key: cord-0008934-mzsoh16g
authors: nan
title: Chapter 4 Picornavirus infections
date: 2008-05-29
journal: Perspect Med Virol
DOI: 10.1016/s0168-7069(08)70012-0
sha: 2720c7d6a7524702708203f86dc6ab4c74af1ef6
doc_id: 8934
cord_uid: mzsoh16g

The oldest member of the Picornaviruses group is polio virus, which was recognized early by clinicians because of its characteristic paralytic disease. This chapter examines the polio virus in regard to its virology, disease, and prevention by vaccines and chemoprophylaxis. Polio has been well controlled in most developed countries using live or inactivated vaccines. Research work has intensified using genetic engineering techniques to produce live attenuated viruses with defined and stable mutations so as to prevent reversion to virulence, and also to produce immunogenic oligopeptides or proteins for a new generation of inactivated polio vaccines. Chemotherapy is therefore not required for polio infections. In contrast, neither vaccines have been developed against rhinovirus infections, nor are the vaccines thought to have a use, unless broadly reacting antigenic determinants can be located. Several interesting but only weakly effective antiviral compounds have been selected against rhinoviruses and this is a major research area at present. Studies continue also with interferon, but because of toxicity problems these look less interesting at present. Sequence and biochemical data is now available for several additional enterovirus strains and this could open new possibilities both with antivirals or vaccines (for example synthetic peptides) in the near future.

chemical characteristics to polio were isolated, which paralysed infant mice, and they were divided into 2 groups dependent upon the different pathology in mice, namely Coxsackie A and B viruses. Thereafter, with the extensive use of tissue cultures in the 1950s, more enteroviruses were isolated which were not pathogenic for animals, or humans such as ECHO viruses (enteric cytopathogenic human orphan) (reviewed by McLean, 1966 , Artenstein et al., 1965 , Karzon et al., 1956 , Grist et al., 1975 . Finally, in the late 1950s viruses were at last isolated from patients with common colds and these were classified as rhinoviruses (Tyrrell, 1965) . Probably in excess of 150 viruses of the latter group circulate in man. Nowadays, all these viruses are designated as 'enterovirus' serotypes (Table 4 .4).

Electron microscopy reveals the enteroviruses to be small spheres with a regular protein capsid structure 18-30 nm in diameter, although only a few members have been studied in any detail. The capsid is composed of 32 capsomeres in a pattern of cubic symmetry (Fig. 4.1) of an icosahedron with 180 polypeptide molecules per capsid. The molecular weight of the RNA approximates to 2-2.8 x lo6 (7500 nucleotides) and sedimentation coefficients of the viruses range from 140 to 160 s.

Most of the protein is encompassed in 4 polypeptides existing in equimolar amounts (VP1, 2, 3, 4) and an RNA capping peptide. In certain of the viruses, which have been studied in more detail, a single large protein VPO is synthesized and then the 4 capsid proteins are derived by a series of proteolytic cleavages (see below, and Cooper, 1977) . After infection and uncoating, the viral RNA is translated to form the giant polyprotein which is cleaved to yield capsid proteins and the viral RNA polymerase. This enzyme, which may exist in two formsto synthesize plus-and minus-strands respectivelyis unique to virus-infected cells, and is responsible for the synthesis of virus-specified RNA using an RNA template.

-----All types All types Types 2,7, and 9 Types 1, 2. 4, 5,7,9, 10. 14, 16, 22 , and 24 Types 9 and 16 Types 2, 4, 9, 16, 8, 10, 16, Types 5, 9, and 16 Type 10 Enterovirus 70 23 and 22

All types All types Types 3-5 Types 1-6 Types 1-5 Types 1-5 ----Types 1-5 Types 1-5 Types 1-5 All types All types -Types 2 and 4 11, 12, 21, 22, 25, 30, 31, and 33 Types 2, 6, 9, 19, 11, 9, $1 4, 16,  18. and 19 -Type 18

All types All types (for additional details see Mirkovic et al. 1973 , Carter 1933 , and Ranzenhofer et al., 1958 form (RF) and a multi-stranded R N A species known as replicative intermediate (RI) . There is evidence that the RI is an intermediate in virus R N A synthesis. I t consists of an R N A molecule which serves as a template for the simultaneous semiconservative synthesis of several R N A molecules (between six and seven for poliovirus) with nucleotide sequences complementary to that of the template. Two types of RI could existthat using a minus-strand R N A to produce plus-strands (positive RI) and that using a plus-strand R N A to produce minus-strands (negative RI). Unlike RI, which is partially sensitive to the action of ribonuclease, RF is highly resistant to this enzyme, and since it is infective it must contain an intact plusstrand, which is hydrogen-bonded to a minus-strand. R N A synthesis switches from exponential to linear synthesis early in infection, the time varying between 1 and 3.5 h after infection and depending upon input multiplicity. This switch implies that the concentration of positive RI (the structure responsible for the synthesis of the major product) is constant throughout the latter period of infection. This could most readily be explained if synthesis of the template of positive RI (the minus-strand) ceased a t the switch point, or alternatively was produced at a rate equal to its degradation. It may be important that the switch occurs at the time when the virus R N A is beginning to be encapsidated to form progeny virus.

T o develop a sensible and effective strategy of immunization or chemotherapy for a particular disease, we need to know features of the virus epidemiology, how the virus is spread in the community, reservoirs, infectivity and how and where the virus replicates and spreads in the host.

The term 'poliomyelitis' comes from polios (grey) and muelos (marrow) and 'polio' has come to be used as a shortened version meaning paralysis. The virus exists as 3 serotypes originally called Brunhilde, Lansing and Leon but now known as types I, 2 and 3 which are easily distinguished by neutralization tests, although some cross-immunity between them occurs (see Fig. 4.2 and Table 4.5) . Second attacks of polio are rare (Bodian, 1951) and, as far as is known, vaccination with one serotype (e.g. type 1) will protect against all members of these type I serotypes, although individual viruses may differ in virulence (Boulger et a] ., 1979), biochemical properties (Romanova et al., 1981) and antigen composition (Nakano et al., 1963 . Polio is a highly infectious disease and infection rates may reach 100% in infant classes. Personal contact is the main method of spread. A seasonal incidence is marked in temperate countries but outbreaks may still occur in winter, whereas in tropical areas it occurs throughout the entire year (Paul, 1955) .

Following initial infection of humans, virus is detected in the throat secretions Thorpe et al., 1982) . (a) Immunoblot processed with rabbit antiserum 115B. Lanes 1. 3 and 5. antibodylabelled proteins; lanes 2. 4 and 6. biosynthetically labelled proteins. Lanes I and 2. poliovirus type 3 ; lanes 3 and 4. poliovirus type 2; lanes 5 and 6, poliovirus type 1. (b) Immunoblot processed with rabbit antiserum MI. Lanes I. 3 and 5. antibody-labelled proteins; lanes 2, 4 and 6. biosynthetically labelled proteins. Lanes I and 2. poliovirus type 3; lanes 3 and 4. poliovirus type 2; lanes 5 and 6, poliovirus type I . Antiserum R50 produced an identical blot (see also Table 4 .5). and faeces as virus replication continues in the tonsil and intestinal wall regions. Most patients excrete virus for 2-3 weeks and more rarely for 6 weeks. The virus then passes to the regional lymph nodes and probably then reaches the bloodstream. At this stage there is no clinical evidence of invasion of the central nervous system (CNS). There is little doubt that the virus travels along neural pathways from the periphery to the CNS and spread becomes neural once the CNS is reached. Certainly, if antibody is present in the blood the virus does not reach the CNS. A basis for a successful immunization is thus apparent already ~ induction of neutralizing antibody in the serum and local IgA antibody in the gut. The incubation period is as long as 2-3 weeks. The most remarkable feature of the pathology of polio is the very high selectivity of the virus for nervous tissue, where it can cause rapid and widespread damage to the central nervous system. The primary changes are to the neurones which can be destroyed. As a response to this nerve cell damage an inflammatory reaction oc- Characteristics of the antibodies to poliovirus 3 antigens used in the 'blotting' test (after Thorpe et al., 1982 

bAutoradiographic bands were scored in order of increasing intensity from + (weak) to ++++ (very intense).

