- Email: [email protected]
The proof of the cloning is in the eating
TIBTECH - JANUARY 1991 [Vol. 9]
--Fig. 1
incorporated into soluble CD4 protein in Chinese hamster ovary cells 4, higher eukaryotic cells are more sensitive to the unnatural amino acid. However, it is clear that if a protein is already cloned and expressed in E. coli, going MAD might just get you that first ascent.
Heavy-atom solution of the phase problem. (a) Geometric representation of a single diffracted X ray from a protein crystal The red circle represents the unknown phase angle (0 to 360 degrees). The radius, Fp, is determined from the square root of the X-ray intensity measurement. Location of a heavy atom diffused into the protein crystal allows calculation of the ampfitude and of the phase of its contribution to the X-ray intensity. This is represented by the vector Fh. The heavy atom changes the diffracted X-ray intensity such that the amplitude is now Fph. Drawing a second circle (black outline) of this diameter, centred at the end of Fh, it can be seen that this cuts the first circle at two points. These are the two choices of phase angle. (b) is the same diffracted X ray as in (a), but now the X rays are close to the absorption edge of the heavy atom h. An additional (anomalous) diffraction occurs which advances the phase angle of Fh and changes its amplitude (Fh + Fh "). This can be calculated as before. Now a third (thicker) circle can be drawn with radius Fph" and this determines the correct choice of phase. In MAD, the heavy atom h is already part of the structure, so that additional wavelengths are used to provide the information obtained in (a) by the isomorphous derivative.
The proof of the cloning is in the eating In 1991, there will be a large number of applications to food-regulatory bodies [e.g. Food and Drugs Administration {FDA) in the USA, and the Ministry of Agriculture, Fisheries and Food (MAFF), in the UK], for approval of genetically engineered living material as food for human consumption. It will be a concerted onslaught, involving microorganisms, plants and higher animals, which will keep the special committees (Government Advisory Committee on Novel Foods, Advisory Committee for Genetic Manipulation) very busy indeed. How will the regulators act and the public react?
ranged so that a change in the animals' diet would greatly increase circulating levels of the hormone. Unlike the famous 'giant' mice z, the pigs did not show dramatic size increases, but instead had very lean muscle tissue - a desirable commercial feature. Unfortunately, the constitutively high levels of growth hormone led to diabetes, sterility and other pathologies. Insufficient attention had been paid to the endocrinologists 1, since release of growth hormone is known to be periodic and not constitutive. However, the potential for modifying livestock was established.
Livestock modification The tip of the iceberg was exposed in 1988-1989. In the USA, thousands of domestic pig embryos were injected with transgenes encoding growth hormones (somatotropins) a. The promoters for these were ar-
Modification of plants, yeast and fish In the world of plants, ICI has produced transgenic tomatoes which express an antisense mRNA, designed to reduce the amount of a cell-wall degrading enzyme. Such tomatoes can be stored, without
~) 1991, Elsevier Science Publishers Ltd (UK) 0167- 9430FJ0/$2.00
References 1 Hendrickson, W. A., Horton, J. R. and LeMaster, D.M. (1990) EMBO J. 9, 1665-1672 2 Bockhoven, C., Schoone, J. C. and Bijvoet, J, M. (1951) Acta Crystallogr. 4, 275-284 3 Yang, W., Hendrickson, W. A., Crouch, R. J. and Satow, Y. (1990) Science 249, 1398-1405 4 Deem K. C., McDougal, J. S., Inacker, R. Folena-Wasserman, G., Arthos, J., Rosenberg, J., Madden, P.J., Axel, R. and Sweet, R.W. (1988) Nature 331, 82-84 MICHAEL GEISOW
Biodigm, 115 Main Street, East Bridgeford, Nottingham NG13 8NH, UK.
