Molecular characteristics and biological properties (genetic markers) of candidate strains for preparation of live influenza virus vaccines

ioReviews / Analyses/ Bulletin of the World Health Organization, 62(3): 493-499 (1984) ©D World Health Orgzanization 1984 Molecular characteristics and biological properties (genetic markers) of candidate strains for preparation of live influenza virus vaccines Y. Z. GHENDON 1 The biological and molecularproperties ofboth virulent and vaccine types ofinfluenza virus strains are reviewed, including their analysis in laboratory animals and in organ tissue cultures, as well as studies of their genome peculiarities. Methods are discussed for ob- taining attenuated donor strains of influenza virus, and the properties of these strains are analysed. The methods of studying the genome composition and the genes carrying mutation lesions in recombinant vaccine strains obtained by recombination of an attenu- ated donor strain with current epidemic influenza virus variants are also reviewed. ATTENUATED DONOR STRAINS Several types of live influenza vaccines have been prepared from strains attenuated by various methods. These strains include the so-called host range (hr) mutants (obtained by being passaged through chick embryos), strains resistant to the inhibitors of normal serum (obtained by passages in the presence of inhib- itors), temperature-sensitive (ts) mutants (obtained by treatment of the virus with mutagens and contain- ing several point mutations in their genomes), and cold-adapted variants (obtained by passage of the virus at a lowered temperature) (19, 28, 35, 38). Recently recombination between an attenuated donor strain and a new variant of influenza virus has been considered the most appropriate method for ob- taining live vaccine strains; the resulting recombinant will inherit genes coding for haemagglutinin and neur- aminidase from the variant, as well as genes carrying mutations responsible for attenuation from the at- tenuated donor strain. ' Chief, Department of General Virology, Research Institute for Viral Preparations, 1st Dubrovskaya Ul. 15, 109088 Moscow, USSR, and Director of the WHO Collaborating Centre for Molecular Biology and Genetics for Epidemic and Vaccine Strains of the Influenza Virus. The A/PR/8/34 (HIN1) strain, considered to be an hr mutant, was found to be unsatisfactory as an at- tenuated donor strain because several of the recom- binants were virulent for humans, although they contained six genes coding for non-glycosylated pro- teins of the A/PR/8/34 strain (38). For this reason, the recombinants from this donor strain require additional attenuation by passages in the presence of serum containing gamma-inhibitors (31). Influenza virus ts mutants with point mutations in two genes also proved to be unacceptable as donors of attenu- ation because of a high rate of reversion of the recom- binants obtained from them (29, 38). At present, the most appropriate attenuated donor strains are the cold-adapted variants, which have been shown to be strongly attenuated in tests on humans, and whose recombinants are stable, weakly reacto- genic, and highly antigenic for humans (19). The development of laboratory techniques for the evalu- ation of attenuation of viruses, which could serve as a basis for the selection of candidate strains for use in the preparation of vaccines is one of the main problems to be solved by investigators who are look- ing for suitable attenuated influenza virus strains with which they could develop live virus vaccines. 4423 -493 Y. Z. GHENDON EVALUATION OF STRAIN PATHOGENICITY The pathogenicity of viruses in general, and of in- fluenza virus in particular, is well known to be a poly- genic characteristic where each gene may be involved in the manifestation or loss of pathogenicity of the virus (12, 32, 36). The genome composition may also be very important for the manifestation of virus pathogenicity since it has been shown that recombin- ation of non-pathogenic parents could yield path- ogenic recombinants (34). Some years ago several laboratory tests were devel- oped correlating with the pathogenicity of the viruses for humans (12). With respect to influenza virus strains, these tests included the ability of the viruses to reproduce