See also Fig. 4 .2 for extra details of serological cross-reactivity. See also Ferguson et al., 1982. curs, with infiltration of surrounding tissue with polymorphonuclear cells and lymphocytes, but paralysis occurs from virus destruction of nerve cells. The distribution of lesions in the CNS seems very similar in most cases of polio and the clinical symptoms depend on the severity of damage to nerve cells (Lassen, 1956) . The brain is normally invaded, particularly in the brain stem area (Baker, 1949) . The virus can also be isolated from extraneural tissues e.g. lymphoid tissue including Peyers patches and mesenteric lymph nodes (Horstmann et al., 1954, Wenner and Rabe, 1951) . The proportion of inapparent to paralytic infections may be as high as 1000 to 1 in children and 75 to 1 in adults. The disease is often described as biphasicminor and major illness. In the minor phase the symptoms are non-specific, with malaise as the common symptom and may clear up after 3-4 days. The major illness may occur without preceding illness and the first symptoms are meningitis, with headache, fever, malaise and vomiting with an abrupt onset (Christie, 1948) . The meningitic phase often concludes in a week. However, in a minority of persons paralysis sets in and first becomes apparent after a couple of days of meningitis symptoms (Fig. 4.3 and 4.4) . In the worst cases nearly every skeletal muscle can be paralysed and the person cannot swallow or breathe properly, whereas in mild cases only a part of one muscle may be affected. Paralysis is a lower motor neurone type with flaccidity of affected muscle and develops quickly. In bulbar poliomyelitis, paralysis of cranial nerves occurs and paralysis of the pharynx is a feature. In fact, the inability to swallow is the main clinical characteristic of bulbar poliomyelitis. Signs of other cranial nerve involvement may be present, facial weakness and paralysis of the flexor muscles of the neck being the commonest. Respiration may be deeply disordered and there may be pharyngeal paralysis (Baker, 1949) .

In cases of paralytic polio, the full extent of paralysis is reached by 72 hours although not until 4 weeks have passed can the clinician make a reasonable prognosis, since by this time most reversible neuronal damage will have disappeared and residual irreversible damage can be assessed. Often great improvement takes place in these weeks but by the end of one month muscles still paralysed will probably remain paralysed. In bulbar polio the outlook is good if the patient is still alive by the 10th or so day since the pharyngeal muscles then begin to show signs of recovery. The basic principles for clinical care are outlined by Christie (1980) with his characteristic flair "to accept into ones care a patient stricken with a disease that may cut him off for all time from a return to normal life, but to ensure at every stage of his illness whether his life is at risk from respiratory failure or a limb is made useless by paralysis, that the exact nature of his disability is analysed as accurately as the medical sciences and clinical skill can measure it and, when that has been done, to bring to his aid all the sovereign art of modern medicine." Fortunately, the development of polio vaccines was given considerable priority in the late 1950s following the new developments of tissue culture technology (reviewed by Enders et al., 1980) . Few clinicians, at least in Europe, USA, Japan, Australia and the USSR have now to diagnose polio or to care for the ravages of the virus in children. The dramatic effects of vaccination on the incidence of polio is illustrated for European countries in Table 4 .6.

Although the clincial effects of polio have been known since 1000 BC and a concise discussion of the disease was published by Underwood in 1789, surprisingly epidemics of polio have only been described in the last 100 years or so. It would seem in part that when sanitation improved, infection was encountered later in life and paralysis became more prominent. Polio vaccine (Melnick, 1954 , Koprowski et al., 1956 , Salk, 1960 , Salk and Salk, 1967 ) has been used so successfully in many countries that the virus and vaccine can be used as an example of a situation where the remote risks of vaccination itself begin to assume major importance (see below). In the US before the introduction of vaccine approximately 20 000 cases of polio-caused paralysis were recorded annually and now this number approximates to 10.

Polio has been controlled successfully by vaccines in many temperate countries of the world (Bottiger et al, 1966 , Nathanson, 1982 but not in many tropical, third world countries (Sabin, 1982 , John and Christopher, 1975 , Swartz et al., 1972 .

There is little precise information concerning the antigenic determinants of polioviruses and their involvement in virus structure and function. Polioviruses have been classified into three serotypes ( I , 2 and 3) according to the ability of type-specific antisera to neutralize infectivity. The virus exists in two different antigenic forms: the D antigen, which is associated with the infectious particles, and the C antigen, which is associated with non-infectious particles (Mayer et al., 1957 , le Bouvier et al., 1957 , Roizman et al., 1959 . As infectivity is associated with D antigen particles, it has been assumed that this immunogen is largely responsible for the production of neutralizing antibodies. Most immunochemical work with polioviruses, as well as with other picornaviruses, has been carried out with intact virus particles in which the capsid proteins are arranged in complex structural configurations, so that the immunological and antigenic properties of individual virus proteins could not be shown. In contrast, for foot-and-mouth disease virus it has been possible to establish that immunization with the trypsin-sensitive protein (VPI) induces neutralizing antibodies (Meloen et al., 1979) . (see section 4.12 on immunogenic peptides of polio VPI .)

Relevant to vaccines, no single common antigen has yet been described for picornaviruses, but, as stated above, we should remember that very little detailed bio- chemistry has been carried out with many of these viruses. Indeed RNA hybridization shows a high degree of homology between certain members and so common amino acid sequences with corresponding antigenic relationships may exist. Certainly, antigenic cross-reactions have been noted among certain serotypes such as coxsackie A3 and A8 for example, between A9 and 1 I coxsackie B viruses. Again with the ECHO viruses, heterotypic cross reactions occur between some viruses, e.g. types 1 and 8, and between 12,29,23 and 22. Thorpe et al. (1982) using the sensitive immunoblot technique have described antigenic relationships between VP 1 of polio types I, 2 and 3. Furthermore, certain monoclonal antibodies directed towards the C antigen of polio type 3 reacted with VPI of this virus. VPI seems to be immunodominant regardless of the form of the immunizing antigen. A recent report (Blondel et al., 1982) suggests that immunization with VPI causes the production of neutralizing antibodies and in this respect the immune response to poliovirus VPI may be similar to that to VPI of foot-and-mouth disease virus and to VP2 of coxsackie virus (Meloen et al., 1979 , Beatrice et al., 1980 . Although homotypic antibodies binding to VP2 were detected with hyperimmune sera, no heterotypic cross-reactivity was observed for this protein.

To some extent views about the use of either killed or live polio vaccines are in a state of flux at present (Melnick, 1978 , Sabin, 1982 . Perhaps not unexpectedly the virologists who hold dominating and contrasting views are Drs. Salk and Sabin (originators of inactivated and attenuated polio virus strains respectively). Also, perhaps not unexpectedly Sabin enthusiastically supports the widespread use of live vaccine whilst Salk confines his interest to killed polio vaccine. Both vaccines have their merits and weaknesses and it may not be possible to decide between them (Tables 4.7 and 4.8). To some extent a judgement on their relative efficacy and usefulness may depend on the purpose of the vaccination campaign and where in the world it is being conducted. If our goal is total eradication of virulent polio virus from the world community (which should be possible, there being no animal reservoir of the virus, and the virus exists as a limited number of 3 serotypes) then inacti- a Some of the disappointing results in the decade after killed vaccine was introduced may have been due in part to problems that may now have been corrected. Present antigens are stronger than the older antigens. (See also Marsden et al., 1980 and Boulger et al., 1979 for a further discussion of assessment of neurovirulence of poliovirus vaccine.) 

