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The ecology of influenza viruses in ducks and analysis of influenza viruses with monoclonal antibodies
Comp. lmmun. Microbiol. infect. Dis., Vol. 3. pp. 155-164. (~ PergamonPress Ltd., Printedin GreatBritain
0147 9571/80/0601~)155$02.00/0
THE ECOLOGY OF INFLUENZA VIRUSES IN DUCKS A N D ANALYSIS OF INFLUENZA VIRUSES WITH MONOCLONAL ANTIBODIES* V. S. HINSHAW, R . G . WEBSTER, W . J. BEAN a n d G . SRIRAM Division of Virology, St Jude children's Research Hospital, 332 N. Lauderdale, P.O. Box 318, Memphis, TN 38101, U.S.A.
Abstract--It has been suggested that influenza A viruses from lower animals may be involved in the origin of human pandemic strains. To test this hypothesis, we have been analyzing the influenza A viruses present in lower mammals and birds. Over the past few years, it has become increasingly evident that a major reservoir of influenza viruses exists in both domestic and feral avian species, particularly ducks. For examiale, studies on migratory ducks in Canada in 1978 showed that 5096 of these healthy birds were infected with influenza viruses. These viruses included 16 different combinations of hemagglutinin and neuraminidase subtypes, including those related to human strains as hemagglutinins, Hav7, H2, and Hswl and neuraminidases, N1 and N2. The duck isolates which were antigenically related to the HswlNl viruses from pigs and man were evaluated by in vivo pathogenicity tests in ferrets, ducks, pigs, and chickens; the results indicated that these avian HswlN1 viruses replicated in both avian and mammalian spoeies, underlining their potential for interspeeies transmission. Since the reassortment of genetic information between human and animal influenza A viruses has been suggested as the mechanism for the origin of new human strains, we also examined the migratory ducks for evidence of mixed infections, i.e. infection with two antigenicaUy distinct viruses to determine if this phenomenon occurs in nature. Antisera to the original isolate was used to select for antigenically different viruses in the original cloacal sample. Mixtures of two and, on rare occasions, three distinct subtypes (e.g., Hav7N2, Hav6N2, and Hav7Neq2) were isolated from one bird. Analysis of the RNAs of these viruses by polyacrylamide gel electrophoresis suggested that genetic reassortment between viruses in the mixedly infected birds was occurring. These findings agree with laboratory studies in which recombinant viruses were isolated from mixedly infected ducks. These data indicate that mixed infections do occur in the natural setting, thus establishing the situation where genetic reassortment between viruses could occur. The avian species, particularly ducks, continue to represent an impressive reservoir of influenza A viruses and may well contribute to the appearance of related influenza viruses in other species, including humans. Key words: Influenza A viruses, ducks, monoelonai antibodies
ECOLOGIE DES VIRUS GRIPPAUX CHEZ LES CANARDS ET ANALYSE DES VIRUS GRIPPAUX AVEC ANTICORPS MONOCLONIQUES R~sumt---On a suggtr~ que les virus grippaux A des animaux inf~rieurs peuvent ~tre impliquts darts I'origine des souches pandtmiques humaines. Pour mettre cette hypoth~se /l l'~preuve nous avons analyst l'influence des virus A prtsents chez les petits mammiftres et les oiseaux. Ces derni~res anntes, il est devenu de plus en plus trident que le principal rtservoir de virus grippaux existe chez les asp~ces aviaires domestiques, plus particulitrement chez les canards. Par exemple, des 6tudes sur les canards migrateurs du Canada en 1978, ont montr~ que 50% de ces oiseaux bien portants &aient infeetts par des virus grippaux. Ces virus comprenaient 16 combinaision difftrentes d'htmaglutinine et de sous-types de neuraminidases, y compris celles en relation avec les souches humaines, telle que les h~maglutinines Hart, H2 et Hswl et les *This work was supported in part by U.S. Public Health Service Research Grant AI 08831 from the National Institute of Allergy and Infectious Diseases and Contract AI 52524 from the National Institutes of Health and by ALSAC. 155
156
V. S. HINSHAW, R, G. WEBSTER, W. J. BEAN and G. SRIRAM
neuraminidases N 1 et N2. Les pr616vements effectues sur les canards qui &aient antigeniquement li6s aux virus HswlNI porcins et humains, furent ~valu6s par des tests de pathog6nicitb in ~,ivo chez les canards, les porcs et les poulets. Les r6sultats ont montr6 que ces virus aviaires H s w l N 1 se produisaient dans les esp6ces avaires et mammif6res, soulignant leur potentiel pour la transmission inter-esp~ces. Depuis que le ressortiment de l'information g6n6tique entre les virus grippaux A humains et animaux a 6t6 suppos6 ~tre un m~canisme pour l'origine de nouvelles souches humaines, nous avons egalement examinb les canards migrateurs pour mettre en 6vidence les infections mixtes, c'est ~t dire l'infection avec deux virus antig6niquement diff6rents si ce ph6nombne se produit en nature.