at lowered and elevated temperatures, thermoresistance on heating, the ability to agglutinate red blood cells, adsorption-elution in red cells, and resistance to the inhibitors in blood serum (12). Studies of a limited number of virus strains revealed some correlation between these properties and path- ogenicity (reactogenicity) for humans, which was, however, not always confirmed by studies on a large number of other strains. Recently, organ cultures and some animals have been used for the laboratory evaluation of the degree of pathogenicity of candidate live vaccine virus strains before use in humans. Investigations carried out by Tyrrell and co-workers (15, 27), using tracheal organ cultures of 15-24-weeks old human embryos, revealed some correlation between the degree of at- tenuation of influenza virus strains for humans (as estimated in trials on volunteers) and the reduction in the activity of ciliated epithelium which is caused by the virus. However, a distinct correlation was observed with only one group of influenza virus recombinants, whereas in the experiments with two other groups of recombinants derived from different parents the correlation was incomplete, and for the recombinants of a fourth group (obtained by crossing A/PR/8/34 and A/England/42/72 strains), as well as for influenza B viruses, no correlation was observed. The use of ferret tracheal organ cultures (17) has shown that two influenza virus strains (A/Hong Kong/45/68 and A/Victoria/3/75), which were responsible for a high mortality rate in the USA, had a stronger ability to inhibit the activity of ciliated epi- thelium than three other strains (A/Scotland/840/74, A/New Jersey/8/76, and A/USSR/90/77), which did not show a significant increase in mortality rate. Studying ts recombinants of A/Udorn/72 influenza virus, Mostow and co-workers (26) found some cor- relation between the degree of attenuation (as tested on volunteers) and a reduction in the activity of ciliated epithelium. However, no distinct correlation was observed with ts recombinants of A/Hong Kong/68 strain. According to the data of Hara et al. (15), a correlation between the degree of attenuation of influenza virus strains for humans and their ability to reduce the activity of ciliated epithelium is less marked in ferret tracheal organ cultures than in human embryo tracheal organ cultures. Boudreault (6) improved the technique for assess- ment of the effect of influenza viruses on the activity of ciliated epithelium in ferret tracheal organ cultures by using reference influenza virus strains, e.g., A/Hong Kong/68(H3N2). A high degree of attenu- ation of this strain was shown in immunization of several hundred volunteers (3). The period during which the activity of ciliated epithelium is reduced by 50%7o following infection of ferret tracheal organ cul- tures with this virus was assumed by Boudreault to have an index of attenuation of 1.0, and the inhibiting activity of other influenza virus strains is then evaluated with respect to this period. This approach was used to study over 20 virulent and attenuated (inhibitor-resistant, cold-adapted) influenza virus strains and their recombinants, and it was found that all the attenuated viruses had an attenuation index of more than 1.0, whereas all the virulent strains (except one) had an attenuation index of less than 1.0 (6, 9). In 1976 it was demonstrated that two influenza virus strains differing in their virulence for humans had different abilities to infect ferrets (37). Later it was reported that influenza virus recombinants, administered intranasally to ferrets at low doses, differed in their virulence towards these animals, and that these differences correlated with the degree of attenuation of the viruses for humans (10). Campbell and co-workers (7) proposed four criteria to evaluate the virulence of influenza virus to ferrets: (i) deter- mination of the 500o infectious dose; (ii) determin- ation of the degree and level of infection in the upper respiratory tract; (iii) determination of the degree and duration of fever; and (iv) determination of the level of infection in the lungs. Using these criteria these re- searchers found that strains A/Finland/4/74(H3N2) and A/Okuda/57(H2N2), which differed in their virulence