Being living viruses, the vaccine viruses do mutate, and in rare instances have reverted toward

Vaccine progeny virus spreads to household contactsa Vaccine progeny virus also spreads to persons in the community who have not agreed to be vaccinated" In certain warm-climate countries, induction of antibodies in a satisfactorily high proportion of vaccinees has been difficult to accomplish unless repeated doses are administered. In some areas, even repeated administration has not been effective. Contraindicated in those with immunodeficiency diseases, and in their household associates, as well as in persons undergoing immunosuppressive therapy neurovirulence sufficiently to cause paralytic polio in recipients or their contacts a Some people consider this spread into the community to be an advantage, but the progeny virus excreted and spread by vaccinees often is a mutated virus. Obviously it cannot be a safety-tested vaccine, licensed for use in the general population.

see Magrath, 1976. vated vaccine must be used in the final thrust, although much earlier work could be carried out with live vaccine or a combination of live and killed vaccine. This is because attenuated viruses may (and do) revert to virulence on occasion (Nathanson, 1982). As mentioned above the genetic determinants of virulence or attenuation for polio virus are not known and it will require extensive biochemical analysis of the genome of a number of attenuated and virulent viruses, mutants and recombinants to establish the genetic basis of virulence. At this stage one could envisage that an attenuated and stable variant could be constructed by using DNA technology (see Chapter 2 ) . On the other hand, it may not be practicable to produce large enough quantities of inactivated vaccine cheaply enough for a total eradication campaign and moreover, if the manufacture of vaccine is extended more widely then escapes of virulent virus in incompletely inactivated vaccine may occur. An early example of such an accident was the Cutter affair in the USA where 92 children contracted paralytic poliomyelitis following immunization with insufficiently inactivated vaccine (Nathanson and Langmuir, 1963) . It could be added however that nowadays, should large scale manufacture of inactivated polio vaccine be reinitiated then viruses would be chosen with less than full virulence to avoid potential problems of incomplete inactivation (Salk et al., 1981) . Genetic engineering techniques could now be explored to produce immunogenic polio proteins and synthetic peptides which would side step any 'failure of inactivation' problems although these synthetic techniques are likely to produce problems of their own (see Chapter 2). We can conclude that both vaccines have had their successes and failures, both vaccines are not in evolutionary blind alleys and can be improved in the near future, and both vaccines probably have a long and useful life and function ahead of them.

Until the 1930s polio virus could only be identified by infection of monkeys and subsequent production of paralytic disease. This property of neurotropism of polio in vivo and early studies in cell culture which were wrongly interpreted as indicating that polio could only replicate in neural tissues delayed work with the virus and, moreover, erroneously led to deductions that droplet infection and subsequent invasion of the CNS via the olfactory nerve endings was the major point of entry of the virus in humans. In fact it could be said that the early studies of 'chemotherapy' were initiated by these observations and sodium alum, zinc sulphate and picric acid were all used unsuccessfully to prevent paralysis (Armstrong and Harrison, 1935 , Sabin et al., 1936 , Olitsky and Sabin, 1937 , Schultz and Gebhardt, 1937 , Armstrong, 1937 . Sabin and Ward (1941) were among the first to appreciate that the alimentary tract was a major site for polio virus replication in man. Studies in the 1940s and 1950s established that although polio virus could be isolated in flies and cockroaches, and antibodies detected in cattle and chimpanzees (Sabin and Ward, 1942 , Melnick and Penner, 1952 , Sabin and Fieldsteel, 1952 , nevertheless the viruses are maintained in nature by human to human transmission. Epidemiological studies in Eskimoes demonstrated that antibodies to polio persisted for at least 40 years in the absence of re-infection and that natural infection gave rise to complete or partial resistance in the intestine. The scientific ground work was therefore laid for the production and application of polio vaccines. Theiler (1941) reported laboratory modification of the neurotropic property of polio and Koprowski et al. (1952) described for the first time that an immune response could be produced in humans following infection with an attenuated (mouse adapted) type 2 polio virus which had reduced intra-cerebral virulence for monkeys. Finally, the clear demonstration by Enders et al. (1952) that all three polio virus types could replicate in cell cultures of non-nervous tissue (see below) and simultaneously lose monkey virulence (Table 4 .9.) opened the way to the extensive laboratory studies leading to the development of successful formalin inactivated and also live attenuated polio vaccines.

Salk commenced work on producing an inactivated polio vaccine with the premise that circulating serum IgG antibody provided protection against the clinical signs of polio and therefore as long as a killed vaccine induced such antibody in suficient quantity it would be effective. (Earlier work in his laboratory had been orientated towards developing live attenuated viruses, but this ceased as soon as it was apparent that tissue culture cultivated formalin-inactivated virus was immunogenic.) His group demonstrated that virus concentration (and hence antigen concentration) was the single most important variable factor in vaccine production, rather than the inactivation process per se. Inactivation with 1/4000 formalin at 35°C at pH 7.0 resulted in a constant rate decline of virus infectivity for the first 2; days. However, even at this early stage inactivation of lots of vaccine varying from 30 to 50 litres was showing some variation between manufacturers, with satisfactory 'base line' virus inactivation in one manufacturer detected at 6-9 days but 13 days at another production plant (12 days was later chosen as safer after the Cutter incident). If inactivation proceeded for too lengthy a period then immunogenicity decreased, and hence the time period was critical. Early experiments in monkeys established that vaccine given in three divided doses was more immunogenic than a single large dose. Table 4 .10 summarizes the data from some of the first trials in children who had no antibody prior to immunization with polio vaccine and the effect of boosting at 7 months and at 20 months (Table  4 .1 1). Quite clearly the era of successful immunization against polio had commenc- ed, although problems of incomplete and variable inactivation of virus were to be noted immediately the vaccine process was scaled up to produce the tens of millions of doses required for mass immunization (Nathanson and Langmuir, 1963) . As regards production of polio vaccine nowadays, human diploid cells are used by some manufacturers and still other cell substrates are being considered for polio vaccine production mainly because of the immediate shortage of monkeys and safety considerations but additional advantages might be increased yields of virus (Van Wezel et al., 1978 , Marel et al., 1981 . Possible new cell substrates are: 1: subcultured monkey cells rather than primary cells. In addition, newly devel-2: primate embryo diploid cells (in addition to human diploid cells). 3: continuous cell lines e.g. Vero (these cells have been suggested because of evi-4: 'suspension' cultures of cbntinuous cell lines using Sephadex beads.

oped techniques of perfusion with trypsin gives increased yields of cells.

dence of absence of tumourigenicity).

Captive bred monkeys have fewer adventitious 'foamy' viruses and so are more suitable as a source of kidney cells than captured animals. Good cell growth of M K cells is obtained in monolayers and in microcarrier cultures for up to 3 passages (approx. 12 cell generations) although the cells stop growing after 7-8 passages. 15-20 monkeys per year will then be required to produce 3-4 x loh doses of vaccine. Although diploid cells have many favourable properties (absence of viruses, strict control on karyotype (Chapter 2)) they require high quality serum for growth and are less suitable for large scale cultivation. Finally, Vero cells may be suitable particularly since polio virus will be inactivated with formalin at the final stages (so inactivating any adventitious or unknown virus) and these cells can be cultivated in microcarrier systems. Criteria for seed polio viruses for inactivated vaccine include stability, immunogenicity, and replication rate but the most important factor is immunogenicity. Type 2 strains (MEFI) with type 3 (Saukett) induce much higher antibody levels than a comparable antigenic mass of type 1 (G. Mahoney) polio. It has also been recognized that antigenic differences exist within polio types and so the antigenic structure of the vaccine strain should be representative of field strains. Type I virus still gives problems of antigenic instability after formalin inactivation. For final vaccine production, cell debris are removed by filtration and the virus suspension concentrated by ultrafiltration and standardized using D antigen concentration, or by single radial diffusion techniques. Gel filtration on Sepharose (to remove remaining serum proteins), followed by ion exchange on DEAE Sephadex is used to remove further impurities. The original inactivation techniques developed by Salk ( ! 955) using formaldehyde at elevated temperatures has proved successful also for purified polio virus suspensions (Van Wezel et a]., 1978). It could be added that attenuated polio viruses could be used nowadays for production of inactivated polio vaccine thus avoiding potential problems of virus escaping inactivation and infecting and paralysing recipients.