Mots-clefs: Virus grippe A, canards, anticorps monoclonal
INTRODUCTION Influenza A viruses are widespread in water fowl in the United States [ 1], particularly in feral ducks [2]. Influenza A viruses are unique among infectious agents infecting man in their frequent and extensive antigenic variation; every 10 to 30 yr antigenically new viruses appear (antigenic shift) and cause major pandemics of disease. In the interim periods, viruses undergo minor antigenic variation (antigenic drift) and cause epidemics of disease. The origin of these pandemic strains which appear suddenly in the human population is not known, but the hypothesis has been developed that some of the 'new' human viruses may arise by recombination between the human strain and an influenza virus from lower mammals and birds I3, 4]. It is because of this possibility that the ecology of influenza in lower mammals and birds is of such interest [6-9]. This paper describes some of the recent findings concerning the ecology of avian influenza A viruses and examines the evidence for a relationship between these viruses and those of humans. Feral ducks as a reservoir of influenza A viruses Our ecological studies over the past four years have concentrated on the study of water birds trapped in the Vermillion River County, Alberta, Canada. Studies have been done during the month of August, after the breeding season and prior to migration when the ducks are assembling in marshalling areas. Some of these ducks migrate down the Mississippi flyway of the United States and others down the Pacific flyway. In co-operation with the Canadian Wildlife Service, tracheal and cloacal samples are collected for viral examination. Studies in these birds have revealed a high frequency of virus isolation from year to year (Table 1), thus in juvenile mallard ducks (Anas platyrhynchos) the frequency of isolation varied from 22% in 1976 to 67% in 1978. The viruses were predominantly isolated from cloacal samples and viruses were also isolated from lakewater and fecal material on the shorelines. Previous studies show that these viruses replicate preferentially in the cells lining the intestinal tract [ 10]. Many different influenza A viruses have been isolated each year; the results from 1978 are presented in Table 2 which shows that 801 influenza A viruses were Table 1. Frequency of influenza A virus isolation from feral ducks in Alberta, Canada Year 1976
1977
1978
22%
26%
67%
% of juvenile mallard ducks yielding virus
(A nasplatyrhynchos)
157
A major reservoir of influenza A viruses in nature
Table 2. Antigenic subtypes of influenza A viruses isolated from Canadian feral ducks in 1978 Antigenic classification
Number of viruses
H2Nav2 HswlN1 HavlNav2 Hav2Nav2 Hav2Neql Hav4N2 Hav4Navl Hav4Neq2 Hav6N2 Hav6Neq2 Hav6Nav5 Hav7Nav2 Hav7N2 Hav7Neq2 Hav7Navl Hav8Nav4
1 4 2 1 4 8 119 21 505 5 1 1 7 157 5 1
isolated and grouped into 16 different combinations of hemagglutinin and neuraminidase. Of particular interest, are the viruses possessing hemagglutinin molecules similar to those found in mammals, these include the H2, Hswl, and Hav7 and viruses possessing the neuraminidases, N1 and N2. Since Hav7 is antigenically very similar to the H3 hemagglutinin subtype of man, it can be seen that the only hemagglutinin subtype of human influenza virus that has not been isolated from avian sources is the HI variety. The predominant influenza A virus present in the feral duck population varies (Table 3). Thus, in 1976 and 1977, the predominant virus subtype was Hav7Neq2, but, in 1978, was Hav6N2. Table 3. Most frequent influenza A virus isolated from feral ducks in Alberta, Canada
Antigenic subtype (number isolated) Total number of influenza A viruses isolated
1976
Year 1977
1978
Hav7Neq2 55
Hav7Neq2 128
Hav6N2 506
106
355
801
The mechanism of survival of the many different influenza A viruses in the feral duck population is not yet resolved, but laboratory studies have shown that some of these viruses are excreted in fecal material for considerable periods of time (Table 4). Ducks infected with A/duck/Alberta/35/76 (HswlNl) continued to shed virus for up to 30 days, showing that the viruses can be maintained for a long period in one bird. Influenza A viruses of different subtypes have also been isolated from ducks during migration suggesting that the viruses continually circulate in the avian population. An alternative possibility for the maintenance of these viruses in the feral duck population is that they may survive in frozen lakes for an extended period, but to date we have not isolated influenza viruses from the lakes in Canada when the ducks were not present.
158
V.S. HINSHAW,R. G. WEBSTER,W. J. BEANand G. SRIRAM Table 4. Prolonged excretion of influenza virus in duck feces Isolation of influenza virus from fecal samples:
Virus strain
A/duck/Alberta/35/76 (HswlN1)
Days after infection
% Positive
1 2 3 4-13 14 15 16 21-28 29 30 31
0 50 85 100 100 80 80 80 20 20 0
Infectivity Titer-log 10/ml
6.30 4.30
3.96 2.50
One-day-old Peking white ducks were infected intratracheally with approximately 10~ EIDs0 of A/duck/Alberta/35/76 (Hsw 1N1). Cloacal samples were collected daily and assayed for virus in embryonated hens' eggs.
Genetic interaction between avian influenza viruses It has been proposed that genetic interaction between different influenza A viruses plays a role in the emergence of some of the human pandemic strains. Convincing'evidence for genetic reassortment has been obtained under laboratory conditions, but has never been convincingly demonstrated in nature. The presence of many different influenza A subtypes in ducks on one lake in Canada provides the opportunity for mixed infections and genetic interaction to occur. With this in mind, during 1978, we studied the samples from ducks for evidence of multiple infections (Table 5). It is apparent from Table 5 that multiple infection of Table 5. Multiple influenza A viruses isolated from individual Canadian feral ducks Number of samples
Antigenic subtypes isolated
13
Hav6N2 Hav7Neq2
3
Hav6N2 H av4 Nav 1 Hav6N2 Hav4Neq2
1
Hav7Neq2 Hav4N2
1
Hav7N2 Hav7Neq2
2
Hav6N2 Hav7N2 Hav7Neq2
A major reservoir of influenza A viruses in nature
159
ducks occurs quite frequently; two different antigenic subtypes were isolated from 19 different samples and on two occasions, three different antigenic subtypes were isolated from the same bird. RNA analysis of the three different viruses from the same bird showed heterogeneity in their migration patterns, suggesting the presence of many different influenza viruses in the same bird. Whether these occurred by recombination between two influenza viruses in the same animal could not be answered, for it is possible that a multitude of different influenza A viruses co-circulate and the ducks could be multiply infected with them. Since it is difficult to answer this question in nature, ducks were experimentally infected with two antigenic subtypes of influenza viruses (Table 6). Ducks infected with the duck/Alberta/35/76 (HswlN1) strain were subsequently infected with duck/Albertaf48/76 (HavlNav2). Fecal samples were examined for parental and recombinant viruses without any selective pressure and it can be seen that recombinant viruses were isolated. RNA analyses of these viruses confirmed the serological findings and showed genetic exchange between many of the RNAs. These studies show that there is a very large pool of different influenza A viruses in feral ducks and that genetic interaction between these viruses occurs readily. Table 6. Recombination between influenza A viruses in the intestinal tract o f ducks
Ducks infected orally with the following influenza A viruses
Viruses isolated from feces at limit dilution* Days after infection
H sw 1N 1
Duck/AlbertaJ35/76 (Hsw 1N 1)
1 2 3-6
0 + +
Duck/Alberta J48/76 (Hav 1Nav2)
7 8 9-17 18
+ + + +
Hav 1Nav2
Hav 1N 1
+ + +
+* +*
*No selection for isolation ofrecombinants. Juvenile mallard ducks (Anasplatyrhynchos)from 4 to 6 months of age were inoculated orally with approximately 107 EIDso of H s w l N 1 and Hay 1Nay2 influenza viruses on the days indicated. Fecal material was collected daily from the ducks and inoculated into embryonated hens' eggs at limit dilutions. The virus isolates were cloned twice and the surface antigens identified with specific antisera in serological tests.
