SCOPE 44 - Introduction of Genetically Modified Organisms into the Environment

10.1 INTRODUCTION

In the mid-1960s my colleagues and I were engaged in experiments on animal virus genetics, using vaccinia virus and a vaccine strain of poliovirus. One of my research students was Joe Sambrook, later to become head of the Tumor Virology Laboratory at Cold Spring Harbor. I asked Sir Macfarlane Burnet to examine his thesis. It was a good thesis and no problems arose with its acceptance, but it prompted Burnet (1966) to write a provocative article in the journal Lancet entitled `Men or molecules? A tilt at molecular biology'. He commented that, although genetic manipulation of bacteriophages was harmless, `its inevitable extension to such readily manipulated viruses as poliomyelitis and vaccinia raises some very serious dangers'. He went on to envisage the possible consequences of the emergence of a genetic variant of poliovirus with high virulence but serologically quite different from polioviruses 1, 2, and 3. If such a virus was produced, he foresaw `the almost unimaginable catastrophe of a "virgin-soil" epidemic of poliomyelitis involving all the populous regions of the world'.

Some years later recombinant-DNA techniques were discovered. Some of the molecular biologists who developed these methods shared Burnet's misgivings, and convened the Asilomar Conference. Their concern was largely with the potential danger to humans of the products of recombinant-DNA techniques utilizing tumor viruses like SV40 and the retroviruses. There were many biochemists at the Asilomar Conference but few epidemiologists, and the result was something of an overreaction, because none of the prominent speakers knew much about how infectious agents spread between vertebrate hosts. We have come a long way since then, and this conference is an attempt to address the question, can organisms modified by recombinant-DNA techniques become established and spread in the environment. and what will be the consequences if they do, to man and his domesticated animals and plants and to ecosystems? Are the consequences likely to be more or less advantageous and more or less harmful to man and to ecosystems than the introduction of organisms changed by other methods, such as conventional plant and animal breeding?

Coming to the topic assigned to me, `Patterns of establishment and spread', I will restrict my talk to consideration of the question: how are animal viruses transmitted from one animal to another and how do they become established and survive in the environment, i.e. in populations of animals? Most of the general principles that I will outline are equally applicable to other infectious agents, such as bacteria and protozoa. After briefly considering some aspects of epidemiological theory (for reviews, see Evans, 1989; Fenner et al ., 1987; White and Fenner, 1986), I will look at some examples with `normal' viruses that may be taken as models of the possible establishment of genetically modified organisms in the environment. I will then discuss live virus vaccines, for most of these are produced by the genetic manipulation of virulent viruses, and finally describe recombinant vaccinia virus vaccines, which are currently being considered for use in human and veterinary medicine, and thus for widespread and large-scale introduction into the environment.

10.2 VERTICAL TRANSMISSION

To become established in the environment, viruses must be transmitted from one animal to another, in an ongoing series of infections. They may be transmitted from one animal to another either vertically or horizontally. `Vertical transmission' means transmission from parents to offspring via the egg or sperm, either as a provirus integrated in the cell's DNA or in mammals, via the placenta or during the birth process. Other modes of transmission are designated `horizontal'. When considering the establishment of any novel virus in the environment, the initial problem is of course horizontal transmission, but if this occurred, vertical transmission could be important in helping to ensure the indefinite maintenance in a vertebrate population of a virus that had been introduced by horizontal transmission. 

10.3 HORIZONTAL TRANSMISSION

Horizontal transmission can occur across any surface of the body: skin, respiratory mucous membrane, genital tract, and, more rarely, the urinary tract or conjunctiva. The route of infection is fairly characteristic of the viral group (Table 10.1), although some viruses are transmitted by more than one route.