During 1953-1954 Sabin and co-workers attempted to attenuate highly virulent polio virus (Mahoney, Y-SK and Leon) by cultivation in cynomolgus kidney tissue cultures. However, it should be realised that several other groups were actively working at the same problem and several potential 'candidate' vaccine strains had been selected (Koprowski et al., 1952 , Enders et al., 1952 , Melnick, 1954 . They soon found that mere passage had no attenuating effect but, significantly, use of high inocula together with rapid passage favoured the overgrowth of virions able to replicate well in non-nervous tissue and the harvests had diminished virulence for monkeys (Table 4 .12). This suggested a mixed population originally and therefore terminal cloning was carried out and non-virulent variants of all three polio types obtained (Sabin, 1955) . A marked diminution in intramuscular and oral infective capacity for cynomolgus monkeys was noted for these strains (Table 4 .13) and these monkeys produced antibodies in response to infection. Sabin had selected a variant of the mouse passaged Mahoney strain (type 1) which was potentially useful as a vaccine strain, and early tentative trials in volunteers confirmed attenuation of these viruses (Table 4 .14).

The WHO Expert Committee on Poliomyelitis in 1957 recommended large scale trials of the newly developed formalin-inactivated vaccine and particularly well controlled trials in the USA (Francis et al., 1957) demonstrated efficacy and safety of the new vaccine. However, Sabin (1955) had by now developed several live attenuated candidate vaccine viruses and since he did not want to interfere with the still progressing trials of Salk vaccine in the USA, he initiated large scale trials in Eur- ope. By this stage the genetic basis of virulence of these new polio strains had been established, at least tentatively, and several phenotypic virological markers which co-segregated with virulence had been observed. Thus, attenuated polio strains often possessed ts or ca properties. In fact it appeared, in retrospect, that since the early tissue culture passage experiments to 'attenuate' these viruses had been carried out at low temperatures of 35"-36" C this may have also increased the selection pressure for ts attenuated virus subpopulations (Sabin, 1958) . Large scale polio immunization was carried out in Europe in collaboration with the large and active virology group in Leningrad and these represent, in retrospect, a classic series of experiments both scientifically and for the benefit of mankind. Initially small groups of children were immunized, but as increasing clinical and laboratory experience showed no adverse effects the campaign was widened. Thus, some of the early clinical trials were carried out with Sabin live vaccine virus in the baltic republics of Latvia, Lithuania and Esthonia (Chumakov et al., 1959) . Twenty seven thousand individuals were immunized with vaccine provided by Sabin but a further 10 million people were fed with first passage material grown in the USSR. The first cycle of immunizations was carried out in January-March 1959 (Table  4 .15 and 4.16). Almost half the susceptible population in Lithuania received live virus vaccine and convincing proof of the highly immunogenic characteristics of the virus was obtained. Trivalent mixtures were also used effectively with children developing antibody to all three types.

In separate studies, Smorodintsev et al. (1959) also used a cautious approach and between April 1957 and April 1958 observed 150 vaccinated children and contacts before vaccinating a further 822 children in May 1958 and 12000 children in January 1959. By March 1959 a further 1.8 x 106children were fed vaccine virus. Table  4 .16 shows illnesses in 10 000 immunized children and non-immunized controls in Riga. N o clinical polio was detected in the vaccinated group or the internal control group.

Early experiments by Smorodintsev in Leningrad also established that no marked increase in virulence occurred on human-human passage of these attenuated virus- es (Smorodintsev et a]., 1959) and the vaccines began to be widely used (Table 4 .17 and 18). However, in these early studies it soon became apparent that problems of vaccine 'take' could occur in countries with poor hygiene and where many other enteroviruses were present in the gut which could interfere with the replication of the polio vaccine virus (Sabin, , 1963 .

Several developed countries have now virtually eradicated polio and these include, for example, the USA, USSR, UK, Scandinavia (Bottiger, 1969) (Fig. 4 .5) and certain other Western and Eastern European countries. However, polio is still com- mon in underdeveloped countries and a quandary for eradication here is that live polio vaccine often works erratically and so is less than satisfactory in these countries (Chandra, 1975 , Swartz et al., 1972 , Hale et al., 1959 ., 1961). Also inactivated polio vaccine may not produce herd immunity which is assumed to be so important, since in these countries, the faecal-oral route of transmission is most common and inactivated polio may only reduce transmission via the pharyngeal route (Gelfand et al., 1959 , Chin et al., 1961 . Therefore, further research is clearly needed before a decision is made on the strategy for the control and eradication of poliomyelitis in developing countries. Meanwhile, however, we might learn from the very useful data on the experience of eradication of polio in certain countries like the USA using live vaccine, or in Scandinavia using inactivated vaccine.

From 1951 to 1977 the incidence of polio in the USA dropped steeply from a level of 10000-20000 cases a year to 10 cases a year. Similar figures were obtained in the UK and Scandinavia (Fig. 4.6) . Naturally occurring poliomyelitis due to indigenous wild poliovirus essentially disappeared from the USA after 1972 (Nathanson, 1982) . Nevertheless, 5-10% of children under 16 years of age in the USA are considered to remain susceptible to polio because vaccine coverage is not 100% (Table 4 .19). This success in eradication of polio in the USA was achieved mainly by the use of attenuated vaccine (Table 4 .20), but the continuing problem with this vaccine is the, albeit small, number of vaccine-associated cases of poliomyelitis (Schonberger et al., 1976) which approximate to 2 paralytic cases per million vaccinees. In the USA with 3.5 million children being immunized yearly 7 paralytic cases are expected, caused by the vaccine virus, of which one third occur in recipients of vaccine and two thirds in contacts, who are mainly adults.

At present it is estimated that 2-5 million children in the USA are still susceptible to polio type I, providing a substantial pool of susceptibles for epidemics to occur. . Sweden and Finland (1950-1976) An example of the problem was the outbreak of polio in an unimmunized pocket of Amish fundamentalist Protestants, where I3 cases of polio occurred (Chapter 17). In the post-eradication era in the USA there is a constant risk of introduction of wild type virus. It would seem unnecessary to change vaccine at present in the USA, but inactivated polio vaccines could be used with benefit in immunocompromised individuals (Davis et a]., 1977) , for adult contacts of children immunized with live vaccine, or for boosting immunity in adults travelling abroad. An important, and as yet incompletely answered question, is whether inactivated polio vaccine could reduce the spread of virus in the alimentary tract or circulation of virus in the community. Early experience with inactivated polio vaccines in the USA in 1960 would suggest that inactivated polio vaccine did inhibit virus circulation (Marine et al., 1962) . Thus, studies of polio outbreaks in single towns with high and low socio-economic groups showed clustering in the latter groups and few cases in the former groups. Also polio virus isolations from sewage were infrequent in the upper class areas but were frequent in the lower class areas. In an outbreak in 1960 on Rhode Island polio clustered in two lower class areas in Pawtucket (a high attack rate of 12.873, whereas other areas remained free from polio. Sewage from 1962 1963 1964 1965 1966 1967 1968 1969 1970 1971 1972 1973 1974 1975 1976 1977 1978 1979 1980 1981 Inactivated ( the lower class area contained type I polio. The most likely interpretation is that the better vaccinated affluent neighbourhoods were not subject to the silent ecological spread of virus at a time when there was an intense epidemic in a low class area.

