sou · GNB3120d2 · 2000-01-01 00:00:00 — Kommittén skall mot bakgrund av sina överväganden och förslag även redovisa eventuellt behov av författningsändringar. Utredningen skall under arbetets gång samråda med , samt med berörda forskargrupper. För kommittén gäller regeringens direktiv till samtliga kommittéer och särskilda utredare om prövning av offentliga åtagande (dir. 1994:23). Kommittén skall också redovisa jämställdhetspolitiska konse- kvenser (dir. 1994:124), regionalpolitiska konsekvenser (dir. 1992:50) samt konsekvenser för brottsligheten och det brottsförebyggande arbetet (1996:49). Kommittén skall ha avslutat sitt arbete . The Virological Risks of Porcine Xeno- transplantation This draft will be published in a modified form by the Office international Epizooties. Xenotransplantation offers the of a major benefit to human society but as for all new therapeutic modalities there are concomitant risks to the individual patient. However, the unique feature of xenotransplantation is that the safety issues transcend concern for the individual and require consideration to be given to the probability of generating variant viruses capable of infecting the general public. Several features of the biology of viruses and xenotransplantation in particular, may favour these events. First xenotransplantation permits porcine viruses to come into intimate contact with human cells in an environment of weakened immunosurveillance. Amongst RNA viruses the lack of repair mechanisms during nucleic acid replication leads to the formation of a quasi-species with a cloud of variants clustered around a consensus sequence. Such variation within the population of viruses aids the rapid selection of strains adapted to replication in human cells. The possibility of genetic interchange between human and porcine viruses through recombination or genetic reassortment is also favoured in this environment and, as the evolution influenza viruses has shown, is a powerful mechanism for the generation of pandemic viruses. The wider ethical issues associated with xenotransplantation are not considered in this article but microbiological safety and its implications for society, are an important feature of an ethical evaluation. In a standard risk evaluation scheme the risk of harm associated with viral contamination involves two main factors, the probability of access and the consequences of access of an agent. However, the dynamic nature of virus evolution in a xenotransplant leads to a high degree of uncertainty in assigning probabilities to the elements of a risk assessment. Consequently application of the precautionary principle has been seen as a more useful approach to assessing the risks of xenotransplantation (1996). At a practical level this involves reviewing viruses known or suspected of infecting pigs and determining which should be excluded from the donor animals. It is not usually feasible to guarantee that an individual pig or donor organ will be free of a particular virus if it is present within the herd so that, exclusion of viruses from the herd is the primary focus of attention. While much is known about viruses that infect pigs many of the viruses of concern in xenotransplantation are not of major significance in veterinary medicine so that new assay systems have had to be developed. 2 Categories of Viruses of Concern The viruses of concern in xenotransplantation can be divided into several functional groups: (1) zoonotic viruses, (2) viruses which replicate in human cells in vitro although they are not regarded as zoonotic, (3) viruses that could be transmitted from porcine to human cells by syncitial spread, (4) viruses that might undergo abortive replication in human cells but could be oncogenic, (5) viruses that have not been shown to replicate in human cells but belong to groups that have shown host range changes, (6) porcine viruses that could damage the transplant under conditions of reduced immunosurveillance, (7) human pathogens that could use the products of human transgenes to infect the transplanted pig organ and may evolve to have new properties, (8) virus infections that indicate that the biosecurity of the facility housing the animals has been breached. These functional groups, but not the individual viruses, are described below. Recommendations on individual viruses that should be excluded from donor pig herds have recently been submitted for publication (Onions et al submitted 1999). The special case of the endogenous porcine retrovirus is described in section 4. These are viruses known to be transmitted from pigs to humans. The influenza A viruses form the most dramatic example of this group. The pig is susceptible to both avian and human strains of influenza A and there is evidence that the pig forms a reservoir in which new genetic reassortments between human and avian strains occurs (Castrucci et al ). The influenza pandemic of 1918-19 is believed to have caused deaths of between 20 to 40 million people and retrospective serological studies indicated that the porcine influenza strain causing disease in pigs was antigenically very similar to the human virus responsible for this catastrophic event. This classical H1N1 swine fever virus is still endemic in the pig population and has been isolated from people in which it can cause severe and occasionally fatal disease (et al ). Currently, classical H1N1 swine influenza virus is present in both Europe and the but in Europe it has largely been replaced by H1N1 strains of avian origin (et al ). In addition "human like" H3N2 viruses are present in pig population and it is these viruses that have undergone genetic reassortment with avian like H1N1 viruses. Several features of their biology favour the interspecies transmission of influenza A viruses. The receptor for viral binding is sialic acid a moiety that is ubiquitous on avian and mammalian cells and the segmented