softening, for prolonged periods. Meanwhile, in the Netherlands, the Dutch yeast company Gist-Brocades gained approval for the use of a genetically engineered yeast in human food. Expression of the maltose-transport and -utilizing gone products was increased by means of a more efficient transcriptional promoter from the same strain of yeast. More carbon dioxide is produced, which decreases the rising time of the dough. It is also worth noting that the same company has been selling recombinant chvmosin {rennin}, for cheese produciion in Europe for two years, and expects FDA approval for sale in the USA shortly. The natural source of chymosin is the cow stomach. As described in a recent TIBTECH review 3, progress has been made with producing transgenic fish, incorporating commercially important features such as improved resistance to disease and to cold. Such new species will certainly be on the regulators' agendas, and possibly soon on their menus. The science which has led to this transgenic cornucopia represents an
6
TIBTECH - JANUARY 1991 [Vol. 9]
,I astounding leap in our understanding of the structures and controlled expression of genes 4. Several authors, some time ago, predicted that the introduction of 'desirable' characteristics in plants and animals would be almost as slow by biotechnology as by classical breeding, because not just one, but many genes would have to be changed. The ambitious animalgenome-cloning programmes, upon which many countries are now embarked, are likely to weaken that very reasonable argument. When all aspects of the genetic engineering of microorganisms, plants and animals are considered, we have a 'green' industry of major economic importance worldwide. Naturally, more than mere economic issues are involved and over the next few years the companies concerned, as well as the consumers, must come to trust the regulators. The omens are quite favourable. The current views of US food safety advisors and the International Food Biotechnology Council (IFBC)*'5 are that food products which are the outcome of genetic manipulation are 'likely to be safe' for consumption. The IFBC also considers that the existing regulatory bodies and practices are sufficient for safety assessment of new foods derived by genetic engineering.
'Green' politics Although Europe is in some ways ahead of the USA in approvals for the 'fruits' of gene manipulation, there are problem areas. In Germany, there is a popular reaction against genetically modified microorganisms, which presumably extends to genetically modified plants and animals. In the USSR, accidents which have occurred at single-cell-protein manufacturing, and the levels of industrial pollution generally, have made obtaining building approval for biotechnology factories nearly impossible. In both the USSR and Germany, 'green' politics are likely to block food biotechnology developments for some time to come. Risk assessment- the rational approach Regulators are naturally conservative, but the 1980s have seen a *The IFBC consists of 30 member companies from the food-processing and biotechnology industries.
/If
'1/(it growing sense of the 'predictability' of the outcome of genetic manipulation and the business of 'risk assessment' associated with it. Regulators appear to be taking an increasingly pragmatic approach to the new technology rather t h a n ' . . , but what if the unthinkable 1 in 1012 actually h a p p e n e d . . ?'. The rational approach is to rank the known risks, and the gene product itself is top of this list. It isn't difficult to see that expression of ricin in a plant crop may confer resistance to insects but will also result in potentially unsafe food. The expression of a protein injurious to human health in animals is less likely, since the animal's own development or health would most probably be affected. This is in any case unlikely, since by their very nature, the sequences of transgenes are completely defined. It could be argued that a hazard might arise indirectly from incorporation of a transgene, through mutation of the inserted DNA or by deletion, rearrangement or inappropriate expression of adjacent genetic material. The rational answer to this concern is that such processes would be 'extremely unlikely' to produce a principle toxic to humans (although there is a potential for adversely affecting the development of the transgenic plant or animal). Gene deletion, mutation, transposition and rearrangement have been occurring naturally, without man's help, for millenia and have given rise to very few toxic species. Those poisonous plants and animals which have arisen have done so under the pressure of natural selection. The
'/
classical methods of plant and animal breeding employed by man for thousands of years have not given rise to lethal cereals or to poisonous meat. Most objective hazards in foodstuffs come from microbial contamination, or from secondary metabolites such as fungal toxins. All are well known because of thei: devastating effects. The transgene itself cannot represent more risk than any other DNA which is consumed regularly. Transgenes are usually inserted in a form in which the vector (bacteria or phage) DNA has been removed, so that the direct transmissable potential has been lost. If the transgenic species is created by means of a retroviral vector, then there is risk of subsequent infection although, usually, vectors are not infective, but propagated by means of a helper cell line which produces a component in which the provirus is defective. Human retroviruses are rather unlikely to receive regulatory approval as vectors used in the transgenesis of food organisms. Ingested, or even injected, viruses which lack coat proteins (i.e. only naked DNA) appear to represent a very low infectivity; the principle concern here is the potential for oncogenesis. This is a controversial area because of diseases like scrapie and bovine spongiform encephalopathy (BSE) where the nature of the infective agent is still in doubt. Similar concerns are faced by regulators considering the potential for harm of tiny amounts of foreign DNA present in therapeutic proteins derived from genetically engineered hosts. Workers who have tried to
TIBTECH- JANUARY 1991 [Vol. 9]
7
assess this risk injected DNA extracted from transformed Chinese hamster ovary (CHO), cell lines. The DNA was non-tumourigenic even when injected in the same species. The selective breeding of plants and animals both for the serious purpose of human food and the relatively frivolous one of winning competitions has continued since records began. Little harm - to humans - has arisen from this activity. Now genetic engineering promises more rapid and focused improvements in the age-old and economically vital processes of fermentation, cereal production, grox~h of fruit and vegetables and animal
husbandry. All responsible scientists and regulators alike have a duty to explain what they are dning, to demystify the science and provide the reassurance that will certainly be demanded. Man is not 'controlling, subjecting or perverting life'. He has the power to make genetic changes in microorganisms, plants and animals of great potential benefit. This power must be u.sed, and be seen to be used, wisely.