More recently there has been some evidence from the Netherlands polio outbreak that even without 100% immunization a barrier is provided to the transmission of polio by inactivated vaccine. Although there was some evidence of infection in unimmunized classmates of cases, widespread transmission of virus did not occur. As mentioned above, it seems quite possible that inactivated polio vaccine has an inhibitory effect on pharyngeal excretion of polio virus. Gelfand et al. (1959) followed families who had received inactivated polio vaccine. When wild polio was introduced into the household previous immunization of children had no influence on virus spread and faecal excretion of virus was the same in vaccinated and unvaccinated persons. In contrast, spread of polio was limited in upper economic households and a possible explanation is that inactivated vaccine reduced excretion of pharyngeal virus, whilst the level of hygiene in these homes was high enough to limit spread of virus by the faecal oral routes.

Paralytic polio still occurs in all countries of the world but the incidence rates vary from a minute 2.6 per 100 million persons per year in the USA to over 200 cases of persisting paralysis per million persons per year in certain African countries. Recent 'Lameness surveys', a technique pioneered by WHO in the Expanded Programme on Immunization (Chapter 2) have changed previous ideas about poliomyelitis in tropical areas. It is now clear that residual paralytic polio in Burma, Egypt, Thailand, Indonesia, Ivory Coast and Brazil, India, Yemen and Niger is higher now than it was during the prevaccine era in the USA and Sweden. The old dogma that in the tropics the infection rate with polio is high but the paralysis rate is low, and that an increased incidence of the paralytic disease was related to emerging epidemics and an improvement in the standard of living reflected in diminishing infant mortality rates, may no longer hold (Sabin, 1982) . Between 50% and 7 5 " i of lameness in certain developing countries has been found to be caused by poliomyelitis virus. In the absence of immunization programmes some 4 out of 1000 school age children will be disabled by polio. This estimate does not take into account those who have been afflicted and died, and those who have recovered (Tarantola, 1982) . Obviously, on a world scale there is a serious imbalance as regards incidence of polio which requires correction as a high priority. The complete list of reasons for the calamatous differences between countries are not absolutely clear and disagreement exists in the current scientific literature. But, an important reason is the absence of a modern medical and social infrastructure in certain developing countries.

Even although the scientific debate may and will continue about the merits and drawbacks in underdeveloped countries of inactivated versus attenuated polio vaccines it is, nevertheless, clear from the studies of Sabin and others that a vigorous and concerted rapid immunization campaign (Sabin, 1982) using oral attenuated polio vaccine will stop polio in tropical and semi-tropical underdeveloped countries. As early as the 1960s Cuba showed the way with annual mass vaccination campaigns on two Sundays of the year for all children under five years of age regardless of the number of doses they might have had previously. Only such annual mass vaccinations make it possible to reach the extremely high proportions of children necessary to break the chain of infection. When vaccination is more spread out in time, wild polio and other enteroviruses remain dominant in the community. As another example, Brazil in 1980 and 1981, organized an army of volunteers to vaccinate almost all children ( 1 8 million) under 5 years of age twice a year on a single day (June and August in 1980 and August and October in 1981). A precipitous drop in reported cases followed (Sabin, 1982) . An alternative option is to use more doses of vaccine. John (1976) found that multiple doses of conventional oral vaccine containing lo5 and 105-5 TCIDSo for types I , 2 and 3 (five or more doses) were required to give more than 90'x seroconversion. but such an approach requires good organization and mass campaigns. Fang-chou et al. (1982) have described how polio has been controlled recently in sub-tropical areas in China using oral attenuated vaccine. In Guangxi autonomous region, with a population of 20 million persons, apparently healthy children were found to be heavily infected with non-polio enteroviruses. Even as late as 1971 several polio epidemics were detected since the polio vaccination rates were still low, especially in rural areas. An intensive campaign was initiated in 1973 using 'barefoot doctors' and special immunization teams in rural areas and the incidence of polio has since declined from 0.41 (1973) to 0.05 (1977) and 0.02 (1979) per I00000 persons. Fang-chou et al. (1982) conclude "the key to elimination of paralytic poliomyelitis from a community is to have a powerful leadership and authority in public health that can provide effective administrational guidance, and measures to train and organize health personnel."

The alternative approach is to use killed polio vaccine. In an early study Beale (1969) showed that vaccine given at 3, 4 and 5 months with a booster at one year gave a high seroconversion rate. The polio was mixed with diphtheria, tetanus and pertussis vaccine, thus protecting against 4 diseases in one immunization course. Immunization in Mali or Indonesia with 40 D units of currently used polio vaccine can result in essentially 100% seroconversion and it is probable that a single such dose at 6 months will provide immunological memory; subsequent contact with natural virus would boost immunity (reviewed by Beale, 1982 and see also Table 4 .21). Krishnan et al. (1983) evaluated the immunogenic efficacy of inactivated polio vaccine in India in infants given a quadruple vaccine incorporating polio with diptheria-pertussis-tetanus. A total of 150 babies aged 6-45 weeks were immunized with three doses of vaccine containing 20, 2 and 3.5 D antigen units of polio types 1, 2 and 3 respectively at intervals of 4-8 weeks and the overall sero-conversion rates to poliovirus types I , 2 and 3 were 99%, 89% and 91 % respectively. An otherwise adverse effect of maternal antibody on the immune response is reduced by increasing the intervals between doses from 4 to 8 weeks.

A total of 203 cases of paralytic polio (most with pure spinal paralysis and 39 cases of spinal involvement although in 93 % of cases one or both lower limbs were paralysed) were reported in the USA during this period and this represents a dramatic decrease from the 1 0 000 cases or so reported each year in the 1950s. Therefore the attack rate of paralytic polio has declined from a high of 13.7 cases per 100000 population (1952) to 0.003 cases per 100 000 population in 1981. Of these 203 cases, 40 were associated with epidemics, 127 were endemic, 22 were imported and 14 were in immune deficient persons. Of particular interest is the group of 86 vaccine-associated cases which were examined: 26 had occurred in recipients of oral polio vaccine, while 45 occurred in household contacts of vaccinees and 15 in non-household contacts. Perhaps, as to be expected, there was no seasonal distribution of cases and most (99 cases) were under 5 years of age. Compared to the 1960s more adult cases were being observed. Analysis of polio virus isolates by oligonucleotide mapping showed that most isolates from vaccine cases were vaccine-like in oligonucleotide spot pattern (see Chapter 17 for more details).

Of the 167 polio cases analyzed carefully, 77 involved polio type 1 , 45 type 2 and 45 type 3. Type I was responsible for all 38 epidemic associated cases but was responsible for only 4 vaccine associated cases, which were more often associated with type 3 or type 2 viruses. A more recent analysis of the epidemiological characteristics of paralytic polio cases in the USA is illustrated in Table 4 .22.

We have left to last in this chapter any indication of potential new developments in polio vaccines, either as regards live or inactivated vaccine. It has taken many years to establish the safety of existing polio vaccines and therefore it is unlikely that any new technical developments will be introduced quickly, unless (and this is unlikely) they represent a striking scientific breakthrough in, for example, imrnunogenicity (i.e. producing cross-protection against a variety of enteroviruses) or a marked reduction in cost (it should be remembered that live polio vaccines are very cheap). A more extensive discussion, with new data, is presented in Chapter 2 and we shall content ourselves here with a brief review of typical recent data with polio immunogenic peptides since this is an area of intense investigation at present. 

Recently Emini et al. (1983) have synthesized five peptides containing amino acid sequences from type 1 poliovirus structural protein VPI. Each of the peptides was able to prime the immune system of rabbits, which on subsequent chalIenge with a single inoculation of whole polio virus produced a long lasting virus neutralizing IgG antibody response. The essential data is summarized in Table 4 .23 and 4.24. The sequences were chosen after a hydrophilicity analysis of protein sequence and a sequence variation analysis obtained by comparing the amino acid sequence of VPI of the three polio serotypes. It was assumed that hydrophilic regions of VPI were more likely to be exposed at the virus surface. Four of the five peptides were recognized by neutralizing monoclonal antibodies raised to the intact type I poliovirus. A single peptide (No. 3) elicited virus neutralizing antibodies when coupled to a carrier protein. Finally, the peptides were able to prime animals who subsequently reacted rapidly when immunized with whole polio virus. The priming was specific in the sense that priming only occurred for the homologous type I virus. It is just possible that priming alone might give sufficient protection against poliovirus infection. A major study of poliovirus antigenic sites has been reported by Minor et al. (1983) . 

