The nucleoprotein as a possible major factor in determining host specificity of influenza H3N2 viruses

VIROLOGY

147,287-294 (1985)

The Nucleoprotein as a Possible Major Factor in Determining Host Specificity of Influenza H3N2 Viruses C. SCHOLTISSEK,**l H. BURGER,* 0. KISTNER,* *In&it&

ftir

Virologie, Justus-Liebig-Universitti TDepartment of Microbiology,

AND

K. F. SHORTRIDGE?

Giessen, Giessen D63O0, Federal Republic of Germany, University of Hong Kong, Hong Kong

and

Received June 7, 1985; accepted August 9, 1985 In an attempt to assess the importance of the nucleoprotein (NP) in the determination of host specificity, a series of experiments was performed on influenza A viruses of the H3N2 subtype. We have examined rescue of mutants of A/FPV/Rostock/34 with temperature-sensitive (ts) lesions in the nucleoprotein (NP) gene by double infection of chick embryo cells with H3N2 strains isolated from different species. The ts mutants could be rescued by all avian H3N2 strains but not by any of the human H3N2 isolates. Only two of the swine H3N2 strains tested were able to rescue our mutants. The NP gene of these two swine isolates resembled the NP gene of the avian strains genetically in the hybridization test. However, their NPs reacted differently with a set of monoclonal antibodies when compared with NPs of avian H3N2 strains. Concerning multiplication in ducks they behaved like the other swine and human strains. The phosphopeptide fingerprints of all swine isolates tested were alike and were different from those of human or avian origin. Our observations are compatible with the idea that human H3N2 strains might not be able to cross the species barrier to birds directly, and possibly also not the other way around, without prior reassortment in pigs, which seem to have a broader host range o 198s Academic PMS, IIIC. concerning the compatibility of the NP gene in reassortants.

Thus on the premise that certain characteristics of the components of a given viContingent upon the hypothesis that rus may reflect its origin and therefore host pandemic influenza has its origin in an anspecificity, a range of viruses was studied imal reservoir (Webster and Laver, 1975; Hinshaw and Webster, 1982), is the need in a series of experiments designed to deto understand what viral genetic factors termine what factors might influence this determine host specificity, or facilitate or property. Particular emphasis was placed limit interspecies transmission. There is on examination of the nucleoprotein (NP) evidence that segment 4 [coding for the gene and the nucleoprotein itself as this hemagglutinin (HA)] may be important for protein has been implicated in earlier tissue tropism (Hinshaw et al, 1983), but studies on host range (Scholtissek et aZ., 1978a). The demonstration that the influno specific gene has been unambiguously associated with host specificity. Indeed, enza virus NP is a phosphoprotein (Priaccumulating evidence suggests that a valsky and Penhoet, 1977,1978,1981; Petri particular gene constellation of a virus de- and Dimmock, 1981; Almond and Felsenreich, 1982), whose phosphorylation pattermines host range and pathogenicity, those genes coding for the polymerase tern appears to determine to what extent a given cell culture may support virus complex seemingly playing an important growth (Kistner et al, 1985) emphasizes the role (Scholtissek et al, 1977, 1979; Rott et aL, 1979; Bonin and Scholtissek, 1983; Tian potential importance of the NP in this phenomenon. et al, 1985). Also the discovery that the prototype human H3N2 virus A/HK/1/68 cannot i Author to whom requests for reprints should be addressed. rescue mutants of fowl plague virus (A/ INTRODUCTION

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0042-6822185 $3.00 Copyright All rights

0 1985 by Academic Press, Inc. of reproduction in any form reserved.