Table 10.1 Some examples of routes of  infection of viruses of different genera

10.4 SPREAD IN THE BODY

Whatever their route of entry, viruses may remain localized, for example in the skin or upper or lower respiratory tract, or they may spread to other parts of the body, usually via the lymphatic system and the blood stream. Viruses introduced by inoculation, either by arthropods or by needle, almost always produce generalized infections. Whether a virus spreads within the animal or not affects the nature of the disease it causes and how it spreads to other animals. Most respiratory viruses multiply only in the respiratory tract and are excreted by sneezing, coughing, etc. A few, such as measles and chickenpox, enter via the respiratory tract and produce a generalized disease, but spread to other animals occurs from lesions produced in the upper respiratory tract during the rash stage of the disease, 14-21 days after infection. Likewise, most viruses that replicate in the alimentary tract remain localized, but a few, like polioviruses and hepatitis A virus, can cause generalized infections.

10.5 SHEDDING

To survive in nature, infectious agents must either be transmitted vertically, in the germplasm or during the birth process, or they must be shed, so that they can infect other individuals by horizontal infection. Shedding usually occurs via the same surface as the virus enters, but it may occur for only a brief period, as in common colds, for several weeks, as in many enterovirus infections, or recurrently, perhaps throughout life, as in herpesvirus and lentivirus infections.

10.6 INAPPARENT INFECTIONS

In closing this introductory section, we need to note the important distinction to be made between 'infection' and `disease'. Survival of a virus in nature depends upon the maintenance of infection; the occurrence of disease is neither required nor necessarily advantageous. Infection without disease is called 'inapparent' or 'subclinical' infection.

Overall, inapparent infections are much more common than those that result in disease; for example in poliomyelitis due to wild-type polioviruses there may be hundreds of inapparent infections for every paralytic case, in arbovirus encephalitis the ratio of inapparent infections to encephalitis is 1000 to 1. Although clinical cases may be somewhat more productive sources of infectious virus than inapparent infections, the latter are usually more numerous and do not restrict the movement of infected individuals, and thus they provide an important method of viral dissemination.

10.7 SURVIVAL OF VIRUSES IN NATURE

I will now review some of the factors that affect the survival of viruses in nature (Table 10.2).

10.7.1 TRANSMISSIBILITY AND VIRULENCE

The best-documented demonstration of how the virulence of a virus directly affects the probability of its survival in nature is myxomatosis of rabbits. In this disease the virus is mechanically transmitted by biting arthropods ('flying pins'), and transmission is most effective when the diseased rabbit maintains highly infectious skin lesions for a long period. Very virulent strains of myxoma virus kill rabbits too quickly for optimum transmission, whereas the lesions caused by highly attenuated strains have too low a concentration of virus particles in the skin lesions for the mouthparts of biting arthropods to become contaminated (Fenner and Ratcliffe, 1965). On theoretical grounds, we predicted that viruses at either extreme of the virulence range would not survive in nature, and this has been confirmed by data from Australia and Europe over the last 35 years (Fenner and Ross, 1990).

10.7.2 CRITICAL COMMUNITY SIZE

The importance of this factor is best exemplified by two common infections of children, measles and chickenpox, which can survive indefinitely in populations of very different sizes, because of different patterns of shedding (Black, 1980). The incubation period of measles is about 12 days and an attack is followed by solid immunity to reinfection. Once a person has had measles and been infectious for about a week at the height of the disease, he or she never becomes infectious again, i.e. recurrent infectivity is unknown. Persistence of measles virus in a community therefore depends upon a continuous supply of susceptible subjects, usually supplied by new births. Measles spreads more readily in winter, and the infection of many susceptibles at this time may cause difficulty in summer, when its transmission is less efficient. In urban areas, where it spreads quickly, as many as 4000 births a year may be needed to sustain the virus (Bartlett, 1960). In rural areas, where it spreads more slowly, a smaller number may suffice, but 1000 births per year seems to be the lower limit (Black, 1966). These numbers of births imply populations of between 200 000 and one million persons. Large epidemics can occur in smaller populations and may persist for a considerable time, but sooner or later `fade-out' occurs and the disease disappears until there is a new introduction.