References
MICHAEL GE1SOW
1 Pursel, V. G., Pinkert, C. A., Miller, K. F., Bolt, D. J., Cambell, R. G., Biodigm, 115 Main Street, East Palmiter, R.D., Brinster, R.L. and Bridgeford, Nottingham NG13 8,~H, Hammer, R.D. (1989) Science 244, UK.
Progress towards rabies control Morag Ferguson Although safe and efficacious tissue-culture-derived rabies vaccines are available in developed countries, much of the w o r l d still depends on vaccines d e r i v e d f r o m n e u r a l tissue which w e r e i n t r o d u c e d h a l f a c e n t u r y ago. C o n s i d e r a b l e a d v a n c e s have been m a d e in our understanding of the molecular biology of r a b i e s virus, and genetically engineered recombinant viruses ( v a c c i n i a - r a b i e s v i r u s glycoprotein) have been developed. These may facilitate the control of rabies in some species by o r a l v a c c i n a t i o n c a m p a i g n s . world: in foxes in Europe and in skunks and raccoons in the USA. However, some countries, mainly islands and those with physical barriers at their frontiers, are free from rabies. The majority of cases of human rabies result from dog bites and the control of the dog population is therefore important. In countries where universal immunization of dogs has been introduced, the decline of rabies in man has paralleled the decline of disease in M. Ferguson is at the Division of dogs.
Rabies is a viral infection of the central nervous system and is transmitted by the bite of infected animals. Not all bites by laboratoryproven rabid animals result in the development of rabies since the virus may fail to become established. However, if clinical signs do develop, the disease is almost invariably fatal. Rabies can occur in all mammals and is enzootic in many parts of the
Virology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hefts EN6 3QG, UK.
1281-1284 2 Palmiter, R. D., Brinster, R. L., Manner, R.E., Trumb,~uer, M.E., Rosenfeld, M. G., Birnber, N. C. and Evans, R. M. (1982) Nature 300, 611-615 3 Chen, T. T. and Powers, D. A. (1990) Trends Biotechnm'. 8, 209-215 4 Palmiter, R. D. ai:d Brinster, R. L. (1986) Annu. Rev. Genet. 20, 465-499 5 Biotechnologies and Food: Assuring the Safety of Foods Produced by Genetic Modification, ,International Food Biotechnology Council, Washington DC, USA.)
The structure of rabies virus Rabies virus can be isolated from the b:'ain or saliva of an infected
~) 1991. Elsevier Science Publishers Ltd (UK) 0167 - 9430190/$2.00
animal by intracerebral inoculation of mice. By repeated passage of wild ('street') isolates, the biological properties of the virus become altered so that the incubation period becomes less variable ('fixed'). The rabies virus is a bullet-shaped particle 170 x 75 nm and has the characteristic morphology of the family Rhabdoviridae (Fig. 1). The virus contains a helical nucleocapsid core comprising singlestranded RNA, nucleoprotein, phosphoprotein and the virion transcriptase. This core is surrounded by an envelope which consists of lipids derived from the host-cell plasma membrane. 'Spikes' projecting from the surface of the virus are composed of a glycoprotein which is the major factor responsible for the induction of virus neutralizing antibodies and which confers protection against lethal infection 1. The sequence of the gene encoding the glycoprotein has been determined 2 and this has enabled an analysis of the antigenic structure of the glycoprotein to be carried out 3. Monoclonal antibodies directed against the glycoprotein of rabies virus have been used to select antigenic variants resistant to neutralization and several independent antigenic sites have been mapped on the glycoprotein. Amino acid substitutions at a single residue within one of these sites have been shown to affect pathogenicity 4. The antigenic diversity of strains isolated from different species and at different geographical locations has
--Fig. 1
incorporated into soluble CD4 protein in Chinese hamster ovary cells 4, higher eukaryotic cells are more sensitive to the unnatural amino acid. However, it is clear that if a protein is already cloned and expressed in E. coli, going MAD might just get you that first ascent.