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FPV/Rostock/l/34, H7Nl) with ts defects in the nucleoprotein gene following double infection of chick embryo fibroblast (CEF) cells at 40” (Scholtissek et aL, 1978a), provided an opportunity to relate biological behavior of the NP gene to the host of origin of the virus. Most of the viruses examined were isolated from avian, porcine, and human hosts in Hong Kong from the geographical region of Southern China, a likely influenza epicenter (Shortridge and Stuart-Harris, 1982). The study was based on H3N2 viruses of the region because of the emergence of the pandemic H3N2 virus through Hong Kong and because of the greater opportunity for interspecies transmission of influenza viruses generally as a consequence of the age-old agricultural techniques practiced in that region (Shortridge, 1983; Shortridge and Stuart-Harris, 1982). MATERIALS

AND

METHODS

Virus strains and cells. The virus strains listed in Tables 1 and 2 were investigated. We are grateful to Dr. A. P. Kendal for providing strains that were not in the collections of the Institutes of Giessen and Hong Kong. The Hong Kong isolates were obtained in the course of surveillance studies of domestic poultry and pigs originating from the geographical region of Southern China including Hong Kong and Taiwan between 1975 and 1982. The avian viruses comprised mainly first (original) or second level passage allantoic fluids or at the most the fourth level. Porcine and human viruses were first or second level fluids except for four isolates from pigs, namely A/swine/HK/3,4,5 and 6/76, which were subsequently passaged in eggs (Shortridge et aL, 1979). For rescue experiments, two mutants of A/FPV/Rostock/34 (FPV) with ts lesions in the NP gene (ts 19 and ts 81) were used (Scholtissek and Bowles, 1975; Scholtissek et ab, 1976). Primary chick embryo fibroblasts were prepared from llday-old chick embryos and used 48 hr after seeding. In a few experiments also MadinDarby canine kidney (MDCK) cells were investigated.

ET AL.

Determination of the phosphopeptidefingerprints of the NP. The procedure has been published recently (Kistner et al., 1985). Briefly, infected CEF monolayers were labeled with [32P]orthophosphate from 2 to 6 hr after infection. The cells were disrupted by RIPA-buffer, the NP was immunoprecipitated by monospecific anti-NP serum, and the precipitate was dissolved in lysis buffer before electrophoresis on polyacrylamide gels. The NP-band was excised and thoroughly digested with trypsin prior to separation of the peptides by two-dimensional thin-layer chromatography and electrophoresis.

Separation of viral proteins labeled in vivo with [S5SJrn,ethirmine by polyxrylamide gel electrophoresis (PAGE). Infected cultures (6 X lo6 cells) were labeled with 5 &i of [35S]methionine (800 Ci/mmol, Amersham, England) from 3 to 6 hr after infection. Thereafter, the cells were washed twice with PBS and disrupted with lysis buffer. Aliquots were applied to 20% polyacrylamide gels and electrophoresed (Bosch et aL, 1979).

Hybridization of nonlabeled cRNA of test strains to “P-labeled RNA segment 5 (NP gene) of the WSN strain. The hybridization technique has been described in detail (Scholtissek et al., 1976). Hybrid molecules were heated for 10 min in 1% formaldehyde at 80” prior to RNase digestion at 20” in order to increase the difference in RNase resistance between highly related strains (Scholtissek et ak, 1976). The A/WSN/33 (HlNl) virus was chosen as the reference since it can be sufficiently labeled by [32P]orthophosphate in vivo in contrast to other viruses as listed in the tables, which grow only to relatively low titers in CEF cells. RNA segment 5 of the WSN strain is genetically more closely related to human than to avian influenza A strains (unpublished results).

Virus quantitation and rescue of ts mutants. Plaque, hemagglutination (HA), and hemagglutination inhibition (HI) tests were performed according to established procedures (Klenk et al, 1972). Rescue experiments were performed by infecting CEF monolayers singly or doubly with the ts mutants and the influenza A

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strains listed in Table 2 at about 10 PFU per cell of each virus. After infection, the cells were incubated for about 15 hr at 33” before a plaque test was performed on the supernatant at the nonpermissive temperature of 40”. If the plaque titers were at least a loo-fold greater than those of occasionally occurring revertants, as determined on cultures infected singly with the ts mutants, rescue to ts+ reassortants was regarded as positive. Intermediate values were not observed (Scholtissek et ah, 1976). Antigenic analysis. Monoclonal antibodies to the NP of A/WSN/33 virus were used as described (van Wyke et ah, 1980). Briefly, infected allantoic fluid harvests were purified and concentrated, disrupted with lysis buffer, and submitted to enzymelinked immunosorbent assays in microwell plates using monoclonal antibodies conjugated with horseradish peroxidase. In order to increase the discrimination of the test, a positive reaction was considered to be an optical density (at 405 nm) threefold greater than that of the optical density of a P31/X63-Ag8 cell culture supernatant, while a negative reaction was an optical density less than three times that of the supernatant. Infection experiments. Two to four local Hong Kong ducks, approximately 3 weeks old, that had been hatched and raised in isolation were used for each virus. Preinfection tracheal and cloaca1 swabs were free of detectable virus and all birds were seronegative for all influenza hemagglutinin subtypes. The birds were infected orally (1 ml) and intranasally (0.5 ml) with freshly harvested infectious allantoic fluid with an HA titer of 1:64. Tracheal and cloacal swabs were taken for up to 21 days postinfection, inoculated into embryonated hen eggs and hemagglutinating isolates were identified with monospecific antisera. Sera collected at this time were submitted to HI test for antibody detection. RESULTS