Table 10.2 Factors affecting the survival of viruses in nature

Chickenpox is also an acute exanthem in which infection is followed by lifelong immunity to reinfection, but it differs radically from measles in the pattern of shedding of the virus. Outbreaks of chickenpox are due to respiratory spread to contacts during the acute stage of the disease, but as in all herpesvirus infections, recurrent shedding occurs, often many years after the initial attack. Varicella virus, after being latent in the sensory nerve ganglia, may be reactivated and cause a disease called zoster (shingles). For this reason, the virus is usually called `varicella-zoster virus'. Although zoster is only about one quarter as infectious as chickenpox (secondary attack rates of 15%, compared with 60-70% for varicella), it can produce chickenpox in susceptible children (Hope-Simpson, 1952, 1954; Gordon, 1962). Since zoster may occur 50 or more years after an attack of chickenpox, varicella-zoster virus requires a dramatically smaller critical community size for indefinite persistence; less than 1000, compared with about 500 000 for measles.

10.7.3 PERSISTENT INFECTIONS

Viral persistence is of considerable epidemiological importance because it is usually, although not always, associated with continuous or recurrent viral shedding, a mechanism that greatly enhances the likelihood of viral survival by providing recurring opportunities for horizontal spread to other host animals. Many viruses characteristically cause persistent infections. Herpesviruses are the best studied; lentiviruses, including the human immunodeficiency viruses, constitute another important group in which virus persists for the life of the host.

10.7.4 ANTIGENIC DRIFT AND SHIFT

The influenza viruses provide the best example of how changes in the antigens involved in evoking a protective immune response may enable a virus to survive in human populations (Murphy and Webster, 1985). Between pandemics, influenza survives in man by sequential mutations in critical antigenic sites in the haemagglutinin which allow the virus to spread in a population in which many individuals have antibodies due to prior infection with an earlier version of the same viral subtype. Such changes in the antigenic structure of the haemagglutinin are called `antigenic drift'. Sometimes reassortants arise by recombination between a human and an animal (swine or avian) influenza A virus which result in the introduction of a novel (for man) haemagglutinin or neurammadase gene. Sometimes such a reassortant spreads in humans, and since the global human population is non-immune, a pandemic of influenza results. This process is called `antigenic shift'; two instances have been documented since the human influenza virus was first isolated in 1933: Asian influenza in 1957 (H1N1 to H2N2) and Hong Kong influenza in 1968 (H2N2 to H3N2).

10.7.5 MULTIPLE-HOST INFECTIONS

Most viral infections of man and domestic animals are maintained in nature within human or domestic animal populations. However, a number of viruses can spread naturally between different species of vertebrate host; the term `zoonosis' is used to describe a multiple-host infection that is naturally transmissible from lower animals to man. Viral infections of livestock may also have wildlife reservoirs. Many such diseases are caused by arboviruses, and can be spread from one species of vertebrate host to another, as well as from one member to another of the same species, by the bite of an infected arthropod.

When arthropods are active, arboviruses multiply alternately in vertebrate and invertebrate hosts. It is more difficult to understand what happens to the virus during the winter months in temperate climates, when the arthropod vectors are frequently inactive. An important mechanism of such 'overwintering' is transovarial transmission from one generation of arthropods to the next. Long known to occur with the tick-borne flaviviruses, this type of vertical transmission has more recently been discovered to occur also with some mosquito-borne bunyaviruses and flaviviruses as well.

10.8 SOME EXAMPLES OF ESTABLISHMENT AND SPREAD IN NEW ENVIRONMENTS

So much for background epidemiology. I will now examine a number of situations which illustrate various facets of the establishment of different viruses in geographically or ecologically novel environments. As is the case with biological invasions of plants and animals, my examples involve viruses that had previously established ecological niches for themselves in other environments; genetically modified viruses may be incapable of spreading in nature under any ecological conditions. Indeed that has been a condition for the licensing of some viral vaccines, e.g., rubella virus vaccine. I will begin with some examples of poxvirus infections.