Heavy-atom solution of the phase problem. (a) Geometric representation of a single diffracted X ray from a protein crystal The red circle represents the unknown phase angle (0 to 360 degrees). The radius, Fp, is determined from the square root of the X-ray intensity measurement. Location of a heavy atom diffused into the protein crystal allows calculation of the ampfitude and of the phase of its contribution to the X-ray intensity. This is represented by the vector Fh. The heavy atom changes the diffracted X-ray intensity such that the amplitude is now Fph. Drawing a second circle (black outline) of this diameter, centred at the end of Fh, it can be seen that this cuts the first circle at two points. These are the two choices of phase angle. (b) is the same diffracted X ray as in (a), but now the X rays are close to the absorption edge of the heavy atom h. An additional (anomalous) diffraction occurs which advances the phase angle of Fh and changes its amplitude (Fh + Fh "). This can be calculated as before. Now a third (thicker) circle can be drawn with radius Fph" and this determines the correct choice of phase. In MAD, the heavy atom h is already part of the structure, so that additional wavelengths are used to provide the information obtained in (a) by the isomorphous derivative.
The proof of the cloning is in the eating In 1991, there will be a large number of applications to food-regulatory bodies [e.g. Food and Drugs Administration {FDA) in the USA, and the Ministry of Agriculture, Fisheries and Food (MAFF), in the UK], for approval of genetically engineered living material as food for human consumption. It will be a concerted onslaught, involving microorganisms, plants and higher animals, which will keep the special committees (Government Advisory Committee on Novel Foods, Advisory Committee for Genetic Manipulation) very busy indeed. How will the regulators act and the public react?
ranged so that a change in the animals' diet would greatly increase circulating levels of the hormone. Unlike the famous 'giant' mice z, the pigs did not show dramatic size increases, but instead had very lean muscle tissue - a desirable commercial feature. Unfortunately, the constitutively high levels of growth hormone led to diabetes, sterility and other pathologies. Insufficient attention had been paid to the endocrinologists 1, since release of growth hormone is known to be periodic and not constitutive. However, the potential for modifying livestock was established.
Livestock modification The tip of the iceberg was exposed in 1988-1989. In the USA, thousands of domestic pig embryos were injected with transgenes encoding growth hormones (somatotropins) a. The promoters for these were ar-
Modification of plants, yeast and fish In the world of plants, ICI has produced transgenic tomatoes which express an antisense mRNA, designed to reduce the amount of a cell-wall degrading enzyme. Such tomatoes can be stored, without
~) 1991, Elsevier Science Publishers Ltd (UK) 0167- 9430FJ0/$2.00
References 1 Hendrickson, W. A., Horton, J. R. and LeMaster, D.M. (1990) EMBO J. 9, 1665-1672 2 Bockhoven, C., Schoone, J. C. and Bijvoet, J, M. (1951) Acta Crystallogr. 4, 275-284 3 Yang, W., Hendrickson, W. A., Crouch, R. J. and Satow, Y. (1990) Science 249, 1398-1405 4 Deem K. C., McDougal, J. S., Inacker, R. Folena-Wasserman, G., Arthos, J., Rosenberg, J., Madden, P.J., Axel, R. and Sweet, R.W. (1988) Nature 331, 82-84 MICHAEL GEISOW
Biodigm, 115 Main Street, East Bridgeford, Nottingham NG13 8NH, UK.