for humans, also differed in virulence for ferrets. However, recombinants of these viruses were examined by the same workers and found to have a similar virulence for ferrets, although these recombi- nants differed in the degree of attenuation, as was shown by trials in a limited number of volunteers. Based on the analysis of the genome composition of the recombinants, these workers are inclined to think that tests in ferrets can reflect the potential patho- genicity of influenza viruses for humans better than trials in a limited number of volunteers. It should be noted that the process of obtaining recombinants may be accompanied by the appearance of additional mu- tations in the genes of the parent strains, which may 494 INFLUENZA VIRUS STRAINS FOR VACCINES be responsible for different degrees of attenuation of the recombinants having similar genome compo- sition. In further studies, Matsuyama and co-workers (23) found a good correlation between virulence for ferrets and the degree of attenuation for humans in three of the recombinants of A/PR/8/34 and A/ England/939/69 influenza viruses, but one recombi- nant which had slight attenuation for humans proved to be highly virulent for ferrets. Differences in viru- lence for ferrets were also observed with the virulent A/Ann Arbor/6/60 strain, with a cold-adapted vari- ant of this strain, and with its cold-adapted recombi- nants (19). Michaels and co-workers (25) and, later on, Mahmud and co-workers (21, 22) discovered that intranasal administration of influenza virus strains (with virulence for humans) to newborn rats (48 hours old) led to virus replication in the nasal turbinates of the animals to high titres. The virus growth also pro- moted bacteraemia and meningitis in newborn rats infected with Haemophilus influenzae type b; in contrast, the attenuated recombinant strains derived from the attenuated donor A/PR/8/34 rarely en- hanced subsequent bacterial infection. But the attenu- ated A/PR/8/34 strain itself, when administered to newborn rats, behaved as a virulent virus. Analysis of a number of wild-type influenza virus strains, of the cold-adapted attenuated A/Ann Arbor/6/60 strain, and of its attenuated cold- adapted recombinant strains showed that the attenu- ated viruses had a reduced ability to reproduce in the nasal turbinates of newborn rats, compared with the virulent parent strains (2, 21, 22). The only exception was the wild-type strain, A/USSR/77(HIN1), which replicated to levels similar to those of the at- tenuated recombinants. The majority of the wild-type influenza virus strains were more able than the attenuated recombinant strains to promote bacter- aemia and meningitis in newborn rats infected with H. influenzae type b, but a number of strains did not differ in these properties. The majority of virulent strains differed from the attenuated viruses in the degree of virus reproduction in the lungs of newborn rats, but there were still exceptions indicating that the degree of virus reproduction in the lungs of newborn rats was not a sufficiently reliable criterion for differ- entiating between virulent and attenuated influenza virus strains for humans. Jennings and co-workers (18) studied over 30 influenza virus strains and recombinants of different origins in newborn rats and found that, on the whole, there was a correlation be- tween the degree of attenuation of viruses for humans and the level of virus reproduction in the nasal turbinates of newborn rats. But the data presented by these authors show that some virulent strains (A/Queensland/72, A/England/72, and A/Hong Kong/68), some weakly virulent strains (A/PR/8/34, A/Okuda/57, and A/Ann Arbor/60), and some at- tenuated strains (clone 6, clone 64c, recombinants 4a2 and RIT 4050) reproduced in the nasal turbinates of newborn rats to nearly equal titres. Evaluation of the ability of the virus to promote infection with H. influenzae in newborn rats showed more marked differences between virulent strains on the one hand, and weakly virulent and attenuated strains on the other. However, in this instance the virulent A/PR/ 8/34 strain and the attenuated recombinant RIT 4050 strain also behaved like virulent viruses. Efforts were made to use hamsters to differentiate between attenuated and