genome of the viruses results in the rapid generation of new variants through reassortment of these strands. Furthermore as in other RNA viruses the error rate of the virion RNA polymerase is the order of 1 in 104 nucleotides per replication cycle so that mutations rapidly accumulate. In the Pennsylvanian avian influenza epidemic of 1983 a wild bird H5N2 strain was introduced into domesticated chickens and within a few months the acquisition of 7 mutations in the haemagglutinin gene resulted in an increase in mortality from to and an increase in the transmissibility of the virus (et al ). Important as these viruses are in human and veterinary medicine xenotransplantation does not increase the risk to the general public. Moreover control of these viruses should be possible; in the case of influenza virus, by ensuring the housing is bird proof and that the staff are vaccinated. 2.2Porcine viruses that replicate in human cells in vitro A number of porcine viruses replicate in human cells in vitro but evidence of their zoonotic potential is weak or absent. For instance, pseudorabies virus (an alpha-herpesvirus) can infect human cells in vitro but there is only anecdotal evidence for its role as a zoonotic virus although, it will cause fatal encephalitis in other species like cattle, cats and dogs. Two broad of activity may affect the capacity of PRV to induce disease in non-porcine hosts, its ability to infect and be released from cells at the mucosal barrier and the extent to which it undergoes latent or lytic infection in neuronal cells. While pseudorabies, like other alpha-herpesviruses binds to glycosaminoglycans, entry is dependent on interaction with other receptors and requires the activity of at least 4 glycoproteins (gB, gD, gH and gL). The second receptor for the human alpha-herpesvirus HSV has recently been identified as a novel member of the tumour TNF/NGF family which is likely to show a degree of species specificity (et al ). Exit of the alpha-herpesviruses is also dependent on viral glycoproteins particularly gE and the critical role of the rate of exit of viruses from cells is indicated by the attenuating effect gE deletion on wild type virus. Xenotransplantation may overcome the natural barriers to infection, particularly the mucosal barrier, and once replication is initiated in human cells further selection for human adapted strains is likely to occur under the conditions of reduced immunosurveillance. Some barriers to infection may not be so easily bypassed; many complex DNA viruses (adenoviruses, herpesviruses and poxviruses) carry genes whose products modulate the host immune response. These products are often homologues of cytokines, cytokine receptors or proteins that downregulate the expression of class I MHC genes. These products are often species specific so that in heterologous hosts the immune response is unimpaired and effectively eliminates the virus. For instance the structural genes and many non-structural genes of canine adenovirus have clearly identifiable homologues in the human adenoviruses (et al ). However the E3 region genes of the human adenoviruses (B, C, D and E subgroups), which contain several genes that inhibit TNF- induced apoptosis (Gooding et al ) or that sequester class I in the endoplasmic reticulum (et al ), have no obvious homologues in the canine virus (et al ). Nevertheless complex viruses have shown species jumps. Adenovirus 76 is an endemic virus in commercial chickens, but its origin was as an adventitious duck virus in live vaccines. 2.3Viruses that may be able to infect human cells by syncitial spread from cell to cell Some enveloped viruses may be able to spread from the xenotransplant to human tissue through formation of syncitia, even if cell free infectious virions are not produced. Enveloped viruses like Paramyxoviridae and Herpesviridae are particularly adept at this mode of spread. The gD glycoprotein of several alpha-herpesviruses is essential for the infectivity of virions but when genomes lacking this glycoprotein are transfected into permissive cells, spread of the infection can occur through cell fusion. In the Paramyxoviridae viral tropism within a host and between hosts can be affected by the tissue specific proteases required to cleave the fusion glycoprotein (Reviewed by Nagai 1993). 2.4Virus that infect human cells, fail to undergo productive replication but may initiate disease Experimental transmission of certain members of the herpesviruses, adenoviruses and polyomavirus families can result in tumour formation outside their natural host. For instance human adenovirus 12 is oncogenic in hamsters as is equine herpes virus 1. These events are associated with abortive replication and with integration of part of the viral genome into chromosomal DNA. Abortive replication of animal adenoviruses in human cells has been observed for several viruses including canine and bovine adenovirus features which makes them potentially interesting as vectors but which may also indicate that wild type virus could be harmful. Other serious, non-neoplastic, diseases have been associated with natural transmission of herpesviruses from one host to another. For example, sheep carry a gamma-herpesvirus OHV-2 which has not been associated with disease in this species. Natural transmission to cattle results in the production of a fatal lymphoproliferative condition, malignant catarrhal fever, but further cattle to cattle transmission does not occur. Partial DNA sequences of two porcine gamma-herpesviruses have recently been described (et al ) and caution will be required in determining the disease potential of these viruses in people. 