10.8.1 MYXOMA VIRUS 

Experiments in Californian lagomorphs

Leporipoxvirus, the genus to which myxoma virus belongs, evolved in parallel with lagomorphs and squirrels in North America, with later extension to South America. Myxoma virus produces a trivial lump in the skin of its natural reservoir hosts, Sylvilagus bachmani in California and S. brasiliensis in Central and South America. The level of adaptation of myxoma viruses for survival in their particular natural hosts is illustrated by comparisons of the behaviour of the Californian strain of myxoma virus in different species of Californian rabbits (Table 10.3) and of different strains of myxoma virus in the natural host of the Californian strain of virus, S. bachmani (Table 10.4).

Table 10.3 shows that the natural reservoir host, S. bachmani, is an effective source of infection for mosquitoes, which produce lesions when they bite any of several species of recipient host. However, when transfer experiments were attempted from the lesions of the recipient hosts, only those mosquitoes that probed through the lesions in S. bachmani produced infection in the very susceptible indicator host, Oryctolagus cuniculus. Experiments with the Californian strain of myxoma virus, a virulent Brazilian strain (Lausanne), and a somewhat attenuated derivative of a Brazilian strain that had occurred naturally in Australia (KM13) showed that only the Californian strain of myxoma virus produced lesions in S. bachmani from which mosquitoes could acquire enough virus particles to transmit infection to the indicator host, O. cuniculus (Table 10.4).

Table 10.3 Failure to transmit Californian myxoma virus by mosquito bite from lesions on all Sylvilagus species except S. bachmani^a

  Table 10.4 Failure to transfer viruses other than Californian myxoma virus from S. bachmani by means of mosquito bites^a

Infection of Oryctolagus cuniculus

By an accident of nature, myxoma virus produces a devastating generalized infection in the European rabbit, Oryctolagus cuniculus, with a case fatality rate of almost 100%. This property led to its use for the biological control of rabbits in Australia and Europe (Fenner and Ratcliffe, 1965). Here I will consider only how myxoma virus became established and spread in these new environments.

Experiments in dry parts of Australia in the 1940s showed that if Australian wild rabbits were infested with the fleas of certain marsupials (Echidnophaga myrmecobii) infection spread within but not between warrens. Results were little better in the first trials undertaken after the Second World War, when virus was introduced into four different sites in the Murray Valley Region in Victoria over the period August-November 1950. In the best documented trial, an introduction in October 1950 went through four generations of transmission, each causing progressively fewer infections. In spite of a number of deaths, the rabbit population rose from 700 to over 1000 and the infection died out (Myers, 1954).

Establishment and spread in the Australian wild rabbit population occurred a few weeks after the conclusion of these unsatisfactory trials in the Murray Valley, when the disease spread over a vast area of southeastern Australia because of the activity of very large populations of rabbit-biting mosquitoes that regarded rabbits as a highly preferred source of blood meals. They picked up virus particles as they probed through skin lesions, and then acted as `flying pins' to transfer them to other rabbits. Earlier failures to establish the disease in Australian wild rabbits were due to faulty choice of location or season for the attempted introduction.

The establishment of myxomatosis in Europe followed quite a different pattern. Hearing of the devastating outbreaks of myxomatosis in Australia, a French pediatrician whose estate was plagued with rabbits obtained a strain of the virus from a friend in Switzerland and on 14 June 1952 inoculated two wild rabbits with the virus. By the end of August all rabbits on his estate were dead and outbreaks had occurred up to 45 km awaymyxomatosis was established in Europe. Thatcherites would say that this success was inevitable, because private enterprise rather than a government authority was involved, but in fact the French doctor had by chance chosen the optimum time to ensure that mosquito transmission would occur. The contrast between the experiences with myxomatosis in Australia and France illustrates that there is a large element of chance in the process of the establishment and spread of an infectious agent in a new environment.

There are still mysteries about how myxoma virus survives through the Australian winter, when mosquitoes are inactive. Myxoma virus probably did and does die out in many localities, but we believe that it persists in some places because of low-level mosquito transmission and the presence of a few rabbits that remain infectious for a long period. The difficulty of survival over the winter period was a powerful selective force for the emergence and perpetuation of viruses that were somewhat attenuated (see above). Because of the mobility of the mosquito vector, myxomatosis can spread over wide areas each summer, if conditions are suitable for the buildup of large mosquito populations. In Britain, and in parts of Australia where they have become established since their introduction in the 1970s, rabbit fleas (Spilopsyllus cuniculi) provide a vector that operates the year round.