Replication of avian influenza viruses in mammalian species If avian influenza viruses play any role in the emergence of strains that infect mammals, it might be anticipated that these viruses should have some potential to infect mammalian species. Previous studies in our laboratory have shown that the HswlN1 viruses isolated from Canadian wild ducks can be adapted to pigs and other studies (Bachmann and Ottis, personal communication) have shown that the HswlN1 virus isolated from ducks in Germany can infect pigs. In order to determine if the avian influenza viruses isolated from Canadian feral ducks could replicate in pigs, initial uncloned viruses were inoculated directly into the species (Table 7). It is apparent from this table that the HswlN1 influenza viruses isolated from ducks can replicate in pigs; the viruses were recovered from the animals for
160
V. S. HINSHAW, R. G. WEBSTER, W. J. BEAN and G. SRIRAM Table 7. Replication of avian influenza viruses in pigs Antigenic subtype
Virus isolation on days post-infection
Disease signs
HswlNl H2Nav2
1,2,3,4~5 1,3, 5
None None
Four different primary isolates of Hsw 1N 1 and one H2Nav2 influenza A virus were inoculated into groups of 2-month-old pigs. Nasal samples were collected daily for influenza virus isolation and the pigs were examined for signs of disease.
each of five consecutive days post-infection, although none of the viruses produced signs of disease. Similar studies with H2Nav2 influenza viruses from ducks showed that these viruses could also replicate in pigs, but none of the viruses produced signs of disease. These studies confirm the earlier findings that the HswlN1 avian strains do have the potential to replicate in pigs. The implications of these findings are that some of the Hsw 1N 1 influenza viruses isolated from pigs may originate from birds and that genetic interaction between mammalian and avian influenza viruses could occur in the mammalian species. The studies also show that H2Nav2 influenza viruses have the potential to replicate in pigs.
Detailed antigenic analysis of the hemagglutinin of the H2 influenza viruses from man and ducks using monoclonal antibod&s Monoclonal antibodies to the hemagglutinin molecule of A/Japan/305/57(H2N2) influenza virus have been prepared in our laboratory by fusing the spleen cells from immunized mice with myeioma cells [11, 12]. The fused antibody producing cells (hybridomas) were cloned in agar and inoculated intraperitoneally into mice to produce ascites tumors. The hybridoma cell lines produce high concentrations of monoclonal antibodies that have the advantage of being completely specific for a particular antigenic site on an antigen. In the present studies, a panel of 11 monoclonal antibodies to the hemagglutinin molecule of A/Japan/305/57 were employed. It should be pointed out that this panel of monoclonal antibodies probably represents only about 25% of the possible number of different monoclonal antibodies that can be obtained to a hemagglutinin molecule. Previous studies using A/PR/8/34 (HON 1) have shown that there are as many as 50 different antigenic determinants on the hemagglutinin molecule of this virus [13]. Many of these determinants are probably overlapping. The number of nonoverlapping antigenic areas on the hemagglutinin molecule has not been resolved, but preliminary investigations suggest that there may be only four 1141.
Analysis of human H2 strains Detailed antigenic analysis of the H2 influenza viruses from man (1957-1968) with monoclonal antibodies reveal several features (Table 8). (1) Antigenic variation can occur at many sites on the hemagglutinin molecule, thus the Japan/170/62 and Leningrad/29/63 strains showed many differences from the A/Japan/305/57 virus. (2) That viruses antigenically indistinguishable from the Japan/305/57 were isolated in
A m a j o r reservoir o f influenza A viruses in nature
161
Table 8. C r o s s - r e a c t i o n s between H 2 N 2 influenza A viruses from h u m a n s with m o n o c l o n a l antibodies H I a n t i b o d y titers to the following influenza viruses (log2)
M o n o c l o n a l antibodies to A/Japan/305/57
~ .-, ~
~ ~
"~ ~=
"~ ~
~ .~.
~ ~°
,~,°
Z
J 28/1 J41/4 J 61/7 J 67/7 J 100/4 J L 7/2 JL 22/4 JB 75/1 J 162/6 J 137/4 JB 112/2
4.0 6.3 4.2 5.2 8.3 5.2 3.6 4.5 9.0 8.2 4.9
* * * * * * * * * * 5.9
3.0 6.7 4.9 5.6 8.0 6.6 2.7 7.9 8.6 8.5 6.7
* * * * * * * * * * *
4.0 * 8.0 1.9 9.5 * * * * * 5.6
6.9 8.5 7.6 7.5 9.9 6.9 4.7 7.6 10 10 5.6
* * 6.9 * 8.5 * * * * * 4.7
* * * * * * * * * * *
The figures show the reciprocal o f the dilution inhibiting four agglutinating doses o f v i r u s in log2 terms.
= < 1.0.
1961 and 1965; the England/I/61 and Moscow/1019/65 strains could not be distinguished from the Japan/305/57 strain with this panel ofmonoclonal antibodies. (3) That some antigenic determinants on the hemagglutinin molecule disappear for several years and then reappear on later strains. Thus, Taiwan/1/64 and Korea/426/68 share identical determinants with A/Japan/305/57 that had been absent on the viruses circulating in the intervening years. The reappearance of antigenically identical viruses raises the question of whether these are laboratory contaminants or whether antigenically identical viruses do reappear from some as yet unknown source, as happened with the A/U.S.S.R./90/77 (H1N1) strain [15].
Analysis of avian H2 strains Detailed antigenic analysis of avian H2 influenza viruses with monoclonal antibodies showed that, although many of these viruses share antigenic determinants with the A/Japan/305/57 strain, none of the avian viruses are identical with the human strain (Table 9). The avian viruses duck/Ontario/77(H2N 1) and duck/Alberta/77 (H2Nav2) share four out of six determinants with the Japan/305/57 strain. The virus isolated from pintail ducks in U.S.S.R. (A/pintail/U.S.S.R./76-H2Nav2) shares three antigenic determinants with the Japan/305/57 strain. In contrast, the A/duck/GDR/72 (H2Nav6) shares none of the six antigenic determinants with the A/Japan/305/57 strain. The reactivity patterns show that the H2 avian influenza viruses are closely related to the Japan/305/57 human strain and that there is less overall antigenic variation in the avian influenza H2 viruses than in the above group of H2 influenza viruses from humans. Although these detailed antigenic analyses show that none of the H2 avian influenza viruses examined to date had a hemagglutinin identical to the donated gene to the human strain in 1957, H2
V. S. HINSHAW,R. G. WEBSTER,W. J. BEANand G. SRIRAM
162
Table 9. Cross reactions betweenavian H2 influenzaviruseswith monoclonal antibodies HI antibody titers to the followingviruses (log2) ~
-
7
Z
~
"
Z
~,
~
~ z -.