virulent (for humans) influ- enza viruses. They showed that temperature-sensitive and cold-adapted attenuated recombinant strains differed from their virulent parents in the degree of reproduction in the lungs of hamsters. However, studies of the recombinants obtained from the attenu- ated strains A/PR/8/34 or A/Okuda/57 revealed no differences in reproduction in hamsters for the strains that differed in their degree of attenuation for humans (1). Squirrel monkeys (5) and mice (24) were also used to examine whether it was possible to determine the degree of attenuation of influenza virus for humans, but so far very few investigations have been carried out with these animals, and any conclusions on whether it is expedient to use them for the evaluation of virulence of influenza viruses for humans would be premature. Thus, organ cultures of human or ferret embryos, as well as ferrets and newborn rats, are the best studied laboratory models that have recently been used for the evaluation of the potential virulence of influenza viruses for humans. The data obtained show some correlation between the laboratory test and the degree of virulence (or attenuation) of the virus for humans in the majority of the strains under study. At the same time, there were exceptions when several wild-type influenza virus strains behaved like attenuated viruses in laboratory tests, while strains with a certain degree of attenuation could be charac- terized as virulent according to laboratory tests. It should be noted, however, that the characterization of influenza virus strains as virulent or as attenuated for humans is relative since, for the vast majority of the strains studied, the degree of virulence was evaluated only in a very limited number of volunteers, and such evaluation did not necessarily reflect the virulence (or attenuation) of the virus. Thus, the laboratory criteria of attenuation available at present should be considered as preliminary and cannot re- place the evaluation of reactogenicity of candidate live vaccine strains in volunteers. Nevertheless, pre- liminary studies of recombinants obtained by cross- ing a donor of attenuation with a wild-type influenza 495 Y. Z. GHENDON virus, using the above tests, are useful in the process of selection of candidate live influenza vaccine strains. However, it is necessary to use both virulent and attenuated parents as reference strains in these trials, and, naturally, both parents should differ distinctly in the laboratory tests. In such cases, only those recombinants that behave in laboratory models like an attenuated parent strain should be selected for further trials with volunteers. USE OF RECOMBINATION AND MOLECULAR METHODS Genetic and molecular biological techniques have recently been used to evaluate the degree of attenu- ation of influenza virus strains. As mentioned above, a recombination technique involving crossing of a donor of attenuation with a new wild-type strain is at present the optimum method for obtaining influenza virus strains for the&preparation of live vaccines. Not only should the donor of attenuation have been well studied, it is also necessary to know what genes in the donor strain have mutations correlating with attenu- ation, and how many such mutations are contained in each gene, since the total number of mutations in the genome and in each gene are directly associated with the degree of attenuation and genetic stability of the attenuated strain. Unfortunately, it is impossible at present to determine the genes carrying hr mutations in influenza virus strains whose attenuation is due to hr mutations (for instance, A/PR/8/34 and A/ Okuda/57), because accurate genetic techniques for the detection of such mutations in the genome of in- fluenza viruses are not yet available. At the same time, it is possible to detect genes carrying some mu- tations responsible for attenuation in the cold- adapted variants because these variants are not only cold-adapted but also possess a ts phenotype, and according to numerous data in the literature (12, 33, 39), ts mutations generally correlate with the loss of virus pathogenicity. The detection of genes contain- ing ts mutations is possible by a recombination tech- nique using a set of ts mutants with precisely deter- mined