10.8.2 COWPOX VIRUS

Cowpox virus, which is thought to be the virus used by Jenner in 1796 to protect children against smallpox, was for a long time recognized only as a disease of cows and persons who milked cows. However, it was and is a rare disease in both cows and humans, and we now know that it also affects domestic cats and a variety of zoo animals (Fenner et al ., 1989). It is quite clear that it does not survive in nature as a disease of any of these hosts; since recurrent infectivity does not occur, infection is followed by lifelong immunity, and all of these animals have slow population turnover times. Cowpox virus has been recovered from wild rodents in Turkmenia and from rat colonies in Moscow and Poland. We now suspect that cowpox virus survives in wild rodents, which have a large population size and rapid turnover, and infection occasionally spreads from these reservoir hosts to the larger animals.

10.8.3 HUMAN MONKEYPOX

In 1970, about eight months after the last case of smallpox in an area of tropical rain forest in Zaire, a case of vesiculopustular disease clinically indistinguishable from smallpox was reported; subsequently other sporadic cases were recognized in several countries in western Africa. Smallpox did not occur as a `sporadic' disease; every case derived from contact with another human case. The occurrence of sporadic cases of any such disease implies a wildlife source; this sporadic disease was in fact due to another species of Orthopoxvirus-monkeypox virus. Intensive studies in the field and in the laboratory have shown that monkeypox virus probably survives in nature in Zaire as a disease of squirrels, and that occasionally chimpanzees, a number of species of monkeys, and man acquire the infection (Jezek and Fenner, 1988). In the context of this conference, the interesting feature of monkeypox is that although human-to-human infection does occur (Table 10.5), it is so inefficient that, unlike smallpox, monkeypox cannot survive as a human infectionit remains a zoonosis that occasionally gives rise to secondary human cases.

Table 10.5 Human monkeypox: occurrence of primary and secondary cases in Zaire, 198284

10.9 LIVE VIRUS VACCINES

Other than Jenner's smallpox vaccine and a few later parallels (turkey herpesvirus for Marek's disease; fibroma virus for myxomatosis) all live virus vaccines have been genetically modified, in that they are attenuated variants of the virulent pathogen concerned. Among those used for human vaccination, the first was Pasteur's `fixed' strain of rabies virus, followed many years later by yellow fever vaccine, then the poliovaccines and later by measles, rubella, and mumps virus vaccines.

With all of these viruses, the element of possible spread from the vaccinated host arose. Yellow fever vaccine does not produce sufficient viraemia to infect mosquitoes, and neither measles nor mumps vaccines, administered by injection or, for measles, by aerosol, are shed from the oropharynx in quantities adequate to infect contacts. This aspect of viral behaviour was of particular importance in designing rubella virus vaccine (Scott and Byrne, 1971), for the objective in this instance was to protect the fetus and one did not wish by accident to infect a pregnant woman who was in contact with a vaccinated child.

10.9.1 POLIOVIRUS VACCINES

From the point of view of this conference, the Sabin oral poliovirus vaccines are of particular interest. Wild-type polioviruses cannot survive in small communities, although their critical community size is smaller than that of measles, because shedding may persist for several weeks. However, they die out in small isolated groups like the Eskimos, but if a strain that causes paralysis is introduced into such a 'virgin-soil' community it may cause a catastrophic outbreak.

In the late 1950s attenuated strains of each of the three serotypes of poliovirus had been developed by Albert Sabin by tissue culture passage and selection for loss of neurovirulence for monkeys. In 1961-2 they were licensed in the United States, and have proved to be spectacularly successful in controlling poliomyelitis. The question I wish to explore today is how well these vaccine viruses, administered each year to millions of children, have survived in nature.