~
~
__ ~
4.0 6.3 5.2 8.7 5.2 3.6
* * * * * *
* 4.9 * * 5.5 3.1
* 2.9 3.0 6.5 * 2.9
* 6.9 4.7 7, I * 2.7
* * * 7,5 * 3.6
* 5.3 3.9 6.7 * 3.2
0/6
3/6
3/6
4/6
2/6
4/6
Monoclonal antibodies to A/Japan/305/57 J 28/1 J 41/4 J 67/7 J 100/4 JL 7/2 JL 22/4 Sites shared with Japan/305/57
~. =z
The figures show the reciprocalof the dilution inhibitingfour agglutinatingdoses of virus in log2terms. * - - < 1.0.
viruses from avian sources continue to be isolated and it may only be a matter of time before an identical hemagglutinin is detected. These findings would suggest that surface antigens of previous h u m a n strains are maintained in avian viruses.
DISCUSSION The above studies show that there is a large pool of influenza A viruses in feral ducks and that these viruses are maintained in this species from year to year, even though the predominant virus may change. These viruses cause no signs of disease in ducks, replicate predominantly in the intestinal tract and are shed and spread through water. It is apparent that genetic interaction occurs readily between the influenza A viruses in the C a n a d i a n duck population, providing a huge pool of different influenza viruses. The model proposed some years ago for the origin of the A / H o n g K o n g / 6 8 (H3N2) strain [81 of h u m a n influenza virus by recombination between an H 2 N 2 and an avian Hav7 virus has to date not been refuted. The above-information lends credence to this model, showing that reassortment of genetic information of influenza viruses can occur readily in nature. The above studies, showing that some avian influenza viruses have the potential to replicate in m a m m a l i a n species, increases the opportunity of genetic interaction between influenza A viruses of m a m m a l i a n and avian species. It should be stressed that genetic reassortment is not the only mechanism for generating new influenza viruses. The other mechanisms include (i) reappearance of influenza viruses such as the A/U.S.S.R./90/77 (H1N1) strain from an, as yet, unexplained source; (ii) the possibility that influenza viruses m a y survive in an integrated form in the D N A of a human, animal, or even lungworm, to be rescued m a n y years later [16]; and (iii) that accumulated
A major reservoir of influenza A viruses in nature
163
point mutations may explain antigenic shift. There is, as yet, no evidence for the latter two possibilities. In addition to the potential importance in the origin of some human influenza viruses, the influenza viruses in feral ducks may also have economic importance in domestic animals. A recent outbreak of influenza in the turkey raising industry in United States was caused by a virus antigenically related to an influenza virus isolated from feral ducks in Canada (Hav4Neq2). To date, there is no conclusive evidence that this virus spreads from feral ducks to turkeys, but since we know that there is an intermixing of the domestic and wild birds, there seems to be no reason why this should not occur. The isolation of antigenically similar influenza A viruses from migrating feral birds from different parts of the world and from domestic species along their migration routes has been well documented for avian species in the Pacific basin [8]. Thus, influenza viruses isolated from shearwaters (Puffins pacificus) on the Great Barrier Reef of Australia have been found in chickens in Hong Kong, in turkeys in California and in wild ducks in Delaware. The Hav6Nav5 influenza virus isolated from these species may be spread by Puffins tenuirostris along its migration route around the Pacific. Similarly, influenza virus (Hav5Nav2) which was isolated in 1961 from common terns (Sterna hirundo) in South Africa and from avian species in eastern U.S.S.R. and the islands of Alaska are related to a virus (Hav5Nl) found in 1959 in a domestic chicken in Scotland. SUMMARY (1) Canadian feral ducks are a natural reservoir of influenza A viruses. These viruses include strains antigenically related to those in mammalian species. (2) Genetic interaction occurs readily between the avian influenza viruses. (3) Some of these viruses have the potential to replicate in mammalian species. (4) Detailed antigenic analysis shows less variation in the avian H2 influenza viruses than in the human H2 strains. (5) These viruses are probably of economic importance as a source of disease in domestic avian species. (6) The above information does not refute the possibility that some pandemic strains of human influenza viruses arrive by reassortment between avian and human strains. REFERENCES 1. Easterday, B. C., The Influenza Viruses and Influenza, Kilbourne, E. D. (ed.), pp. 449-481. AcademicPress, New York (1975). 2. Hinshaw,V. S., Webster, R. G. and Turner, B., J. gen. ViroL 41, 115 (1978). 3. Webster, R. G. and Laver, W. G., The Influenza Viruses and Influenza, Kilbourne, E. D. (ed.), pp. 209-314. Academic Press, New York (1975). 4. Scholtissek,C., Hoyningen,V. and Rott, R., Virology 89, 613-617 (1978). 5. Stuart-Harris, C. H. and Sehild, G. C. Influenza: the Viruses and the Disease. Arnold, London (1976). 6. Lvov, D. K., Ecologyof viruses. Collectionof papers at the D. I. IvanovskyInstitute of Virology,Moscow (1976). 7. Shortridge, K. F., Webster, R. G., Butterfield, W. K. and Campbell, C. H., Science, N.Y. 196, 1454-1455 (1977). 8. Kaplan,M. M. and Webster, R. G., Scient. Am. 237, 88-106 (1977). 9. Zakstelskaya,L. Ya., Evstigneeva, N. A., Isachenko, V. A., Shenderovitch,S. P. and Efimova,V. A., Am. J. Epidemiol. 90, 400-405 (1969). 10. Webster, R. G., Yakhno, M., Hinshaw, V. S., Bean, W. J. and Murti, K. G., Virology 84, 268-278 (1978).
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11. 12. 13. 14. 15.
V. S. HINSHAW, R. G. WEBSTER, W. J. BEANand G. SR1RAM
Kohler, G. and Milstein, C., Nature, Lond. 256, 495 (1975). Koprowski, H., Gerhard, W. and Croce, W., Proc. hath. Acad. Sci. (U.S.A.) 74, 2985 (1977). Gerhard, W., Topics Infect. Dis. 3, 15 (1978). Yewdell, J. W., Webster, R. G. and Gerhard, W., Nature, Lond. 279, 246 (1979). Zhdanov, V. M., Zakstelskaya, L. Ya., Isachenko, V. I., Reznik, V. I., Andreyev, V. P., Lvov, D. K., Yakhno. M. A., Braude, N. A., Pysina, T. V. and Podchernyaeva, R. Ya., Lancet 1, 294 (1978). 16. Laver, W. G. and Webster, R. G., Br. Med. Bull. 35, 29 (1979).