genes carring a ts mutation. In the USA, attempts have been made to apply a complementation-recombination technique to detect genes carrying ts mutations in the cold-adapted variant of A/Ann Arbor/6/60 (H2N2) influenza virus by using ts mutants of human influenza virus strains, but no distinct results were obtained (19). In our experiments with cold-adapted variants of A/Leningrad/134/57 (H2N2) and A/Krasnodar/ 101/59 (H2N2) strains using ts mutants of fowl plague virus with known genes carrying ts mutations, the results showed accumulation of ts mutations in the virus genome during passages at a lowered temperature. It was also found that the attenuated donor strain, from which the recombinants for live vaccine for adults are obtained and which had been passaged 17 times at a lowered temperature (A/Lenin- grad/ 134/17/57), apparently contains ts mutations in genes 1, 5, and 7, whereas the donor of attenuation used to prepare recombinants for live vaccine for children, which had been passaged 47 times at a lowered temperature (A/Leningrad/ 134/47/57), contains ts mutations in genes 1, 2, 5, 7 and 8 (13, 20). The reason for the obscure results obtained by the U.S. investigators, using a recombination technique to analyse genes carrying ts mutations in the cold- adapted variant of A/Ann Arbor/6/60 strain, is un- known. Probably it is related to differences in the technique used and, in particular, to the different sets of ts mutants used in these tests. It is possible that the ts mutants of fowl plague virus are more suitable for such analyses than the ts mutants of human influenza viruses. If recombinants are not formed in the recombi- nation process, it does not necessarily follow that the strain under study contains a ts mutation in the cor- responding gene; and if recombinants are formed, it does not mean that there are no mutations in this gene. Intragenic complementation, extragenic sup- pression, and other genetic and non-genetic inter- actions may adversely affect the interpretation of the results. Nevertheless, when appropriate controls are used, a recombination technique may yield very use- ful information on the presence of ts mutations in the genome of cold-adapted strains. Analysis of mutations in the genome of attenuated donor strains has also been carried out by molecular methods of investigation, such as analysis of virion RNA using electrophoresis, hybridization or oligo- nucleotide mapping, etc. The use of a number of such methods has revealed mutations in all the genes of the cold-adapted variant of A/Ann Arbor /6/60 (8). However, at present it is difficult to judge what changes of this kind are actually associated with virus attenuation. Currently there is no evidence to indicate the num- ber and type of mutations that should be contained in the genome as a whole, and in each individual gene of the attenuated donor strain, for such a strain to be sufficiently attentuated and completely stable on reproduction in the vaccinees. It is supposed that, optimally, mutations correlating with attenuation should be contained in each gene coding for non- glycosylated proteins. When such a strain is used as live vaccine for mass immunization of humans, it can- not become a source of virulent genes that could cause the appearance of highly virulent strains resulting from recombination with wild-type influenza viruses circulating in the human population. Nevertheless, 496 INFLUENZA VIRUS STRAINS FOR VACCINES the possibility that too many mutations in the genome of the donor strain may lead to hyperattenuation and, consequently, to a low immunogenicity cannot be excluded. This problem needs further detailed studies. Use of molecular genetic techniques for analysis of recombinants derived from well-studied donors of attenuation may be of great importance for the selection of candidate live influenza vaccine strains. According to the available data, mutations, even in a single gene, of influenza virus (particularly a ts mutation) may result in the loss of pathogenicity of the virus (14, 33); thus, a recombinant that inherited only one gene containing a ts mutation from a cold- adapted parent turned out to be areactogenic for volunteers (13). Nevertheless, it can be assumed that a completely genetically