When we talk about the polioviruses we are discussing three distinct viruses, both for wild-type and vaccine strains. I will concentrate my remarks on poliovirus type 1 and the corresponding vaccine strain, because more detailed information is now available about these as a result of recent sequencing studies of a large number of isolates (Rico-Hesse et al ., 1987). 

Poliovirus type 1

In selecting for vaccine strains, Sabin was concerned with two factors: absence of neurovirulence and capacity to immunize after feeding. In both respects the Sabin type 1 virus is an excellent vaccine; it has eliminated endemic wild-type 1 from the United States (Kim-Farley et al., 1984) and it caused only one vaccine-associated case for every 7 to 19 million first doses of vaccine administered (Nkowane et al., 1987) (the range in possible frequency arises from the fact that more than one serotype of vaccine virus was recovered from 9 out of the 14 cases that yielded type 1 vaccine virus). I have already made one comment of interest for this audience, namely that type 1 poliovaccine eliminated wild-type 1 poliovirus from the United States. A similar result has been reported from the Scandinavian countries, where inactivated poliovirus vaccines have eliminated endemic wild polioviruses. This was an immunological exclusion, not one based on interference or some kind of intraspecific competition of the vaccine and wild-type strains.

1. At least in cultured cells, the yield is far below that of wild viruses; for type 1, the ratio is about 1:10 000.
2. The vaccine strains are physically more fragile than wild-type strains and are more easily degraded by heat and other environmental insults. 
3. They shed for short periods onlyfor type 1 for only 2 weeks, compared with 1216 weeks for wild type.

In the developed countries using oral poliovirus vaccines, the only polioviruses now isolated from sewage are vaccine derived. This is due to the fact that in such countries enterovirus infection is seasonal (summer-autumn), and most new susceptibles are vaccinated before the season begins. Since vaccination is given the year round, vaccine viruses are constantly being excreted and are regularly found in sewage. Analysis of these excreted viruses shows that they have all undergone mutational changes and quite a number are intratypic recombinants (Rico-Hesse et al., 1987). However, none of them survives in nature anything like as well as wild polioviruses.

10.9.2 RECOMBINANT VACCINIA VIRUS VACCINES

There is a great deal of interest these days in the production of new types of vaccine. Three main stratagems are being explored: antigens produced by recombinant-DNA methods in E. coli, yeasts, or animal cells, synthetic peptides that are identical to critical antigenic sites, and vaccines constructed by introducing relevant genes into a vector virus or bacterium, which is then administered as a live vaccine. I will discuss only the latter, and of the variety of vectors available, only vaccinia virus, the agent used for smallpox vaccination (for reviews see Fenner et al., 1989; Moss and Flexner, 1987; Mackett and Smith, 1986).

Vaccinia virus, which multiplies in the cytoplasm of the cell, has its own promoters. Recombinant vaccinia viruses can be constructed according to the protocol illustrated in Figure 10.1, by simultaneous vaccinia virus infection and transfection of cells with a plasmid containing the relevant foreign gene, a vaccinia promoter, and flanking fragments of the vaccinia virus thymidine kinase (TK) gene. Homologous recombination incorporates the foreign gene into the vaccinia virus genome and the recombinant viruses are selected for their TK^- character.

A large number of genes from a variety of viruses and protozoa have been incorporated into such vectors. They are expressed when cells are infected and they immunize experimental animals. As used in the smallpox eradication programme, smallpox vaccine had great advantages over all other human vaccines used in tropical developing countries. It was extremely heat stable, it could easily be produced, even in developing countries, and it could be administered by a bifurcated needle or a jet injector without the need for a syringe (Fenner et al., 1988). Recombinant vaccinia virus vaccines have all these advantages. The disadvantage of smallpox vaccine was that occasionally it caused severe complications, at rates of 310 per million primary vaccinations. Whether this is acceptable depends upon the risk against which protection is soughtit was certainly worth taking when and where smallpox had a case-fatality rate of 25%.