0147 9571/80/0601~)155$02.00/0
THE ECOLOGY OF INFLUENZA VIRUSES IN DUCKS A N D ANALYSIS OF INFLUENZA VIRUSES WITH MONOCLONAL ANTIBODIES* V. S. HINSHAW, R . G . WEBSTER, W . J. BEAN a n d G . SRIRAM Division of Virology, St Jude children's Research Hospital, 332 N. Lauderdale, P.O. Box 318, Memphis, TN 38101, U.S.A.
Abstract--It has been suggested that influenza A viruses from lower animals may be involved in the origin of human pandemic strains. To test this hypothesis, we have been analyzing the influenza A viruses present in lower mammals and birds. Over the past few years, it has become increasingly evident that a major reservoir of influenza viruses exists in both domestic and feral avian species, particularly ducks. For examiale, studies on migratory ducks in Canada in 1978 showed that 5096 of these healthy birds were infected with influenza viruses. These viruses included 16 different combinations of hemagglutinin and neuraminidase subtypes, including those related to human strains as hemagglutinins, Hav7, H2, and Hswl and neuraminidases, N1 and N2. The duck isolates which were antigenically related to the HswlNl viruses from pigs and man were evaluated by in vivo pathogenicity tests in ferrets, ducks, pigs, and chickens; the results indicated that these avian HswlN1 viruses replicated in both avian and mammalian spoeies, underlining their potential for interspeeies transmission. Since the reassortment of genetic information between human and animal influenza A viruses has been suggested as the mechanism for the origin of new human strains, we also examined the migratory ducks for evidence of mixed infections, i.e. infection with two antigenicaUy distinct viruses to determine if this phenomenon occurs in nature. Antisera to the original isolate was used to select for antigenically different viruses in the original cloacal sample. Mixtures of two and, on rare occasions, three distinct subtypes (e.g., Hav7N2, Hav6N2, and Hav7Neq2) were isolated from one bird. Analysis of the RNAs of these viruses by polyacrylamide gel electrophoresis suggested that genetic reassortment between viruses in the mixedly infected birds was occurring. These findings agree with laboratory studies in which recombinant viruses were isolated from mixedly infected ducks. These data indicate that mixed infections do occur in the natural setting, thus establishing the situation where genetic reassortment between viruses could occur. The avian species, particularly ducks, continue to represent an impressive reservoir of influenza A viruses and may well contribute to the appearance of related influenza viruses in other species, including humans. Key words: Influenza A viruses, ducks, monoelonai antibodies
ECOLOGIE DES VIRUS GRIPPAUX CHEZ LES CANARDS ET ANALYSE DES VIRUS GRIPPAUX AVEC ANTICORPS MONOCLONIQUES R~sumt---On a suggtr~ que les virus grippaux A des animaux inf~rieurs peuvent ~tre impliquts darts I'origine des souches pandtmiques humaines. Pour mettre cette hypoth~se /l l'~preuve nous avons analyst l'influence des virus A prtsents chez les petits mammiftres et les oiseaux. Ces derni~res anntes, il est devenu de plus en plus trident que le principal rtservoir de virus grippaux existe chez les asp~ces aviaires domestiques, plus particulitrement chez les canards. Par exemple, des 6tudes sur les canards migrateurs du Canada en 1978, ont montr~ que 50% de ces oiseaux bien portants &aient infeetts par des virus grippaux. Ces virus comprenaient 16 combinaision difftrentes d'htmaglutinine et de sous-types de neuraminidases, y compris celles en relation avec les souches humaines, telle que les h~maglutinines Hart, H2 et Hswl et les *This work was supported in part by U.S. Public Health Service Research Grant AI 08831 from the National Institute of Allergy and Infectious Diseases and Contract AI 52524 from the National Institutes of Health and by ALSAC. 155
156
V. S. HINSHAW, R, G. WEBSTER, W. J. BEAN and G. SRIRAM
neuraminidases N 1 et N2. Les pr616vements effectues sur les canards qui &aient antigeniquement li6s aux virus HswlNI porcins et humains, furent ~valu6s par des tests de pathog6nicitb in ~,ivo chez les canards, les porcs et les poulets. Les r6sultats ont montr6 que ces virus aviaires H s w l N 1 se produisaient dans les esp6ces avaires et mammif6res, soulignant leur potentiel pour la transmission inter-esp~ces. Depuis que le ressortiment de l'information g6n6tique entre les virus grippaux A humains et animaux a 6t6 suppos6 ~tre un m~canisme pour l'origine de nouvelles souches humaines, nous avons egalement examinb les canards migrateurs pour mettre en 6vidence les infections mixtes, c'est ~t dire l'infection avec deux virus antig6niquement diff6rents si ce ph6nombne se produit en nature.