Since the vaccine has been so successful in preventing the disease of polio, rather little new work has been initiated in recent years to study specific inhibitors of polio virus replication, although important studies were carried out earlier with guanidine and hydroxybenzimidazole compounds (see below). In contrast, inhibitors against rhinoviruses have been sought extensively, although without success until recently (reviewed by Tyrrell et al., 1983 , Reed, 1980 . Little work has been carried out with rhinovirus vaccines, mainly because of the awesome problems of multiplicity of serotypes.

Early work by Hollinshead and Smith (1958) described the inhibitory effects of 2 (M -hydroxybenzy1)benzimidazole (HBB) (Fig. 4 .7) on polio type 2 replication in mice: death of the animals was prevented. Early tissue culture experiments established that the compound exerted an antiviral effect in vitro, was not simply virucidal and had no toxic effect on uninfected tissue culture cells (Tamm and Nemes, 1959) . This compound was one of the first antiviral molecules to be discovered. However, HBB showed some unwanted characteristics which were to become familiar to specialists working with later antiviralsdrug resistant virus mutants could be isolated with relative ease, and the compound had a very narrow antiviral spectrum more or less limited to enteroviruses (Table 4 .25).

HBB acts during the first 2-5 hours after polio virus infection of cells, although the precise point of action is not known. Virus adsorption, penetration and uncoating, however, are not affected and virus RNA and protein synthesis are not inhibited. Virus macromolecular synthesis is thought to be the target site for inhibition, and cessation of virus RNA synthesis eventually stops viral protein synthesis. An interpretation of the rather conflicting data is that HBB inhibits the synthesis of a virus directed RNA polymerase, which in turn halts replication of virus RNA leading to inhibition of synthesis of viral capsid polypeptides. Alternatively, other experiments suggest that HBB inhibits viral RNA synthesis, thereby inhibiting replication of infectious virus.

HBB significantly delays the development of enterovirus CPE in MK cells, although the cells ultimately succumb to virus induced CPE, presumably because of 

Other Coxsackie viruses Rhinoviruses virus induced cell protein and RNA shut off. Production of infectious virus is very significantly inhibited by HBB in vitro with > 8.0 log,, LD,,/ml inhibition of certain enteroviruses. However, as with many antivirals much less antiviral effect is noted in vivo.

A large number of derivatives of HBB have been investigated for virus inhibitory effects (Tamm et al., 1969) and it has been found that the hydroxybenzyl group at position 2 of the benzimidazole ring is of critical importance. Also, replacement of the benzimidazole nucleus of HBB by an imidazole ring results in complete loss of antiviral activity. Certain substituted derivatives such as 1 -propyl, 1 $-methylpropyl and I-phenyl-HBB are more active and selective in vitro than HBB itself. Thus, the selective virus inhibitory activity of HBB is dependent upon the overall geometry of the molecule and most structural modifications lead to a loss of viral inhibitory activity and selectivity.

As early as 1961 Rightsel et al. published details of the inhibition of polio virus by guanidine and soon afterwards the by now familiar drug resistant or even drug dependent mutants were detected (Loddo et al., 1963) .

Many enteroviruses are inhibited in vitro by guanidine (see Table 4 .26 and Fig.  4 .8) but nevertheless the antiviral spectrum of the compound is severely restricted to RNA viruses.

Guanidine has no direct virucidal effect and enterovirus attachment and penetration are not inhibited. (Crowther and Melnick, 1961) . Inhibiting effects begin midway through the virus latent phase and continue through the reproduction cycle. The principal site of guanidine action appears to be the synthesis of viral RNA and more than one step may be affected (reviewed by Tershak et al., 1982, Caliguiri and Tamm, 1968) . Polio RNA polymerase is not inhibited in vitro, presumably because initiation of viral RNA synthesis does not occur in cell free extracts but only results in completion of RNA chains in the process of synthesis during extraction of the crude enzyme (all hough more recently pure preparations of picornavirus polymerase that utilize exogenous viral RNA have been obtained and could now be tested). Inititation of synthesis of viral RNA seems to be depressed by guanidine, and release of completed RNA chains is restricted. Current data would point to the initiation step of viral RNA synthesis as the site of action of guanidine (Tershak et al., 1982) . In addition, guanidine exerts mild conformational changes in viral proteins and specifity could be due to a specific amino acid sequence in picornaviruses compared to cell proteins or proteins of other viruses. It is possible that synthesis and encapsidation of viral RNA both require capsid subunits and Vpg, and any interaction with guanidine would cause multiple aberrations.

Guanidine, in combination with HBB has been used to successfully treat echovirus type 9 and coxsackie A9 infections in newborn mice, although neither compound alone was effective (Eggers, 1982) . Theoretical levels of inhibitor were 200 pM HBB and 2mM guanidine.

Several acyclic &diketones were shown to inhibit the replication of equine picornaviruses (Diana et al., 1977) . Arildone, 4-[(2-chloro-4-methoxy)phenoxy]hexyl-3,5heptadione, emerged as a promising candidate antiviral, having broad spectrum in vitro antiviral activity against equine rhinovirus and interestingly HSV (Table 4 .27. 28). The compound arildone is composed of a /?-diketone separated from the substituted benzene ring by an alkyl chain of 6 carbons. It is stable to heat but virtually insoluble in water, which is a drawback. Substituents of the benzene ring contributed lipophilicity and substitution and addition of more hydrophobic substituents decreased the antiviral activity of the compound. Shorter and longer alkyl chains decreased antiviral activity (reviewed by McSharry and Pancic, 1982) .

The compound is thought to inhibit polio replication by preventing intra-cellular uncoating of the virion (McSharry et al., 1979) perhaps by stabilizing capsid proteins so that they cannot undergo conformational changes required for uncoating and release of virion RNA. The isolation of drug resistant mutants of polio also suggests a direct antiviral effect on the virus. Certainly arildone appears to stabilize polio in the presence of heat and alkali (Table 4 .29).

Arildone was assessed for its ability to prevent paralysis and death in mice infected intracerebrally with a lethal dose of human poliovirus type 2 (McKinlay et al., 1982) . Intraperitoneal administration of arildone suspended in gum tragacanth prevented paralysis and death in a dose-dependent manner (minimal inhibitory dose = 32 mg/kg, twice daily) and protected animals from virus challenges in excess of twenty 50% lethal doses (Table 4 .30). Oral medication with arildone solubilized in corn oil was similarly effective in preventing poliovirus-induced paralysis and death. Arildone was therapeutically effective even when intraperitoneal medication was delayed for 48 h postinfection. Analysis of the virus titres in the central nervous system tissues of animals infected with 200 50% lethal doses demonstrated that arildone reduced titres in the brain and spine by approximately 3 and 4 log,,PFU per g of tissue, respectively, implying that direct inhibition of virus replication was responsible for host survival. However, this animal model depends on direct inoculation of polio into the CNS and therefore the relevance of the study to human polio must await further study. It should be noted that the acute oral LDSo of arildone in mice is in excess of 8 g/kg and monkeys and rats medicated daily for 5 weeks with 1000 mg/kg showed no toxic reactions.

In summary, these compounds excite little interest with clinicians because of the success of polio vaccines and relative lack of interest in other enteroviruses. Their main use at present appears to be in laboratory studies of enterovirus replication, including genetic markers. 