With our increasing knowledge of the molecular biology of vaccinia virus, it is possible to improve the performance of such vaccines by three types of stratagem: lowering the virulence of the virus, increasing the expression of the inserted gene, and possibly by increasing the immune response in the vaccinated subject. Considerable improvements have been obtained by the use of some of these approaches. The use of the vaccinia virus TK gene for insertion leads to a dramatic reduction in the virulence of the virus and hence its propensity to cause complications (Buller et al., 1985). Indeed, there is a risk that TK^- mutants may be overattenuated and not multiply enough to produce a good immune response. This risk may be partially counteracted by greatly increasing the expression of the inserted gene. One way of doing this is by the production of a vaccinia virus recombinant by infection of cells with two recombinants, one with the foreign gene flanked by the T7 bacteriophage polymerase promoter and another containing the T7 polymerase gene (Fuerst et al., 1987). This has resulted in a tenfold increase in expression of the inserted gene. Another possibility, still being explored, is that the insertion of genes for lymphokines may dramatically augment the host's immune response. So far only interleukin-2 has been studied, but thymus-deficient 'nude' mice and heavily irradiated mice infected with vaccinia containing interleukin-2 were protected against a million times the usual lethal does of vaccinia virus (Ramshaw et al., 1987).

Figure 10.1 Method of constructing a recombinant vaccinia virus carrying a selected gene from another source (virus, protozoan, or eukaryote). TK = thymidine kinase gene of vaccinia virus; BudR = bromodeoxyuridine. (Courtesy Dr B. Moss.) 

Efforts will soon be made to use such vaccines, so that consideration needs to be given to their potential for spread. During the period when smallpox vaccination was widely practiced, contact infection of vaccinated persons occasionally occurred, and could .be serious if eczematous children were affected (Fenner et al., 1988). The occurrence in cows of `cowpox' due to vaccinia virus acquired from vaccinated humans has been described on a number of occasions, but only one outbreak of vaccinia in man has been reported, in association with a self-limited outbreak of vaccinia virus-induced `cowpox' (Lum et al., 1967).

The only known instance of the probable escape of vaccinia virus into the environment and its maintenance in nature over a period of years relates to buffalopox in India. For many years, veterinarians have described a disease that they called buffalopox among water buffalos in India, Egypt, and the USSR (Lal and Singh, 1977). Most outbreaks could be traced to the transfer of infection from persons vaccinated against smallpox to the buffalos that they milked, and transfer between buffalos was effected by milkers, or sometimes by sucking calves. However, smallpox vaccination was discontinued world-wide in 1980, yet as recently as 1986 outbreaks of buffalopox have been reported in Maharashtra State in India. All strains of `buffalopox' virus that have been studied have been identified as vaccinia virus, but their restriction patterns differ slightly from those of strains used for smallpox vaccination in India (M. Richardson and K. Dumbell, personal communication, 1987). The implication is that vaccinia virus is surviving in nature in some places in India and causing recurrent outbreaks of buffalopox. It is said that buffalopox is a much more chronic infection than cowpox, but I find it hard to believe that it is surviving in buffalos, perhaps with humans as intermediate hosts. The problem merits proper epidemiological investigation.

Even with this precedent, I believe that the new recombinant vaccinia virus vaccines should be safe, because they are much less virulent than the strains used for smallpox vaccine and are correspondingly less likely to spread from one animal to another.

10.10 CONCLUSIONS

Experience with the introduction of animal viruses into geographically and ecologically novel environments confirms experience with biological invasions of plants and animals, namely that the risk of establishment and spread under such conditions is unpredictable; chance factors, including timing, are critical. Although insect viruses are used for the control of some insect pests, deliberate large-scale introductions of viruses of vertebrates in situations in which they may be released into the environment will occur most frequently in association with immunization against infectious diseasesthose of man, of domestic animals, and (with rabies virus) of wildlife. Provided that care is taken in the selection and design of the viruses used, whether they are ordinary live virus vaccines or vectors for genes from other pathogens, I believe that the benefits of these activities should greatly exceed the risks, but each case will have to be examined on its own merits.

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