Mots-clefs: Virus grippe A, canards, anticorps monoclonal
INTRODUCTION Influenza A viruses are widespread in water fowl in the United States [ 1], particularly in feral ducks [2]. Influenza A viruses are unique among infectious agents infecting man in their frequent and extensive antigenic variation; every 10 to 30 yr antigenically new viruses appear (antigenic shift) and cause major pandemics of disease. In the interim periods, viruses undergo minor antigenic variation (antigenic drift) and cause epidemics of disease. The origin of these pandemic strains which appear suddenly in the human population is not known, but the hypothesis has been developed that some of the 'new' human viruses may arise by recombination between the human strain and an influenza virus from lower mammals and birds I3, 4]. It is because of this possibility that the ecology of influenza in lower mammals and birds is of such interest [6-9]. This paper describes some of the recent findings concerning the ecology of avian influenza A viruses and examines the evidence for a relationship between these viruses and those of humans. Feral ducks as a reservoir of influenza A viruses Our ecological studies over the past four years have concentrated on the study of water birds trapped in the Vermillion River County, Alberta, Canada. Studies have been done during the month of August, after the breeding season and prior to migration when the ducks are assembling in marshalling areas. Some of these ducks migrate down the Mississippi flyway of the United States and others down the Pacific flyway. In co-operation with the Canadian Wildlife Service, tracheal and cloacal samples are collected for viral examination. Studies in these birds have revealed a high frequency of virus isolation from year to year (Table 1), thus in juvenile mallard ducks (Anas platyrhynchos) the frequency of isolation varied from 22% in 1976 to 67% in 1978. The viruses were predominantly isolated from cloacal samples and viruses were also isolated from lakewater and fecal material on the shorelines. Previous studies show that these viruses replicate preferentially in the cells lining the intestinal tract [ 10]. Many different influenza A viruses have been isolated each year; the results from 1978 are presented in Table 2 which shows that 801 influenza A viruses were Table 1. Frequency of influenza A virus isolation from feral ducks in Alberta, Canada Year 1976
1977
1978
22%
26%
67%
% of juvenile mallard ducks yielding virus
(A nasplatyrhynchos)
157
A major reservoir of influenza A viruses in nature
Table 2. Antigenic subtypes of influenza A viruses isolated from Canadian feral ducks in 1978 Antigenic classification
Number of viruses
H2Nav2 HswlN1 HavlNav2 Hav2Nav2 Hav2Neql Hav4N2 Hav4Navl Hav4Neq2 Hav6N2 Hav6Neq2 Hav6Nav5 Hav7Nav2 Hav7N2 Hav7Neq2 Hav7Navl Hav8Nav4
1 4 2 1 4 8 119 21 505 5 1 1 7 157 5 1
isolated and grouped into 16 different combinations of hemagglutinin and neuraminidase. Of particular interest, are the viruses possessing hemagglutinin molecules similar to those found in mammals, these include the H2, Hswl, and Hav7 and viruses possessing the neuraminidases, N1 and N2. Since Hav7 is antigenically very similar to the H3 hemagglutinin subtype of man, it can be seen that the only hemagglutinin subtype of human influenza virus that has not been isolated from avian sources is the HI variety. The predominant influenza A virus present in the feral duck population varies (Table 3). Thus, in 1976 and 1977, the predominant virus subtype was Hav7Neq2, but, in 1978, was Hav6N2. Table 3. Most frequent influenza A virus isolated from feral ducks in Alberta, Canada
Antigenic subtype (number isolated) Total number of influenza A viruses isolated
1976
Year 1977
1978
Hav7Neq2 55
Hav7Neq2 128
Hav6N2 506
106
355
801
The mechanism of survival of the many different influenza A viruses in the feral duck population is not yet resolved, but laboratory studies have shown that some of these viruses are excreted in fecal material for considerable periods of time (Table 4). Ducks infected with A/duck/Alberta/35/76 (HswlNl) continued to shed virus for up to 30 days, showing that the viruses can be maintained for a long period in one bird. Influenza A viruses of different subtypes have also been isolated from ducks during migration suggesting that the viruses continually circulate in the avian population. An alternative possibility for the maintenance of these viruses in the feral duck population is that they may survive in frozen lakes for an extended period, but to date we have not isolated influenza viruses from the lakes in Canada when the ducks were not present.
158
V.S. HINSHAW,R. G. WEBSTER,W. J. BEANand G. SRIRAM Table 4. Prolonged excretion of influenza virus in duck feces Isolation of influenza virus from fecal samples:
Virus strain
A/duck/Alberta/35/76 (HswlN1)
Days after infection
% Positive
1 2 3 4-13 14 15 16 21-28 29 30 31
0 50 85 100 100 80 80 80 20 20 0
Infectivity Titer-log 10/ml
6.30 4.30
3.96 2.50
One-day-old Peking white ducks were infected intratracheally with approximately 10~ EIDs0 of A/duck/Alberta/35/76 (Hsw 1N1). Cloacal samples were collected daily and assayed for virus in embryonated hens' eggs.
Genetic interaction between avian influenza viruses It has been proposed that genetic interaction between different influenza A viruses plays a role in the emergence of some of the human pandemic strains. Convincing'evidence for genetic reassortment has been obtained under laboratory conditions, but has never been convincingly demonstrated in nature. The presence of many different influenza A subtypes in ducks on one lake in Canada provides the opportunity for mixed infections and genetic interaction to occur. With this in mind, during 1978, we studied the samples from ducks for evidence of multiple infections (Table 5). It is apparent from Table 5 that multiple infection of Table 5. Multiple influenza A viruses isolated from individual Canadian feral ducks Number of samples
Antigenic subtypes isolated
13
Hav6N2 Hav7Neq2
3
Hav6N2 H av4 Nav 1 Hav6N2 Hav4Neq2
1
Hav7Neq2 Hav4N2
1
Hav7N2 Hav7Neq2
2
Hav6N2 Hav7N2 Hav7Neq2
A major reservoir of influenza A viruses in nature
159
ducks occurs quite frequently; two different antigenic subtypes were isolated from 19 different samples and on two occasions, three different antigenic subtypes were isolated from the same bird. RNA analysis of the three different viruses from the same bird showed heterogeneity in their migration patterns, suggesting the presence of many different influenza viruses in the same bird. Whether these occurred by recombination between two influenza viruses in the same animal could not be answered, for it is possible that a multitude of different influenza A viruses co-circulate and the ducks could be multiply infected with them. Since it is difficult to answer this question in nature, ducks were experimentally infected with two antigenic subtypes of influenza viruses (Table 6). Ducks infected with the duck/Alberta/35/76 (HswlN1) strain were subsequently infected with duck/Albertaf48/76 (HavlNav2). Fecal samples were examined for parental and recombinant viruses without any selective pressure and it can be seen that recombinant viruses were isolated. RNA analyses of these viruses confirmed the serological findings and showed genetic exchange between many of the RNAs. These studies show that there is a very large pool of different influenza A viruses in feral ducks and that genetic interaction between these viruses occurs readily. Table 6. Recombination between influenza A viruses in the intestinal tract o f ducks
Ducks infected orally with the following influenza A viruses
Viruses isolated from feces at limit dilution* Days after infection
H sw 1N 1
Duck/AlbertaJ35/76 (Hsw 1N 1)
1 2 3-6
0 + +
Duck/Alberta J48/76 (Hav 1Nav2)
7 8 9-17 18
+ + + +
Hav 1Nav2
Hav 1N 1
+ + +
+* +*
*No selection for isolation ofrecombinants. Juvenile mallard ducks (Anasplatyrhynchos)from 4 to 6 months of age were inoculated orally with approximately 107 EIDso of H s w l N 1 and Hay 1Nay2 influenza viruses on the days indicated. Fecal material was collected daily from the ducks and inoculated into embryonated hens' eggs at limit dilutions. The virus isolates were cloned twice and the surface antigens identified with specific antisera in serological tests.
