrophenyl phosphate substrate after a 30 min 1. Mata, L., Lizano, C., Hernández, F., Mohs, E., Herrero, L., Peñaranda, M. E., Gamboa, F., Le6n, J. Med. Hosp. Inf. (Méx.), 1977, 5, 955. 2. Yolken, R. H., Kim, H. W., Clem, T., Wyatt, R. G., Kalica, A. R., Chanock, R. M., Kapikian, A. Z. Lancet, 1977, ii, 163.