PAGE of H3N2 Virus Proteins

In order to ascertain that many of the strains as listed in Table 2 were indeed

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H3N2

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separate viruses and not independent isolates of otherwise identical viruses, PAGE patterns of [35S]methionine-labeled cells infected with the various avian, porcine, and human virus strains were determined. On the basis of significant differences in migration rates of the NP, the nonstructural protein 1, and membrane protein the avian and porcine H3N2 strains could be placed into at least four different groups each. The human strains all exhibited an identical migration pattern. Thus, it is not excluded that some of the viruses under study are independent isolates of otherwise identical viruses. Hybridization with cRNA of H3N2 Strains to VRNA Segment 5 (NP Gene) of the WSN Strain

After hybridization of 32P-labeled vRNA segment 5 of the WSN strain with a surplus of nonlabeled cRNA of the strains listed in Table 1, the samples were heated for 10 min at 80” in 1 X SSC containing 1% formaldehyde in order to render the test as sensitive as possible (Scholtissek et al, 1976). As can be seen from that table, the human and swine Sw6 strains are genetically more TABLE

1

GENETIC RELATEDNESS OF H3N2 STRAINS TO RNA SEGMENT 5 (NP GENE) OF THE A/WSN/33 STRAIN

H3N2 isolate A/HK/1/68 A/HK/5/83 A/sw/HK/6/76 A/sw/HK/126/82 A/sw/HK/127/82 A/duck/HK/64/76 A/duck/HK/115/77 A/duck/HK/662/79 A/duck/HK/940/80 A/chicken/HK/8/76

% RNase resistance” after hybridization 31 28 28 17 17 18 16 18 16 17

LI% RNase resistance was measured by heating the RNA double strands at 80” in 1 X SSC containing 1% formaldehyde prior to RNase treatment (Scholtissek et al, 1976). The strains are abbreviated in the text as follows: Sw6 = ~/sw/HK/ty76. -

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ET AL.

closely related to the WSN strain than are the avian strains and the swine viruses Sw126 and Sw127. The genetic relatedness of segment 5 of Sw126 and Sw127 compared with that of the avian viruses suggests that the NP gene of these two swine isolates may have an avian derivation.

In a few experiments MDCK cells were investigated for rescue of ts 19 and ts 81. All swine isolates tested, which were unable to rescue the ts mutants on CEF, were able to do so on MDCK cells as was demonstrated formerly for A/HK/1/68 (Scholtissek et al., 1978a).

Rescue of FPV ts 19 and ts 81 Mutants by Double Infection of CEF Cells with H3N2 Viruses

Phosphopeptide Patterns of H3N2 Nucleo-

Scholtissek et al. (1978a) have shown that the NP of the human H3N2 virus, A/HK/ l/68, cannot rescue ts mutants of FPV with lesions in the NP gene following infection of CEF cells at 40”, while most of the other prototype human viruses are able to do so (Scholtissek et ab, 1976). This property was used to explore further viruses of the H3N2 subtype isolated from different species. Seventeen of the 19 nonavian viruses tested including the reference human virus, A/HK/1/68, failed to rescue either of the mutants (Table 2). In contrast, all 15 viruses of avian origin or source and the two porcine isolates, Sw126 and Sw127, did. Thus, there is a clear correlation between their ability to rescue the mutants and the genetic relatedness of their NP genes. TABLE