2.5Porcine viruses that are not known to replicate in human cells but which belong to groups that have shown significant changes in tropism or host range One of the most problematic viruses in pig herds is the porcine parvovirus. This small unenveloped single stranded DNA virus is one of the most resistant viruses in the environment and coupled with its ability to cross the placenta makes it very difficult to eradicate. Relatively minor mutational change in the genomes of the autonomous parvoviruses have been associated with dramatic host range changes or changes of virulence and tropism. In 1978 a new pandemic virus infection of dogs which resulted in high morbidity and mortality spread round the world. The origin of this virus (CPV type 2) is almost certainly from a related parvovirus of carnivores most probably the feline parvovirus (FPV). Experimental mutation of the FPV genome indicated that 2 critical amino acid changes within the surface loops of the capsid protein were sufficient to confer on FPV the canine host range, a CPV specific neutralising epitope and heamagglutination characteristics of CPV (et al ). The restriction of FPV replication in canine cells is post entry as both CPV and FPV bind to and enter canine cells to at the same rate (Horiuchi et al ). Following the original identification of CPV type 2 the virus has become further adapted to replication in the dog and two variants CPV 2a and 2b are now endemic in the canine population. Moreover recent infections of cats with parvovirus now involve these viruses (Parish pers com). In pigs four broad pathogenic types of parvovirus (PPV) have been distinguished, non-pathogenic strains (e.g. NADL-2), strains pathogenic to non-immunocompetent foetuses, highly pathogenic strains causing death in immunocompetent foetuses (Kresse) and dermatitis in adults and finally enteric strains. There is a high degree of identity between prototype, non-pathogenic strains and highly virulent strains with only 5 amino acids in the VP2 capsid protein distinguishing the Kresse strain from NADL-2 (Bergeron et al ). The possibility of host range changes in this virus should not be discounted particularly as PPV is able to replicate in non-porcine cells in vitro a property determined by both the capsid and NS genes (Vasudevacharya and Compans 1992). The porcine cornaviruses have also shown dramatic changes in tissue tropism. The Transmissible gastroenteritis virus (TGEV) causes severe enteric disease but in the 1980s a new porcine respiratory coronavirus, (PRCV) spread rapidly through European pig population and was determined to be a mutant of TGEV containing a large deletion in the spike protein (et al ). In North , PRCV strains appear to have evolved independently and where more virulent than their European counterparts. TGEV and PRC belong to the closely related antigenic group 1 coronaviruses which include the human respiratory coronavirus and the feline and canine coronaviruses. Genetic interchange may occur between this group of viruses and there is evidence that one serotype of the feline coronavirus has undergone recombination with the canine coronavirus during its evolution. The high frequency of recombination in coronaviruses may be related to the unusual mechanism of coronavirus replication involving discontinuous transcription and associated polymerase jumping (Reviewed in Lai 1992). 2.6Porcine viruses that may damage the xenotrans- plant under reduced immunosurveillance Some viruses may not pose a public health risk or be of consequence in to the health of the transgenic pig but, under the conditions of reduced immuno-surveillance, could damage the xenotransplanted organ. Such viruses are likely to be those that establish latent infections in their host of which the porcine cytomegalovirus is the prime example. 2.7Transmission of human viruses to the xenotrans- plant Some human viruses may be able to infect the xenotransplant and damage its integrity. These can be divided into several categories: viruses known to infect pigs like human influenza, viruses where there is serological evidence of infection which could be productive or abortive, e.g. human adenoviruses and rhinoviruses and viruses that have been experimentally transmitted to pigs like human isolates of hepatitis E virus. The complexity of the latter situation is revealed by the recent description of a naturally occurring porcine hepatitis E virus that is capable of causing disease in people (Meng et al ). A further complication in xenotransplantation is that the complement regulating transgenes may also serve as receptors for human viruses; decay-accelerating factor CD55 has been identified as the receptor for echovirus 7 (et al ) and CD46 is one of the receptors required by measles virus to infect cells. However, while some murine cells expressing human CD46 transgenes may be permissive for measles virus, other cells and transgenic mice expressing this human protein have not been shown to support measles virus replication even when the viruses attach to and fuse with the cells (Gerlier et al ). Certain virus families like the hepadnaviridae or certain virus groups like the morbilliviruses have not been reported in the pig. However new virus diseases are being recognised or are recently emerging like Menangle and Nipah viruses (Philbey et al , Centers for Disease Control and Prevention 1999). There is no simple solution to the question "what are the dangers of the viruses you have not identified?" but one approach is to identify risk categories of viruses and then attempt to demonstrate that the virus genus or family is absent using redundant PCR and related techniques. For instance, redundant PCR has been used to identify the porcine cytomegalovirus in tissue and in turn the amplified sequence has enabled the development of specific assays for this virus (. Similarly representational difference analysis may be useful in some contexts to exclude the presence of exogenous DNA (or RNA) in a target tissue. For instance in a particular line of pigs, the target organs within a donor breeding herd could be used to screen for unknown agents by RDA (Lisitsyn et al , Chuang et al ). Our experience of RDA indicates that there is extensive work load involved in sequence analysis but it is probably worth considering as a final validation of the quality of a particular line. The probability of detecting novel viruses by this technique might be enhanced by immunosuppressing the pigs before analysis. 