stable recombinant possessing the same degree of attenuation characteristic of the donor strain would have inherited all the genes coding for non-glycosylated proteins from the attenuated donor strain, while genes coding for the haemag- glutinin and neuraminidase would have been derived from the wild-type virus strain. The genome composition of recombinants derived from various attenuated donor strains is evaluated using analysis of electrophoretic mobility of virion RNA segments (8, 30), as well as analysis of cRNA/vRNA hybrids (11, 13, 16). Some of the re- combinants inherited all six genes coding for non- glycosylated proteins from the attenuated donor (8, 13, 23), and these recombinants could be considered as optimum candidates for use as live vaccine strains. Nevertheless, there is evidence that some recombi- nants obtained by crossing of attenuated and virulent strains were virulent for volunteers, although they had inherited six genes coding for non-glycosylated proteins from the attenuated donor (4, 38), and that recombination of two non-pathogenic influenza virus strains may yield a highly pathogenic recombinant (34). It is therefore essential that recombinants which have inherited six genes coding for non-glycosylated proteins from an attenuated donor strain should first be evaluated in laboratory models and then in volun- teers. Thus, on the one hand, there are at present no absolutely reliable laboratory tests for evaluating the potential virulence of influenza virus strains on humans, and trials of reactogenicity in a limited num- ber of volunteers can yield only relative estimates of the degree of virus attenuation. On the other hand, efficient and safe influenza prophylaxis requires the large-scale use of live influenza vaccines. From the above considerations, it can be concluded that careful trials of all candidate live influenza vaccine strains should be carried out both by laboratory tests and in volunteers. Since the technique of recombination of an attenu- ated donor strain with new wild-type influenza virus strains is the most appropriate method for obtaining live vaccine strains, it is necessary firstly to carry out detailed studies of the attenuated donor strain. This strain should be tested in laboratory models (e.g., ferret tracheal organ cultures, ferrets, and newborn rats) and compared with the virulent parent virus; the genes having mutations should be determined; and the reactogenicity, antigenicity, and genetic stability should be evaluated in a sufficient number of vaccinees. The possibility that different attenuated donor strains will be required for producing live vaccines intended for use in adults and children can- not be ruled out. Recombinants derived from a well-studied attenu- ated donor strain should, first of all, be characterized by a ts phenotype (when cold-adapted donors are used) and by the appropriate genome composition; all recombinants which inherited 5 or 6 genes coding for non-glycosylated proteins from the attenuated donor strain should be selected for further studies. One should also make sure that the genes inherited from the attenuated donor strain retained the mutation changes characteristic of the parent strain. The selected recombinants should be tested in laboratory models (ferret tracheal organ cultures, ferrets, and newborn rats) for comparison with the parent strains. Those recombinants, which behave in these tests like the parent attenuated strains, should then be tested in a limited number of volunteers for reactogenicity, antigenicity and genetic stability. Only after this has been verified should the strain be allowed for large- scale use as live vaccine. RU RESUME CARACTERISTIQUES MOLECULAIRES ET PROPRIETES BIOLOGIQUES (MARQUEURS GENETIQUES) DES SOUCHES A PRIORI UTILISABLES POUR LA PREPARATION DE VACCIN ANTIGRIPPAL VIVANT Les cultures d'organes d'embryon humain ou d'embryon de furet constituent, parallelement aux furets et aux rats nouveau-nes, les modeles de laboratoire les mieux etudies de tous ceux qu'on a recemment utilises pour evaluer la viru- lence potentielle des virus grippaux vis-a-vis de l'homme. Pour la majorite des souches examinees, on a observe une certaine correlation entre l'6preuve de laboratoire et le degre de virulence (ou