proteins

Since the phosphopeptide fingerprints of NPs were strain specific and have been correlated with growth of an influenza virus in a certain host cell (Kistner et ah, 1985) we have prepared such fingerprints of %P-labeled NPs of the H3N2 strains Sw3, Sw4, Sw127, D24, HKl, and HK5 propagated in CEF cells (data not shown). The fingerprints of HK5 exhibited 3 labeled phosphopeptides, which according to the nomenclature by Kistner et al. (1985) are numbered with 2, 3, and 4. All the other NPs including A/HK/1/68 had only two phosphopeptides (2 and 3). After mixing corresponding samples prior to separation phosphopeptides 3 always comigrated while there were small differences in migration of the phosphopeptides 2. The distances between phosphopeptide 2 and 3 of 2

RESCUE OF ts 19 AND ts 81 WITH H3N2 STRAINS ISOLATED FROM HUMANS, PIGS, OR BIRDS H3N2 isolate

Rescue

A/Hong Kong/l/68 A/Texas/l/77 A/Colorado/l/77 A/Wyoming/l/78 A/Lackenheath AFB/387/78 A/Hong Kong/5183 A/Hong Kong/14183 A/Hong Kong/26/83 A/sw/HK/3/76 A/sw/HK/4/76 A/sw/HK/5/76 A/sw/HK/6/76 A/sw/HK/22/77 A/sw/HK/72/77 A/sw/HK/81/78 A/sw/HK/82/78 A/sw/HK/126/82 A/sw/HK/127/82

+ +

H3N2 isolate A/duek/HK/7/75 A/duck/HK/24/75 A/duck/HK/64/76 A/duck/HK/115/77 A/duck/HK/245/77 A/duck/HK/315/78 A/duck/HK/326/78 A/duck/HK/335/78 A/duck/HK/662/79 A/duck/HK/940/80 A/goose/HK/10/76 A/chicken/HK/8/76 A/chieken/HK/39/78 A/duck pond water/HK/502E/79 A/duck feces/HK/975/81

Rescue + + + + + + + + + + + + + + +

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the human and duck NPs were slightly larger when compared with the porcine isolates Sw3, Sw4, and Sw127. Thus, the phosphopeptide fingerprints of the NPs correlated with the species from which the H3N2 viruses were isolated except that HK5 exhibited an additional phosphopeptide. Antigenic Variation of the H3N2 NP: Analysis with Monoclmal Antibodies to the NP of A/ WSN/33

Whereas all except one of the avian viruses tested reacted with all of the monoclones in the panel, the human and swine viruses, apart from two, did so only with three monoclones, namely 3/l, 5/l, 7/3 (Table 3). The two swine viruses that did not, namely Sw126, and Sw127, reacted with monoclone 15014 as did the avian viruses, but not with monoclone 7/3. Avian virus D64 exhibited an aberrant behavior in that it did not react with monoclone 7/3. Infection

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DISCUSSION

A series of characteristics of influenza H3N2 viruses isolated from man, pig, and poultry in the geographical region of Southern China and their NPs was studied in an attempt to relate them to the host of origin. These characteristics are summarized in Table 3. It can be seen that strains

As domestic ducks are the major reservoir of influenza viruses in the region of TABLE TO SHOW GROUPING

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Southern China (Shortridge, 1981, 1982), these poultry were infected with representative viruses from the three categories of host under study to gain insight into the role of the NP in host specificity. Isolations could not be made from the trachea or cloaca of ducks infected with any of the human or porcine viruses including Sw127 (Table 3). In contrast, avian viruses could be isolated for up to 18 days p.i. from both sites, there being no marked differences in the duration of shedding from trachea or cloaca. However, antibodies to both human and porcine viruses were detected in the serum of the ducks inoculated with these viruses although virus shedding from the trachea and cloaca could not be detected. It was also noted that the levels of these antibodies were generally higher than those detected in the birds infected with viruses of avian origin.

of Domestic Ducks

SUMMARY OF THE CHARACTERISTICS

H3N2

3

OF A RANGE OF HUMAN, PORCINE, AND AVIAN OF CHARACTERISTICS WITH HOST OF ORIGIN

H3N2

VIRUSES

Host of origin of virus Pig

Pig Man

Poultry

Characteristic investigated

All isolates

All except Sw126 and Swl27

All isolates

Sw126 and SW127

Hybridization to WSN segment 5: % RNase resistance

28-31%

28%

17%

17..18%

Ability to rescue FPV ts mutants

-

-

+

+

3/l, 5/l, 7/3

3/l, 5/l, l/3

3/l, 5/l, 7/3,150/4, 46914 (all)’

3/l, 5/l, 150/4

+

+

+ +

+

Binding to monoclonal antibodies to WSN NP: Monoclones bound Ability of virus to infect ducks Virus excretion Serum antibody

a Virus D&l NP was not bound by monoclone 7/3.