Replication of avian influenza viruses in mammalian species If avian influenza viruses play any role in the emergence of strains that infect mammals, it might be anticipated that these viruses should have some potential to infect mammalian species. Previous studies in our laboratory have shown that the HswlN1 viruses isolated from Canadian wild ducks can be adapted to pigs and other studies (Bachmann and Ottis, personal communication) have shown that the HswlN1 virus isolated from ducks in Germany can infect pigs. In order to determine if the avian influenza viruses isolated from Canadian feral ducks could replicate in pigs, initial uncloned viruses were inoculated directly into the species (Table 7). It is apparent from this table that the HswlN1 influenza viruses isolated from ducks can replicate in pigs; the viruses were recovered from the animals for
160
V. S. HINSHAW, R. G. WEBSTER, W. J. BEAN and G. SRIRAM Table 7. Replication of avian influenza viruses in pigs Antigenic subtype
Virus isolation on days post-infection
Disease signs
HswlNl H2Nav2
1,2,3,4~5 1,3, 5
None None
Four different primary isolates of Hsw 1N 1 and one H2Nav2 influenza A virus were inoculated into groups of 2-month-old pigs. Nasal samples were collected daily for influenza virus isolation and the pigs were examined for signs of disease.
each of five consecutive days post-infection, although none of the viruses produced signs of disease. Similar studies with H2Nav2 influenza viruses from ducks showed that these viruses could also replicate in pigs, but none of the viruses produced signs of disease. These studies confirm the earlier findings that the HswlN1 avian strains do have the potential to replicate in pigs. The implications of these findings are that some of the Hsw 1N 1 influenza viruses isolated from pigs may originate from birds and that genetic interaction between mammalian and avian influenza viruses could occur in the mammalian species. The studies also show that H2Nav2 influenza viruses have the potential to replicate in pigs.
Detailed antigenic analysis of the hemagglutinin of the H2 influenza viruses from man and ducks using monoclonal antibod&s Monoclonal antibodies to the hemagglutinin molecule of A/Japan/305/57(H2N2) influenza virus have been prepared in our laboratory by fusing the spleen cells from immunized mice with myeioma cells [11, 12]. The fused antibody producing cells (hybridomas) were cloned in agar and inoculated intraperitoneally into mice to produce ascites tumors. The hybridoma cell lines produce high concentrations of monoclonal antibodies that have the advantage of being completely specific for a particular antigenic site on an antigen. In the present studies, a panel of 11 monoclonal antibodies to the hemagglutinin molecule of A/Japan/305/57 were employed. It should be pointed out that this panel of monoclonal antibodies probably represents only about 25% of the possible number of different monoclonal antibodies that can be obtained to a hemagglutinin molecule. Previous studies using A/PR/8/34 (HON 1) have shown that there are as many as 50 different antigenic determinants on the hemagglutinin molecule of this virus [13]. Many of these determinants are probably overlapping. The number of nonoverlapping antigenic areas on the hemagglutinin molecule has not been resolved, but preliminary investigations suggest that there may be only four 1141.
Analysis of human H2 strains Detailed antigenic analysis of the H2 influenza viruses from man (1957-1968) with monoclonal antibodies reveal several features (Table 8). (1) Antigenic variation can occur at many sites on the hemagglutinin molecule, thus the Japan/170/62 and Leningrad/29/63 strains showed many differences from the A/Japan/305/57 virus. (2) That viruses antigenically indistinguishable from the Japan/305/57 were isolated in
A m a j o r reservoir o f influenza A viruses in nature
161
Table 8. C r o s s - r e a c t i o n s between H 2 N 2 influenza A viruses from h u m a n s with m o n o c l o n a l antibodies H I a n t i b o d y titers to the following influenza viruses (log2)
M o n o c l o n a l antibodies to A/Japan/305/57
~ .-, ~
~ ~
"~ ~=
"~ ~
~ .~.
~ ~°
,~,°
Z
J 28/1 J41/4 J 61/7 J 67/7 J 100/4 J L 7/2 JL 22/4 JB 75/1 J 162/6 J 137/4 JB 112/2
4.0 6.3 4.2 5.2 8.3 5.2 3.6 4.5 9.0 8.2 4.9
* * * * * * * * * * 5.9
3.0 6.7 4.9 5.6 8.0 6.6 2.7 7.9 8.6 8.5 6.7
* * * * * * * * * * *
4.0 * 8.0 1.9 9.5 * * * * * 5.6
6.9 8.5 7.6 7.5 9.9 6.9 4.7 7.6 10 10 5.6
* * 6.9 * 8.5 * * * * * 4.7
* * * * * * * * * * *
The figures show the reciprocal o f the dilution inhibiting four agglutinating doses o f v i r u s in log2 terms.
= < 1.0.
1961 and 1965; the England/I/61 and Moscow/1019/65 strains could not be distinguished from the Japan/305/57 strain with this panel ofmonoclonal antibodies. (3) That some antigenic determinants on the hemagglutinin molecule disappear for several years and then reappear on later strains. Thus, Taiwan/1/64 and Korea/426/68 share identical determinants with A/Japan/305/57 that had been absent on the viruses circulating in the intervening years. The reappearance of antigenically identical viruses raises the question of whether these are laboratory contaminants or whether antigenically identical viruses do reappear from some as yet unknown source, as happened with the A/U.S.S.R./90/77 (H1N1) strain [15].
Analysis of avian H2 strains Detailed antigenic analysis of avian H2 influenza viruses with monoclonal antibodies showed that, although many of these viruses share antigenic determinants with the A/Japan/305/57 strain, none of the avian viruses are identical with the human strain (Table 9). The avian viruses duck/Ontario/77(H2N 1) and duck/Alberta/77 (H2Nav2) share four out of six determinants with the Japan/305/57 strain. The virus isolated from pintail ducks in U.S.S.R. (A/pintail/U.S.S.R./76-H2Nav2) shares three antigenic determinants with the Japan/305/57 strain. In contrast, the A/duck/GDR/72 (H2Nav6) shares none of the six antigenic determinants with the A/Japan/305/57 strain. The reactivity patterns show that the H2 avian influenza viruses are closely related to the Japan/305/57 human strain and that there is less overall antigenic variation in the avian influenza H2 viruses than in the above group of H2 influenza viruses from humans. Although these detailed antigenic analyses show that none of the H2 avian influenza viruses examined to date had a hemagglutinin identical to the donated gene to the human strain in 1957, H2
V. S. HINSHAW,R. G. WEBSTER,W. J. BEANand G. SRIRAM
162
Table 9. Cross reactions betweenavian H2 influenzaviruseswith monoclonal antibodies HI antibody titers to the followingviruses (log2) ~
-
7
Z
~
"
Z
~,
~
~ z -.