d'attenuation) du virus a l'6gard de 497 498 Y. Z. GHENDON l'homme. I1 existe pourtant des exceptions, la souche sau- vage se comportant alors de la meme fa$on qu'une souche attenuee dans les epreuves de laboratoire, et inversement. Par consequent, les crit&es d'attenuation en laboratoire dont on dispose a l'heure actuelle doivent etre consideres comme preliminaires; ils ne sauraient remplacer 1'evaluation sur des volontaires du pouvoir reactogene des souches envi- sagees pour la production de vaccin vivant. La recombinaison d'une souche donatrice attenuee et d'un nouveau variant de virus grippal de type sauvage est consideree comme la methode la mieux appropriee pour obtenir une souche vaccinale vivante. Les souches dona- trices attenuees convenant le mieux sont des variants de virus grippal adapte au froid dont le genome contient un certain nombre de genes codant pour des proteines non glycosylees et porteurs de mutations ts. Ces souches dona- trices doivent etre mises a l'epreuve sur modeles de labora- toire et comparees au virus parental virulent; il faut, en outre, determiner les genes modifies a la suite d'une mutation et evaluer, chez un nombre suffisant de sujets vaccines, le pouvoire reactogene de ces souches ainsi que leur antigenicite et leur stabilite genetique. Les produits de recombinaison obtenus a partir d'une souche donatrice attenuee bien connue doivent etre caracterises par un pheno- type ts et par leur composition genomique; on retiendra pour la suite des etudes les recombinants qui ont herite de la souche donatrice attenuee 5 ou 6 genes codant pour les proteines non glycosylees. REFERENCES 1. ABOU-DONIA, H. ET AL. Growth of influenza A viruses in hamsters. Archives of virology, 65: 99-107 (1980). 2. ALl, M. ET AL. Infant rat model of attenuation for recombinant influenza viruses prepared from cold- adapted attenuated A/Ann Arbor/6/60. Infection and immunity, 38: 610-619 (1982). 3. BEARE, A. S. & BYNOE, M. L. Attenuation of human influenza viruses. British medical journal, 4: 198-201 (1969). 4. BEARE, A. S. ET AL. Trials in man with live recombinants made from A/PR/8/34(HON1) and wild H3N2 influenza viruses. Lancet, 2: 729-732 (1975). 5. BERENDT, R. F. & HALL, W. C. Reaction of squirrel monkeys to intratracheal inoculation with influenza A/ New Jersey/76 (swine) virus. Infection and immunity, 16: 476-479 (1977). 6. BOUDREAULT, A. Live influenza vaccine: screening of attenuated virus strains by a 50%o ciliary activity inhi- bition test in organ cultures of ferret trachea. Canadian journal of microbiology, 25: 279-284 (1979). 7. CAMPBELL, D. ET AL. Comparisons of virulence of influ- enza virus recombinants in ferrets in relation to their be- haviour in man and their genetic constitution. Journal of general virology, 44: 37-44 (1979). 8. Cox, N. J. ET AL. Genetic and biochemical analysis of the A/Ann Arbor/6/60 cold-adapted mutant. In: Tot, C. & Nayak, D., ed., Genetic variation among influenza viruses (ICN-UCLA Symposia on Molecular and Cellular Biology, Vol. 22). New York, Academic Press, 1981, pp. 639-652. 9. DIAz-RODRIGUEz, P. & BOUDREAULT, A. Inhibition of ciliary activity in organ cultures of ferret trachea in reference to genetic and biological characters of influ- enza virus strains. Canadian journal of microbiology, 28: 809-814 (1982). 10. FENTON, R. J. ET AL. Differential response of ferrets to infection with virulent and avirulent influenza viruses: a possible marker of virus attenuation. Archives of vir- ology, 55: 55-66 (1977). 11. FLORENT, G. Gene constellation of live influenza A vaccines. Archives of virology, 64: 171-173 (1980). 12. GHENDON, Y. Z. [Genetics of human and animal viruses.] Moscow, Nauka, 1967 (in Russian). 13. GHENDON, Y. Z. ET AL. Analysis of genome compo- sition and reactogenicity of recombinants of cold- adapted and virulent virus strains. Journal of general virology, 53: 215-224 (1981). 14. GHENDON, Y. Z. ET AL. Correlation between ts pheno- type and pathogenicity of some animal viruses. Archiv fur die gesamte Virusforschung, 42: 154-159 (1973). 15. HARA, K. ET AL. Growth and pathogenicity of influenza viruses in organ cultures of ciliated epithelium. Archiv fur die gesamte Virusforschung, 44: 227-236 (1974). 