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fall into two broad groups, one comprising all human and most of the porcine isolates, the other the avian strains. However, two viruses isolated from pigs, Sw126 and Swl27, had characteristics exhibited by both of the groups. Since available evidence suggests that H3N2 viruses are transmitted from man to pig (Kundin, 1970; Shortridge et al, 1977) it is not too surprising that most of the porcine H3N2 strains should resemble the human H3N2 viruses. However, a question arises on the origin of the Sw126 and Sw127 isolates. Rescue of mutants of FPV with a ts lesion in the NP gene might provide an insight into the possible origin of the NP of a given H3N2 virus, and might allow an interpretation of what constitutes an “avian” or “nonavian” NP. However, human and avian influenza viruses comprising other subtypes need to be tested in this respect in order to see whether this observation can be generalized. According to our results on rescue (Table 2) and hybridization (Table 1) the Sw126 and Sw127 isolates contain an “avian-like” NP. If a reverse system of mutants of human H3 strains with a ts lesion in the NP gene were available to test whether the “avian-like” gene is compatible in reassortants for growth in human cells, such a system might help to resolve the enigma of the origin of the NP gene of Sw126 and Swl27 viruses. Bean (1984) has shown recently a certain species specificity of the NP by comparing RNA segment 5 of influenza virus strains isolated from different species by the hybridization technique. Furthermore, the NP and membrane (M) protein genes were shown to be associated with restriction of replication in the squirrel monkey respiratory tract using reassortants between the A/mallard/NY/78 (H2N2) and A/ Udorn/72 (H3N2) viruses (Tian et ak, 1985). These more recent findings augment the concept of a particular gene constellation influencing host specificity (see under Introduction), the NP apparently being the most significant factor. In this context it should be mentioned that ts mutants of FPV with lesions in RNA segments 1,2, 3, 6, or 8 can be rescued on CEF cells by the A/Hong Kong/l/68 (H3N2) and/or A/

ET AL.

Singapore/l/57 (H2N2) viruses (Scholtissek et ak, 1976; unpublished results). In infection experiments representative viruses from the two groups (including the Sw127 virus) were inoculated into ducks. Only the avian viruses were isolated from the trachea and cloaca indicating replication in the respiratory and intestinal tracts, respectively. The human and porcine viruses must have multiplied somewhere else in the duck to have produced serum antibody. In this respect, the Sw127 viruses behaved like human and porcine viruses. Although circumstances did not permit infection of pigs with these viruses to consolidate such views, the likelihood exists that host specificity and tissue tropism are integral parts of the same phenomenon, i.e., the survival of virus in nature. There was a certain correlation between phosphopeptide fingerprints of the NPs and species specificity as might have been expected from recent comparative studies (Kistner et al., 1985). In this respect Sw127 behaved like the other swine viruses. The fact that the surface glycoproteins can be exchanged quite easily during the antigenic shift (Scholtissek et al, 1978b), and that influenza viruses with almost identical surface glycoproteins (H3N2) can be isolated at the same time within the same area from different species might be interpreted to mean that the genes coding for these surface glycoproteins are able to cross the species barrier with certain ease. However, from our studies, the NP gene appears to be fairly species specific. It was interesting to detect aberrant viruses such as Sw126 and Sw127, which appeared to have characteristics of both groups. This implied that they may be hybrids-products of reassortment between porcine or human and avian viruses. The recognition that these two viruses have hemagglutinins closely related to the earliest human H3N2 strains of 1968 and neuraminidase to much later ones (Shortridge et ah, unpublished data) support this. Thus, both viruses, which according to PAGE patterns might be independent isolates of the same virus, could be viruses with an avian NP on the way to becoming adapted to the porcine