Minor respiratory infection (common cold) is one of the commonest viral diseases in the UK and other temperate and even sub-tropical countries. In addition, they cause exacerbation of chronic conditions such as asthma, chronic bronchitis or cardiac failure (reviewed by Andrewes, 1962 , Hope-Simpson, 1958 . We have mentioned above that, at present, few cross-reacting antigenic determinants have been detected among this group of 150 viruses and hence development of effective vaccines would be difficult. Since the viruses are all closely related as regards physico-chemical properties and presumably biochemistry receptor sites etc., it would appear more logical to place an emphasis on development of antiviral drugs or interferon. Most pharmaceutical groups searching for new antivirals include typical rhinoviruses in their antiviral screening programme and most of the latter work is done in tissue culture systems because of the absence of an animal model of human rhinoviruses. Suitable compounds are tested in volunteers for pharmacological properties and any nontoxic compounds are then tested at, for example, the Common Cold Research Unit at Harvard Hospital,'Shlisbury, or in comparable units in Leningrad and Houston. Volunteers in the UK unit are aged from 18-50 years and on arrival at the unit are given a medical examination and a preliminary serum sample is taken and subsequently used to estimate the level of any anti-virus antibody (Beare and Reed, 1977) . During the experimental period volunteers are isolated in pairs in their own self-contained apartments and no physical contact is allowed with other persons ( Fig. 4.9) . Subjective symptoms and objective criteria are used to grade reactions induced by experimental viruses ranging from very mild (trivial reactions), mild (local symptoms), moderate (both local and constitutional symptoms) and severe (febrile and other unpleasant reactions necessitating bed rest). The number of paper handkerchiefs used each day is counted and may often increase from a baseline of 3 per day to 35 per day during the symptoms of a cold. After 3 days quarantine, antiviral medication is started and the volunteers are then infected with rhinovirus intranasally (Fig. 4.10) . Nasal washings are taken for several days thereafter for virus reisolation studies. Drugs may be administered locally by spray or orally. In the latter case there must be evidence that enough compound reaches the secretions of the upper respiratory tract. On the other hand, mucociliary clearance mechanisms are efficient and may rapidly reduce the local concentration of a nasally administered drug. Reed (1980) has summarized earlier trials in volunteers with both specific antivirals and interferon (Tables 4.31 and 4.32) (see also Chapter 3). In general, guanidinetype molecules have given disappointing results in volunteers but more recently a benzimidazole has been shown to have mild antiviral effects (enviroxime or 2amino-I -(isopropyl sulphonyl)-6-benzimidazole phenyl ketone oxime (Table 4 .33, Fig. 4.1 I) . The compound completely suppressed plaque formation in vitro by rhinoviruses types 3, 9, 14 and 31 (Table 4 .33). Moreover, enviroxime inhibited a fur- Fig. 4.9 . Accommodation at the Salisbury Common Cold Unit (after Beare and Reed. 1977) ther 12 rhinovirus serotypes by at least 3.0 log,,,units at 0.5 ,ug/ml. Enviroxime was equally effective in human embryo fibroblasts and HeLa cells and also in human embryo nasal trachea organ culture at concentrations of 0.2 pg/ml. In clinical trials (Table 4 .34) both intranasal and oral administration of the compound was used. and the severity of colds was reduced (Fig. 4.12) although at the borderline of statistical significance. We should remember also that volunteers were given a minimum virus challenge in the presence of a maximum (near toxic) level of drug. Even under these conditions the antiviral effect was not striking, but it nevertheless represents the first synthesized antiviral coumpound to have detectable anti-rhinovirus effects in man.

However, trials of enviroxime have also been carried out by Hayden and Gwaltney (l982) , who were unable to detect any antiviral effect (Table 4 .35). Intranasal administration of enviroxime by aerosol spray was associated with drug levels in nasal secretions that 1 h later averaged 750-fold higher than those inhibitory for rhinoviruses in vitro (0.2 pglml). However, administration of intranasal enviroxime (one spray per nostril, five times per day) to susceptible volunteers, beginning 1 day before and continuing for 4 days after virus exposure, did not significantly reduce infection or illness due to experimentally induced rhinovirus type 39 infection. The combined results of two separate trials yielded an infection rate of IOOo,, for 21 pla- cebo-treated and 89% for I9 enviroxime-treated subjects. Approximately one-half of the volunteers in each group had seroconversion to the challenge virus. Overall, 52% of the placebo-treated and 53% of the enviroxime-treated subjects developed colds. No significant differences in symptom scores, nasal mucus weights, or numbers of nasal tissues used were observed between the two groups. Two-thirds of the enviroxime-treated volunteers noted intranasal irritation immediately after sprays, as compared with only one-third of the placebo-treated subjects.

Another recently investigated compound, dichloroflavan, (Fig. 4.13 ) was shown to have no detectable anti-rhinovirus effects in volunteers (Fig. 4 .14, Table 4 .36) although the compound has anti-rhinovirus activity in vitro (Table 4 .37), but this could be attributed to pharmacology problems with perhaps only low concentrations reaching the nasal mucosa.

A nitrobenzene derivative, MDL-860, was found to inhibit plaque formation, cyto- pathic effect, or both in 11 of 12 picornaviruses (Table 4 .38) at concentrations which did not affect cell growth (Torney et al., 1982) . The compound did not directly inactivate the virus and inhibited actinomycin D-resistant 7H-uridine uptake in cells infected with coxsackie virus All or rhinovirus 1-A, whereas incorporation into uninfected cells was not inhibited. With three picornaviruses (echovirus type 12, poliovirus type 2, and rhinovirus type l-A) made photosensitive with neutral red, MDL-860 did not appear to cause a significant reduction in their loss of photosensitivity (uncoating) during the first 3 h of infection. The authors concluded that MDL-860 appeared to inhibit some early event in virus replication, after uncoating, which is required for synthesis of viral RNA.

In multiple growth cycle experiments, 1 pg of MDL-860 per ml caused a reduction in virus yield of at least 1.0 log,, 50% tissue culture infectious doses per 0.2 R o 09-01 79 (4',5-dihydroxy-3,3',7-trimethoxyflavone), isolated from a Chinese medicinal herb, was found to have potent antiviral activity by . It selectively inhibited the replication of human picornaviruses, such as rhinoviruses and Coxsackie viruses in tissue culture (Table 4. (Table 4 .41). The critical time for the inhibition of rhinovirus replication by the compound was 2 to 4 h after virus adsorption, i.e., in the early stages of virus replication. It markedly inhibited coxsackie virus and rhinovirus RNA synthesis in infected HeLa cells, but not in a cell-free system using the RNA polymerase complex isolated from the infected cells. In the infected cells, the RNA polymerase complex was not formed in the presence of the drug and therefore, it was suggested that Ro 09-0179 interferes with some process of viral replication which occurs between viral uncoating and the initiation of viral RNA synthesis.

Furthermore, studies of various analogs related to 4',5-dihydroxy-3,3',7-trimethoxyflavone led to the identification of 4'-ethoxy-2'hydroxy-4,6'-dimethoxychalcone (Ro 09-0410), a new and different type of antiviral agent . Ro 09-0410 had a high activity against rhinoviruses but no activity against other picornaviruses. Of 53 rhinovirus serotypes tested, 46 were susceptible to the compound in HeLa cell cultures. The concentration of Ro 09-0410 inhibiting 50% of the types of rhinovirus was about 0.03 pglml, whereas the 50% cytotoxic concentration was 30 pglml. Ro 09-0410 inactivated rhinoviruses in direct dose-, time-, and temperature-dependent fashion. We can conclude that the inactivation may be associated with the binding of the agent to some specific site of the rhinovirus capsid. Finally, we end up near the beginning of attempts at chemotherapy with rhinoviruses, namely with interferon. Merigan et al. (1973) showed that 14 x lo6 units (50 pg approximately) of partially purified human leukocyte interferon could protect volunteers against infection with rhinovirus type 4. At the time this experiment used up a great proportion of the total world supply of interferon and the pretreatment and treatment was prolonged! This classic experiment has now been repeated but "Groups of three mice were infected intraperitoneally with about 10 LD500f coxsackievirus B1 and were treated orally four times with Ro 09-0298 at 1, 2, 5, and 19h after infection using highly purified and ten-fold more interferon, and using rhinovirus type 9 (Scott et al., 1982) . The data is summarized in Table 4 .42. Symptoms were markedly reduced but virus infection and seroconversion were not prevented and it was possible that interferon only dehyed the onset of the cold. There is still a long way to go before the common cold is vanquished!