16. HAY, A. J. ET AL. Procedures for characterization of the genetic material of candidate vaccine strains. Develop- ments in biological standardization (Basel), 39: 15-24 (1977). 17. HOKE, C. H. ET AL. Comparison of several wild-type influenza viruses in the ferret tracheal organ culture system. Reviews of infectious diseases, 1: 946-952 (1979). 18. JENNINGS, R. ET AL. The replication of type A influenza viruses in the infant rat: a marker for virus attenuation. Journal of general virology, 49: 343-354 (1980). 19. KENDAL, A. P. ET AL. Development of cold-adapted re- combinant live, attenuated influenza A vaccines in the USA and USSR. Antiviral research, 1: 339-365 (1981). 20. LISOVSKAYA, K. V. ET AL. Analysis of ts mutations of cold-adapted influenza A/Leningrad/ 134/57 virus vari- ants. Acta virologica, 25: 415-417 (1981). 21. MAHMUD, M. I. A. ET AL. The infant rat as a model for assessment of the attenuation of human influenza viruses. Journal of medical microbiology, 12: 43-54 (1979). 22. MAHMUD, M. I. A. ET AL. Influenza virus infection of newborn rats: virulence of recombinant strains pre- pared from a cold-adapted, attenuated parent. Archives of virology, 61: 207-216 (1979). 23. MATSUYAMA, T. ET AL. Aspects of virulence in ferrets exhibited by influenza virus recombinants of known genetic constitution. Journal ofinfectious diseases, 141: 351-361 (1980). 24. MCCAHON, D. & SCHILD, G. C. Segregation of anti- genic and biological characteristics during influenza virus recombination. Journal of general virology, 15: 73-77 (1972). INFLUENZA VIRUS STRAINS FOR VACCINES 499 25. MICHAELS, R. H. ET AL. Influenza virus infection in newborn rats: a possible marker or attenuation for man. Journal of medical virology, 2: 253-264 (1978). 26. MOSTOW, S. R. ET AL. Temperature-sensitive mutants of influenza virus. XIII. Evaluation of influenza A/Hong Kong/68 and A/Udorn/72 ts and wild-type viruses in tracheal organ cultures at permissive and restrictive tem- peratures. Journal of infectious diseases, 136: 1-37 (1977). 27. MOSTOW, S. R. & TYRELL, D. A. J. The behaviour in vitro of attenuated recombinant influenza viruses. Archiv fur die gesamte Virusforschung, 43: 385-392 (1973). 28. MURPHY, B. R. & CHANOCK, R. M. Present status of live influenza A virus vaccine. Monographs in paedi- atrics (Basel), 11: 1-20 (1979). 29. MURPHY, B. R. ET AL. Production and level of genetic stability of an influenza A virus temperature-sensitive mutant containing two genes with ts mutations. Infec- tion and immunity, 37: 235-242 (1982). 30. PALESE, P. & RITCHEY, M. B. Live attenuated influenza virus vaccines. Strains with temperature-sensitive defects in P3 protein and nucleoprotein. Virology, 78: 183-191 (1977). 31. RYTEL, M. W. ET AL. New live attenuated influenza A/England/42/72(H3N2) vaccine (Alice): reactogen- icity, immunogenicity and protection efficacy. Journal of infectious diseases, 132: 652-659 (1975). 32. SCHOLTISSEK, C. The genome of the influenza virus. Current topics in microbiology and immunology, 80: 139-169 (1978). 33. SCHOLTISSEK, C. ET AL. Correlation of pathogenicity and gene constellation of an influenza A virus (fowl plague). I. Exchange of a single gene. Virology, 81: 74-80 (1977). 34. SCHOLTISSEK, C. ET AL. Correlation of pathogenicity and gene constellation of influenza A viruses. II. Highly neurovirulent recombinants derived from non-neuro- virulent or weakly neurovirulent parent virus strains. Virology, 95: 492-500 (1979). 35. STUART-HARRIS, C. The present status of live influenza virus vaccine. Journal of infectious diseases, 142: 784-793 (1980). 36. SWEET, C. & SMITH, H. Pathogenicity of influenza virus. Microbiological reviews, 44: 303-330 (1980). 37. TOMS, G. L. ET AL. The behaviour in ferrets of two closely related clones of influenza virus of different virulence for man. Journal of experimental pathology, 57: 37-48 (1976). 38. TYRRELL, D. A. ET AL. Development and use of influ- enza vaccines. Bulletin of the World Health Organiz- ation, 59: 165-212 (1981). 39. TOLPIN, M. D. ET AL. Evaluation of a phenotypic re- vertant of the A/Alaska/77-ts-lA2 reassortant virus in hamsters and in seronegative adult volunteers: further evidence that the temperature-sensitive phenotype is responsible for attenuation of ts-1A2 reassortant viruses. Infection and immunity, 36: 645-650 (1982).