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host. All we can surmise is that pigs may be more tolerant to the replication of H3N2 viruses irrespective of the derivation of the NP gene, which would be compatible with the idea that pigs are the “mixing vessels” for the creation of new human pandemic strains by reassortment (antigenic shift). All in all, these findings using H3N2 viruses derived from different hosts of origin in a region, in which the opportunity for interspecies transmission is perhaps greatest (Shortridge and Stuart-Harris, 1982), provide an insight into factors that may be relevant to host specificity at both the virus and host levels. They underline the importance of the NP; however, we might assume that more than a single factor influences host specificity. ACKNOWLEDGMENTS The skillful technical assistance of Mrs. Karin Mtiller and Mr. S. Suen is gratefully acknowledged. Thanks are due to Dr. R. Webster for the provision of monoclonal antibodies. Financial support was provided in part by the Sonderforschungsbereich 47 of the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie to C.S. and K.F.S. and by National Institute of Health Grant AI 02649 to K.F.S. REFERENCES ALMOND, J. W., and FELSENREICH, V. (1982). Phosphorylation of the nucleoprotein of an avian influenza virus. J. Germ Viral. 60, 295-305. BEAN, W. J. (1984). Correlation of influenza A virus nucleoprotein genes with host species. Virology 133, 438-442. BONIN, J., and SCHOLTISSEK, C. (1983). Mouse neurotropic recombinants of influenza A viruses. Arch. ViroL 75.255-268. BOSCH, F. X., ORLICH, M., KLENK, H.-D., and ROTT, R. (1979). The structure of the hemagglutinin, a determinant for the pathogenicity of influenza viruses. Virology 95,197-207. HINSHAW, V. S., and WEBSTER, R. G. (1982). The natural history of influenza A viruses. In “Basic and Applied Influenza Research” (A. S. Beare, ed.), pp. 79-104. CRC Press, Boca Raton, Fla. HINSHAW, V. S., WEBSTER, R. G., NAEVE, C. W., and MURPHY, B. R. (1983). Altered tissue tropism of human-avian reassortant influenza viruses. Virology 128,260-263. KISTNER, O., MVLLER, H., BECHT, H., and SCHOLTISSEK, C. (1985). Phosphopeptide fingerprints of nucleoproteins of various influenza A virus strains grown in different host cells. J. Gen ViroL 66,465-472.

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SHORTRIDGE,K. F. (1983). Pandemic influenza: Ap- TIAN, S.-F., BUCKLER-WHITE,A. J., LONDON,W. J., plication of epidemiology and ecology in the region RECLE,L. J., CHANOCK,R. M., and MURPHY, B. R. of Southern China to prospective studies. In “Origin (1985). Nucleoprotein and membrane protein genes are associated with restriction of replication of inof Pandemic Influenza Virus” (W. G. Laver, ed.), pp. 191-200. Elsevier Science, New York. fluenza A/mallard/NY/78 virus and its reassortants in squirrel monkey respiratory tract. J. Viral. 53, SHORTRIDGE,K. F., CHERRY,A., and KENDAL, A. P. (1979). Further studies on the antigenic properties 771-775. of H3N2 strains of influenza A viruses isolated from WEBSTER,R. G., and LAVER, W. G. (1975). Antigenic variation of influenza viruses. In “The Influenza Viswine in Southeast Asia. J. Gen Viral 44,251-254. SHORTRIDGE,K. F., and STUART-HARRIS,C. H. (1982). ruses and Influenza” (E. D. Kilbourne, ed.), pp. 269An influenza epicentre? Lancet 2,812-813. 314. Academic Press, New York. SHORTRIDGE,K. F., WEBSTER,R. G., BUTTERFIELD, VAN WYKE, K. L., HINSHAW, V. S., BEAN, W. J., and W. K., and CAMPBELL,C. H. (1977). Persistence of WEBSTER,R. G. (1980). Antigenic variation of influHong Kong influenza virus variants in pigs. Science enza A virus nucleoprotein detected with mono(Washington, D. C.) 196,1454-1455. clonal antibodies. J. Viral. 35,24-30.