~
~
__ ~
4.0 6.3 5.2 8.7 5.2 3.6
* * * * * *
* 4.9 * * 5.5 3.1
* 2.9 3.0 6.5 * 2.9
* 6.9 4.7 7, I * 2.7
* * * 7,5 * 3.6
* 5.3 3.9 6.7 * 3.2
0/6
3/6
3/6
4/6
2/6
4/6
Monoclonal antibodies to A/Japan/305/57 J 28/1 J 41/4 J 67/7 J 100/4 JL 7/2 JL 22/4 Sites shared with Japan/305/57
~. =z
The figures show the reciprocalof the dilution inhibitingfour agglutinatingdoses of virus in log2terms. * - - < 1.0.
viruses from avian sources continue to be isolated and it may only be a matter of time before an identical hemagglutinin is detected. These findings would suggest that surface antigens of previous h u m a n strains are maintained in avian viruses.
DISCUSSION The above studies show that there is a large pool of influenza A viruses in feral ducks and that these viruses are maintained in this species from year to year, even though the predominant virus may change. These viruses cause no signs of disease in ducks, replicate predominantly in the intestinal tract and are shed and spread through water. It is apparent that genetic interaction occurs readily between the influenza A viruses in the C a n a d i a n duck population, providing a huge pool of different influenza viruses. The model proposed some years ago for the origin of the A / H o n g K o n g / 6 8 (H3N2) strain [81 of h u m a n influenza virus by recombination between an H 2 N 2 and an avian Hav7 virus has to date not been refuted. The above-information lends credence to this model, showing that reassortment of genetic information of influenza viruses can occur readily in nature. The above studies, showing that some avian influenza viruses have the potential to replicate in m a m m a l i a n species, increases the opportunity of genetic interaction between influenza A viruses of m a m m a l i a n and avian species. It should be stressed that genetic reassortment is not the only mechanism for generating new influenza viruses. The other mechanisms include (i) reappearance of influenza viruses such as the A/U.S.S.R./90/77 (H1N1) strain from an, as yet, unexplained source; (ii) the possibility that influenza viruses m a y survive in an integrated form in the D N A of a human, animal, or even lungworm, to be rescued m a n y years later [16]; and (iii) that accumulated
A major reservoir of influenza A viruses in nature
163
point mutations may explain antigenic shift. There is, as yet, no evidence for the latter two possibilities. In addition to the potential importance in the origin of some human influenza viruses, the influenza viruses in feral ducks may also have economic importance in domestic animals. A recent outbreak of influenza in the turkey raising industry in United States was caused by a virus antigenically related to an influenza virus isolated from feral ducks in Canada (Hav4Neq2). To date, there is no conclusive evidence that this virus spreads from feral ducks to turkeys, but since we know that there is an intermixing of the domestic and wild birds, there seems to be no reason why this should not occur. The isolation of antigenically similar influenza A viruses from migrating feral birds from different parts of the world and from domestic species along their migration routes has been well documented for avian species in the Pacific basin [8]. Thus, influenza viruses isolated from shearwaters (Puffins pacificus) on the Great Barrier Reef of Australia have been found in chickens in Hong Kong, in turkeys in California and in wild ducks in Delaware. The Hav6Nav5 influenza virus isolated from these species may be spread by Puffins tenuirostris along its migration route around the Pacific. Similarly, influenza virus (Hav5Nav2) which was isolated in 1961 from common terns (Sterna hirundo) in South Africa and from avian species in eastern U.S.S.R. and the islands of Alaska are related to a virus (Hav5Nl) found in 1959 in a domestic chicken in Scotland. SUMMARY (1) Canadian feral ducks are a natural reservoir of influenza A viruses. These viruses include strains antigenically related to those in mammalian species. (2) Genetic interaction occurs readily between the avian influenza viruses. (3) Some of these viruses have the potential to replicate in mammalian species. (4) Detailed antigenic analysis shows less variation in the avian H2 influenza viruses than in the human H2 strains. (5) These viruses are probably of economic importance as a source of disease in domestic avian species. (6) The above information does not refute the possibility that some pandemic strains of human influenza viruses arrive by reassortment between avian and human strains. REFERENCES 1. Easterday, B. C., The Influenza Viruses and Influenza, Kilbourne, E. D. (ed.), pp. 449-481. AcademicPress, New York (1975). 2. Hinshaw,V. S., Webster, R. G. and Turner, B., J. gen. ViroL 41, 115 (1978). 3. Webster, R. G. and Laver, W. G., The Influenza Viruses and Influenza, Kilbourne, E. D. (ed.), pp. 209-314. Academic Press, New York (1975). 4. Scholtissek,C., Hoyningen,V. and Rott, R., Virology 89, 613-617 (1978). 5. Stuart-Harris, C. H. and Sehild, G. C. Influenza: the Viruses and the Disease. Arnold, London (1976). 6. Lvov, D. K., Ecologyof viruses. Collectionof papers at the D. I. IvanovskyInstitute of Virology,Moscow (1976). 7. Shortridge, K. F., Webster, R. G., Butterfield, W. K. and Campbell, C. H., Science, N.Y. 196, 1454-1455 (1977). 8. Kaplan,M. M. and Webster, R. G., Scient. Am. 237, 88-106 (1977). 9. Zakstelskaya,L. Ya., Evstigneeva, N. A., Isachenko, V. A., Shenderovitch,S. P. and Efimova,V. A., Am. J. Epidemiol. 90, 400-405 (1969). 10. Webster, R. G., Yakhno, M., Hinshaw, V. S., Bean, W. J. and Murti, K. G., Virology 84, 268-278 (1978).
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11. 12. 13. 14. 15.
V. S. HINSHAW, R. G. WEBSTER, W. J. BEANand G. SR1RAM
Kohler, G. and Milstein, C., Nature, Lond. 256, 495 (1975). Koprowski, H., Gerhard, W. and Croce, W., Proc. hath. Acad. Sci. (U.S.A.) 74, 2985 (1977). Gerhard, W., Topics Infect. Dis. 3, 15 (1978). Yewdell, J. W., Webster, R. G. and Gerhard, W., Nature, Lond. 279, 246 (1979). Zhdanov, V. M., Zakstelskaya, L. Ya., Isachenko, V. I., Reznik, V. I., Andreyev, V. P., Lvov, D. K., Yakhno. M. A., Braude, N. A., Pysina, T. V. and Podchernyaeva, R. Ya., Lancet 1, 294 (1978). 16. Laver, W. G. and Webster, R. G., Br. Med. Bull. 35, 29 (1979).