softening, for prolonged periods. Meanwhile, in the Netherlands, the Dutch yeast company Gist-Brocades gained approval for the use of a genetically engineered yeast in human food. Expression of the maltose-transport and -utilizing gone products was increased by means of a more efficient transcriptional promoter from the same strain of yeast. More carbon dioxide is produced, which decreases the rising time of the dough. It is also worth noting that the same company has been selling recombinant chvmosin {rennin}, for cheese produciion in Europe for two years, and expects FDA approval for sale in the USA shortly. The natural source of chymosin is the cow stomach. As described in a recent TIBTECH review 3, progress has been made with producing transgenic fish, incorporating commercially important features such as improved resistance to disease and to cold. Such new species will certainly be on the regulators' agendas, and possibly soon on their menus. The science which has led to this transgenic cornucopia represents an
6
TIBTECH - JANUARY 1991 [Vol. 9]
,I astounding leap in our understanding of the structures and controlled expression of genes 4. Several authors, some time ago, predicted that the introduction of 'desirable' characteristics in plants and animals would be almost as slow by biotechnology as by classical breeding, because not just one, but many genes would have to be changed. The ambitious animalgenome-cloning programmes, upon which many countries are now embarked, are likely to weaken that very reasonable argument. When all aspects of the genetic engineering of microorganisms, plants and animals are considered, we have a 'green' industry of major economic importance worldwide. Naturally, more than mere economic issues are involved and over the next few years the companies concerned, as well as the consumers, must come to trust the regulators. The omens are quite favourable. The current views of US food safety advisors and the International Food Biotechnology Council (IFBC)*'5 are that food products which are the outcome of genetic manipulation are 'likely to be safe' for consumption. The IFBC also considers that the existing regulatory bodies and practices are sufficient for safety assessment of new foods derived by genetic engineering.
'Green' politics Although Europe is in some ways ahead of the USA in approvals for the 'fruits' of gene manipulation, there are problem areas. In Germany, there is a popular reaction against genetically modified microorganisms, which presumably extends to genetically modified plants and animals. In the USSR, accidents which have occurred at single-cell-protein manufacturing, and the levels of industrial pollution generally, have made obtaining building approval for biotechnology factories nearly impossible. In both the USSR and Germany, 'green' politics are likely to block food biotechnology developments for some time to come. Risk assessment- the rational approach Regulators are naturally conservative, but the 1980s have seen a *The IFBC consists of 30 member companies from the food-processing and biotechnology industries.
/If
'1/(it growing sense of the 'predictability' of the outcome of genetic manipulation and the business of 'risk assessment' associated with it. Regulators appear to be taking an increasingly pragmatic approach to the new technology rather t h a n ' . . , but what if the unthinkable 1 in 1012 actually h a p p e n e d . . ?'. The rational approach is to rank the known risks, and the gene product itself is top of this list. It isn't difficult to see that expression of ricin in a plant crop may confer resistance to insects but will also result in potentially unsafe food. The expression of a protein injurious to human health in animals is less likely, since the animal's own development or health would most probably be affected. This is in any case unlikely, since by their very nature, the sequences of transgenes are completely defined. It could be argued that a hazard might arise indirectly from incorporation of a transgene, through mutation of the inserted DNA or by deletion, rearrangement or inappropriate expression of adjacent genetic material. The rational answer to this concern is that such processes would be 'extremely unlikely' to produce a principle toxic to humans (although there is a potential for adversely affecting the development of the transgenic plant or animal). Gene deletion, mutation, transposition and rearrangement have been occurring naturally, without man's help, for millenia and have given rise to very few toxic species. Those poisonous plants and animals which have arisen have done so under the pressure of natural selection. The
'/
classical methods of plant and animal breeding employed by man for thousands of years have not given rise to lethal cereals or to poisonous meat. Most objective hazards in foodstuffs come from microbial contamination, or from secondary metabolites such as fungal toxins. All are well known because of thei: devastating effects. The transgene itself cannot represent more risk than any other DNA which is consumed regularly. Transgenes are usually inserted in a form in which the vector (bacteria or phage) DNA has been removed, so that the direct transmissable potential has been lost. If the transgenic species is created by means of a retroviral vector, then there is risk of subsequent infection although, usually, vectors are not infective, but propagated by means of a helper cell line which produces a component in which the provirus is defective. Human retroviruses are rather unlikely to receive regulatory approval as vectors used in the transgenesis of food organisms. Ingested, or even injected, viruses which lack coat proteins (i.e. only naked DNA) appear to represent a very low infectivity; the principle concern here is the potential for oncogenesis. This is a controversial area because of diseases like scrapie and bovine spongiform encephalopathy (BSE) where the nature of the infective agent is still in doubt. Similar concerns are faced by regulators considering the potential for harm of tiny amounts of foreign DNA present in therapeutic proteins derived from genetically engineered hosts. Workers who have tried to
TIBTECH- JANUARY 1991 [Vol. 9]
7
assess this risk injected DNA extracted from transformed Chinese hamster ovary (CHO), cell lines. The DNA was non-tumourigenic even when injected in the same species. The selective breeding of plants and animals both for the serious purpose of human food and the relatively frivolous one of winning competitions has continued since records began. Little harm - to humans - has arisen from this activity. Now genetic engineering promises more rapid and focused improvements in the age-old and economically vital processes of fermentation, cereal production, grox~h of fruit and vegetables and animal
husbandry. All responsible scientists and regulators alike have a duty to explain what they are dning, to demystify the science and provide the reassurance that will certainly be demanded. Man is not 'controlling, subjecting or perverting life'. He has the power to make genetic changes in microorganisms, plants and animals of great potential benefit. This power must be u.sed, and be seen to be used, wisely.