3Derivation and maintenance of virus free lines A primary advantage of the pig in xenotransplantation is that there is a body of experience in generating specific pathogen free animals which have a "normal enteric bacterial flora". (et al , and 1999). The original founder herd containing a transgene can be used to derive a donor breeding herd by hysterotomy or hysterectomy followed by artificial weaning of the piglets. In practice, in order to generate the numbers of animals required for xenotransplantation it will probably be necessary to have an intermediate amplifying or multiplier herd. The amplifying herd is a useful stage to improve the health status of the pigs and it would be feasible to consider vaccination to break the cycle of infection of viruses that are difficult to eradicate like the porcine parvovirus. The donor breeding herd may then be derived from the amplifying herd by hysterotomy or hysterectomy. Vaccination within the donor breeding herd is not usually advisable as one of the key tools in monitoring the health status of the pigs will be their serological status. If vaccines have to be used they should either be traditional inactivated vaccines, subunit vaccines or delivered by single cycle vectors. These vaccines need to be accompanied by serological assays that discriminate between vaccinated pigs and pigs exposed to wild type virus. The donor pig may in turn be derived by segregated early weaning and its variants or, if additional security is warranted, by hysterotomy. These animals may then be maintained in groups or multiples of groups until required for donation. Once an SPF herd is established maintaining its status is dependent on the quality of the housing and the standard operating procedures within the facility. The staff are the most critical element and of particular importance are their training and motivation. It will be necessary for them to have limits on their contact with a wide range of other animals including pets, to agree to vaccination against a range of viruses and to be prepared to work in masks and protective clothing. Consequently the introduction of automated feeding and cleaning to limit operator contact with the pigs should be a key goal. Vaccination should be used with caution, however, as influenza vaccines may mask symptoms while permitting virus shedding. The transmission of many viruses of concern is by rodents, birds and in certain regions by arthropod vectors so that the design of the must incorporate a high degree of biosecurity. spread is also a factor for some viruses and while long distance transmission has been recorded for foot and mouth disease virus it is usually more restricted as in the case of Porcine Reproductive Respiratory Syndrome virus. This feature of viral transmission emphasises the requirement for the housing to be in areas distant from other pig units. Monitoring the status of the pigs will be necessary at several stages in the breeding pyramid and will be intense in the breeding donor herd and donor cohorts. Serological screening forms the main diagnostic tool but for some viruses like porcine cytomegalovirus, PCR or other techniques are required for diagnosis of infection. An important feature of the operating procedures will be the course of action to be followed if a virus is reintroduced. In many instances this may require the slaughter of all the pigs within the same contained air-space a feature which should be borne in mind when designing the facility. The most intense monitoring will be focused on the donor pig cohorts. In addition to serology sentinel pigs should be removed during the growth phase to permit more detailed analysis including PCR analysis of tissues and in vitro culture for porcine viruses including viruses capable of replicating on human cells. Collectively these procedures will ensure the freedom from the great majority of viruses of concern. Certain problem viruses remain, those that cross the placenta like the porcine cytomegalovirus (PCMV), parvovirus and circovirus, viruses that remain latent like PCMV, and the special case of the genetically acquired porcine endogenous retrovirus. 4The porcine endogenous retrovirus (PoERV) Retroviruses belonging to the Gammaretrovirus group (formely type-C oncovirus group) are associated with the induction of leukaemias, lymphomas and other degenerative conditions (Onions 1994). A feature of the replication of retroviruses is the integration of a DNA copy of their genome into chromosomal DNA. evolutionary history, retroviral genomes have become integrated into the germ line so that some are transmitted as Mendelian elements termed endogenous proviruses (Coffin 1982). Many of these proviruses are defective and are unable to be expressed as functional viruses while others can be reactivated to produce infectious virions. In other instances the endogenous sequences are not expressed as infectious virus but may serve as substrates for recombination with exogenous retroviruses. For instance we and others have shown that a cat to cat transmitted retrovirus, feline leukaemia virus subgroup A (FeLV-A) can undergo recombination with endogenous retroviral elements to generate a new subgroup called FeLV-B (et al ). The subgroups of retroviruses are defined by the receptor used to enter cells which is governed by the major envelope glycoprotein (SU). DNA probes to regions outside the envelope gene will detect viruses belonging to all members of the group. 4.2 The porcine endogenous virus 4.2.1 range of porcine endogenous viruses