This infection caused by enterovirus 70 now has a unique position in virology as one of the two human viruses which are pandemic in their epidemiology (with influenza still heading the 'record' as the classic example). The virus infection was first described by Chatterjee et al. (1970) in Ghana as a disease with an incubation period as short as 24 h and was called Apollo 11 disease in the locality because the outbreak coincided with the time of Apollo I 1 landings on the moon. Within 3 years outbreaks had occurred in Japan (Kono, 1975 ) and a new picornavirus was isolated from the conjunctival scrapings. Enterovirus 70 has now spread almost around the world. (Hierholzer et al., 1975 , Likar et al., 1975 , Mirkovic et al., 1973 . Although Apollo 11 disease is most usually rather benign with complete recovery within 2 weeks, some neurological complications have nevertheless been described affecting about 1 in 10 000 cases. Most of the paralysed cases are men and typically the neurological phase begins 2-3 weeks after the onset of conjunctivitis with an acute hypotonic asymmetrical proximal paralysis of the lower limbs with fever, malaise and nerve root pains. Cranial nerve paralysis is often noted. Paralysis may be permanent in up to 50% of those affected and hence in the near future the virus may be an important candidate for vaccine development or chemoprophylaxis. Epidemics have reached enormous proportions (Table 4 .43) with over a million cases, for example, in Calcutta in 1971. The disease is most frequently seen in young adults and is not common in children, although serological evidence shows that infection occurs no less frequently in children (Table 4 .44). Under conditions of poor hygiene and overcrowding rapid and extensive spread can occur. Enterovirus 70 is seldom found in the faeces of patients. The viruses are rather fastidious from a cultivation point of view but may be isolated in human diploid fibroblasts and organ cultures of human embryonic conjunctiva. They are not pathogenic for suckling mice. Sensitive kinetic neutralization tests have shown that serological differences occur among isolates of enterovirus 70 (Higgins, 1982) .

Apollo I 1 disease is an excellent example of how a new disease can occur in man not only as a result of a previously unknown virus (e.g. Marburg virus) but also when members of a group of viruses behave in a previously undescribed fashion, with coxsackie A24 and enterovirus 70 causing disease not previously recognized as characteristic of the enteroviruses.

At present no vaccine or specific treatment exists. In fact even if a live or inactivated vaccine were to be developed it is not clear which route of administration, for exam- ple, would be most effective. Specific antivirals could prevent the secondary paralysis syndrome, especially when the rather long interval between conjunctivitis and paralysis is considered, but a large number of persons would have to be treated to prevent a single case of paralysis. As with certain arboviruses (see Chapter 5 ) resources might be more usefully diverted to altering and improving hygiene and living conditions in developing countries where the highest attack rates have been recorded.

Polio has been well controlled in most developed countries using ive or (in a ew countries) inactivated vaccines. Research work has intensified using genetic engineering techniques to produce live attenuated viruses with defined and stable mutations so as to prevent reversion to virulence, and also to produce immunogenic oligopeptides or proteins for a new generation of inactivated polio vaccines. Chemotherapy is therefore not required for polio infections. In contrast, no vaccines have been developed against rhinovirus infections and nor are vaccines thought to have a use, unless broadly reacting antigenic determinants can be located. Several interesting but only weakly effective antiviral compounds have been selected against rhinoviruses and this is a major research area at present. Studies continue also with interferon, but because of toxicity problems these look less interesting at present.

Sequence and biochemical data is now available for several additional enterovirus strains (including the pandemic enterovirus type 70 and hepatitis A) and this could open new possibilities both with antivirals or vaccines (e.g. synthetic peptides) in the near future. At present little attention has been paid to preventing the spread of many important enteroviruses of the coxsackie and ECHO virus group, although some of these viruses can result in serious neurological sequelae, resembling polio viruses in this regard.

Harben lectures: the common cold

Experience with the picric acid-alum spray in the prevention of poliomyelitis in

Clinical and epidemiological features of Cox-Baker, A.B. (1949) Neurological signs of bulbar poliomyelitis

Control of poliomyelitis: Killed or living vaccine

The study of antiviral compounds in volunteers

Induction of neutralizing antibodies by the coxsackievirus 83 virion polypeptide VP2

The structural polypeptide VPI of poliovirus type 1 elicits neutralizing antibodies. Compte rendu hebdomadaire des seances de I'Acadkmie des sciences

Second attacks of poliomyelitis in human beings in relation to immunity. virus types and virulence

Sero-immunity to poliomyelitis virus in Sweden 10 years after introduction of vaccinations with inactivated poliovirus vaccine. A pilot study

The immune response to vaccination with inactivated poliovirus vaccine in Sweden

Comparative monkey neurovirulence of Sabin type I11 poliovirus vaccines

Action of guanidine on the replication of polio virus RNA. Virology Carter, A.H. (1933) Bornholm disease

Reduced secretory antibody response to live attenuated measles and poliomyelitis vaccines in malnourished children

An epidemic of acute conjunctivitis in Ghana

Poliomyelitis in Des Moines, Iowa in 1959. The influence of Salk vaccination on the epidemic pattern and the spread of the virus in the community

keys by instillation of alum into the nostrils

103-1 11. sackie group B virus infection. Ann intern Med 63, 597. delphia. dichloroflavan (BW 683C). a new antirhinovirus compound

Infectious Diseases: Epidemiology and Clinical Practice

Preliminary report on mass oral immunization of population against poliomyelitis with live virus vaccine from A.B. Sabin's attenuated strains

The strategy of the picornavirus genome: some implications for chemotherapy

Studies on the inhibitory effect of guanidine on poliovirus multiplication in cell culture

Chronic progressive poliomyelitis secondary to vaccination of an immunodeficient child

Antiviral activity of some B-diketones I. Aryl alkyl diketones. In vitro activity against both RNA and DNA viruses

I 977) Comparative diagnosis of picornavirus (Enterovirus and Rhinovirus) infections

Chemotherapy of Viral Infections

Alteration in pathogenicity for monkeys of brunhilde strain of poliomyelitis virus following cultivation in human tissues

The cultivation of the poliomyelitis viruses in tissue culture. Nobel lecture, delivered at the Karolinska Institute

Priming for and induction of antipoliovirus neutralising antibodies by synthetic peptides

Poliomyelitis in China

Monoclonal antibodies specific for the Sabin vaccine strain of poliovirus 3 . Lancet i

Poliovirus specific primer dependent RNA polymerase able to copy poly (A)

Evaluation of the 1954 Field Trial of Poliomyelitis Vaccine

Studies on the development of natural immunity to poliomyelitis in Indians. IV. Natural infections with polioviruses following immunization with a formalin inactivated vaccine

The epidemiology of enteroviruses

Large-scale use of Sabin type 2 attenuated poliovirus vaccine in Singapore during a type 1 poliomyelitis epidemic

Interference patterns encountered when using attenuated poliovirus vaccines

Prophylactic activity of intranasal enviroxime against experimentally induced rhinovirus type 39 infection

Serosurvey for 'acute haemorrhagic conjunctivitis' virus (enterovirus 70) antibodies in the southeastern United States. with review of the literature and some epidemiologic implications

Coxsackie-virus A24 and acute haemorrhagic conjunctivitis

Enterovirdl conjunctivitis and its neurological complications

Viremia in human poliomyelitis

Anti-picornavirus Flavone Ro 09-0179

Direct and specific inactivation of rhinovirus by Chalcone Ro 09-0410

Antibody response of infants in tropics to five doses of oral polio vaccine

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