References
MICHAEL GE1SOW
1 Pursel, V. G., Pinkert, C. A., Miller, K. F., Bolt, D. J., Cambell, R. G., Biodigm, 115 Main Street, East Palmiter, R.D., Brinster, R.L. and Bridgeford, Nottingham NG13 8,~H, Hammer, R.D. (1989) Science 244, UK.
Progress towards rabies control Morag Ferguson Although safe and efficacious tissue-culture-derived rabies vaccines are available in developed countries, much of the w o r l d still depends on vaccines d e r i v e d f r o m n e u r a l tissue which w e r e i n t r o d u c e d h a l f a c e n t u r y ago. C o n s i d e r a b l e a d v a n c e s have been m a d e in our understanding of the molecular biology of r a b i e s virus, and genetically engineered recombinant viruses ( v a c c i n i a - r a b i e s v i r u s glycoprotein) have been developed. These may facilitate the control of rabies in some species by o r a l v a c c i n a t i o n c a m p a i g n s . world: in foxes in Europe and in skunks and raccoons in the USA. However, some countries, mainly islands and those with physical barriers at their frontiers, are free from rabies. The majority of cases of human rabies result from dog bites and the control of the dog population is therefore important. In countries where universal immunization of dogs has been introduced, the decline of rabies in man has paralleled the decline of disease in M. Ferguson is at the Division of dogs.
Rabies is a viral infection of the central nervous system and is transmitted by the bite of infected animals. Not all bites by laboratoryproven rabid animals result in the development of rabies since the virus may fail to become established. However, if clinical signs do develop, the disease is almost invariably fatal. Rabies can occur in all mammals and is enzootic in many parts of the
Virology, National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Hefts EN6 3QG, UK.
1281-1284 2 Palmiter, R. D., Brinster, R. L., Manner, R.E., Trumb,~uer, M.E., Rosenfeld, M. G., Birnber, N. C. and Evans, R. M. (1982) Nature 300, 611-615 3 Chen, T. T. and Powers, D. A. (1990) Trends Biotechnm'. 8, 209-215 4 Palmiter, R. D. ai:d Brinster, R. L. (1986) Annu. Rev. Genet. 20, 465-499 5 Biotechnologies and Food: Assuring the Safety of Foods Produced by Genetic Modification, ,International Food Biotechnology Council, Washington DC, USA.)
The structure of rabies virus Rabies virus can be isolated from the b:'ain or saliva of an infected
~) 1991. Elsevier Science Publishers Ltd (UK) 0167 - 9430190/$2.00
animal by intracerebral inoculation of mice. By repeated passage of wild ('street') isolates, the biological properties of the virus become altered so that the incubation period becomes less variable ('fixed'). The rabies virus is a bullet-shaped particle 170 x 75 nm and has the characteristic morphology of the family Rhabdoviridae (Fig. 1). The virus contains a helical nucleocapsid core comprising singlestranded RNA, nucleoprotein, phosphoprotein and the virion transcriptase. This core is surrounded by an envelope which consists of lipids derived from the host-cell plasma membrane. 'Spikes' projecting from the surface of the virus are composed of a glycoprotein which is the major factor responsible for the induction of virus neutralizing antibodies and which confers protection against lethal infection 1. The sequence of the gene encoding the glycoprotein has been determined 2 and this has enabled an analysis of the antigenic structure of the glycoprotein to be carried out 3. Monoclonal antibodies directed against the glycoprotein of rabies virus have been used to select antigenic variants resistant to neutralization and several independent antigenic sites have been mapped on the glycoprotein. Amino acid substitutions at a single residue within one of these sites have been shown to affect pathogenicity 4. The antigenic diversity of strains isolated from different species and at different geographical locations has