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83 (H5N2) influenza virus associated with acquisition of virulence
VIROLOGY
149,165-173
(1986)
Molecular Changes
in A/Chicken/Pennsylvania/83
Associated ROBERT
G. WEBSTER,l
with Acquisition
YOSHIHIRO
Received
September
of Virulence
KAWAOKA,
St. Jude Children’s Research Hospital, Department Research Hospital, 332 North Lauderdale,
(H5N2) Influenza Virus WILLIAM
AND
of Viyologl/ and Molecular P.O. Box 318, Memphis,
18, 1985; accepted
November
J. BEAN,
Biology, St. Jude Tennessee 38101
Children
JR. k
14, 1985
One of the unresolved questions concerning the acquisition of virulence by the A/Chicken/ Pennsylvania/83 (H5N2) influenza virus is which gene segments other than the hemagglutinin (HA) showed changes that were relevant. To answer this question, reassortants were made possessing the hemagglutinin gene of the virulent virus and the seven other genes from the avirulent parent. Since both the virulent and avirulent H5N2 strains are antigenically almost indistinguishable, it was necessary to transfer the genes of interest to a “carrier” virus before the appropriate reassortment could be selected. The gene compositions of the reassortants was established by a combination of sequence analysis and migration on polyacrylamide gels. These analyses established that the avirulent influenza virus present in April 1983 possessed seven of the eight gene segments necessary for virulence; mutation(s) in the HA gene were required for acquisition of virulence. Other viruses such as A/Seal/Mass/l/80 (H7N7) could provide the other genes necessary for virulence. Two changes in the HA have been associated with the acquisition of virulence; these are at amino acid residues 23 and 78 (H3 numbering) (Y. Kawaoka and R. G. Webster, Virology, 146, 130-137 (1985)). Isolation of an amantadine-resistant avirulent revertant virus provided the opportunity to determine which of the two amino acid changes in HA is critical. Sequence analysis of the revertant virus revealed amino acid changes at residues 23 in HA1 and 40 in HA2 (H3 numbering). The change at residue 23 of HA1 is probably associated with reversion to avirulence, of cleavability of HA, and inability to plaque in tissue culture without trypsin; while the change at residue 40 of HA2 may be associated with the amantadine-resistant phenotype. These studies establish that a single critical point mutation in the hemagglutinin gene of the avirulent A/Chicken/Pennsylvania/l/ 83 (H5N2) was probably all that was required to produce the highly virulent Chicken/ Pennsylvania virus; the avirulent virus already possessed the other genes necessary for VirUknCe.
0 1986 Academic
Press. Inc.
INTRODUCTION
The highly virulent H5N2 influenza A virus that occurred in chickens in Pennsylvania in October 1983 first appeared in an avirulent form in April 1983 (Bean et al., 1985). This provided a unique opportunity to study the molecular changes in a virus that occur during the acquisition of virulence. Studies to date on this virus have concentrated on the molecular changes in the hemagglutinin (HA) and neuraminidase (NA) genes (Kawaoka et ah, 1984; ’ Author addressed.
to whom
requests
for reprints
should
be
165
Deshpande et al., 1985). The acquisition of virulence by A/Chicken/Pennsylvania/ 13’70183 (H5N2) [Ck/1370] has been associated with ability to plaque in tissue cultures and cleavage of HA into HA1 and HA2 in the absence of trypsin and reduction in molecular weight of HA1 commensurate with loss of a carbohydrate side chain (Kawaoka et al., 1984). Sequence analysis of the hemagglutinin genes of the avirulent and virulent viruses demonstrated four amino acid differences between the two viruses. Subsequent analysis of the HA genes from several virulent and avirulent H5N2 viruses showed that there were two amino acid differences be0042-6822/86 Copyright All rights
$3.00
0 1986 by Academic Press, Inc. of reproduction in any form reserved.
166
WEBSTER,
KAWAOKA,
tween the virulent and avirulent H5N2 viruses, at residues 23 and 78 (H3 numbering, Wilson et ah, 1981) in the hemagglutinin molecule (Kawaoka and Webster, 1985). One of these sequence changes (residue 23) is in the middle of the stalk and results in the loss in the virulent virus of a potential carbohydrate side chain in the vicinity of the cleavage site between HA1 and HA2, while the change at 78 is in an antigenic region (site E). The studies to date have not resolved which of the two sequence changes is critical for acquisition of virulence. Comparison of the deduced amino acid sequences of the neuraminidase genes from the virulent and avirulent strains show four amino acid changes; one in the transmembrane segment, one in the stalk, and two in the head (Deshpande et al., 1985). The NA of both the virulent and avirulent H5N2 viruses possessed a short (stubby) stalk with a 20-amino acid deletion as compared with other influenza viruses containing N2. The aim of the present study is to determine which gene segments of the A/ Chicken/Pennsylvania/l/83 (H5N2)[Ck/l] in addition to the HA are required for virulence and which of the amino acid substitutions in HA is essential for virulence. We will establish that the avirulent H5N2 influenza virus present in April 1983 possessed all of the gene segments necessary for virulence, and that a single point mutation in the hemagglutinin gene was probably all that was required for the acquisition of virulence. The present studies show that the neuraminidase from either the virulent or avirulent parent in association with the HA from the virulent virus is fully virulent. There is no evidence that the changes in NA were associated with acquisition of virulence. MATERIALS
AND
METHODS
Viruses and viral RNA. The avirulent Ck/l (H5N2) virus isolated from the index case in April 1983 (Kawaoka et al,, 1984) and the virulent Ck/13’70 virus isolated in October 1983, were used in these studies. The other influenza viruses used were from the repository at St. Jude Children’s Re-
AND
BEAN
search Hospital and are given in the tables. Viruses were grown in U-day-old embryonated chicken eggs and were purified by differential sedimentation through 25-70% sucrose gradient in a Beckman SW 28 rotor. Virion RNA was isolated by treatment of purified virus with proteinase K and sodium dodecyl sulfate, followed by extraction with phenol:chloroform (1:l) as described previously (Bean et aZ., 1980). The viruses used in this study were handled in a P3 containment laboratory that was approved for such use by the United States Department of Agriculture. Specific antibodies. Antisera specific for the isolated HA and NA antigens of the reference strains of influenza A viruses were prepared in goats (Webster et al., 1974). Monoclonal antibodies to the HA and NA of many of the viruses used in this study were prepared by the method of Kohler and Milstein (1976) as described by Webster et aZ. (1982). Serological tests. HA titrations and hemagglutination inhibition tests were performed in microtiter plates with receptordestroying enzyme-treated sera (Palmer et al., 1975). NA titrations and NA inhibition tests were done by the procedure of Aymard-Henry et al. (1973). Reaasortant viruses. Reassortant influenza viruses were prepared in chicken embryos as described (Webster, 1970). When necessary the virulent Ck/1370 (H5N2) virus was partially inactivated with uv light (>99% reduction in infectivity). This minimized the contribution of genes from the uv inactivated parent. Reassortants were selected in embryonated eggs using either monospecific antisera or monoclonal antibodies specific for the HA or NA of the virus.
Isolation of amuntadine-resistant avirulent revertant viruses. Two adult white leghorn chickens were infected by dropping 0.1 ml of virus containing lo4 EID5,, of virulent Chick/Penn virus into the nasal cleft. Two days after infection, the birds were put in contact with two uninfected birds and all were given 0.01% amantadine in their drinking water. Tracheal and cloaca1 swabs were taken daily, inoculated into embryonated eggs, and the virus yields
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VIRUS
167
were tested for amantadine sensitivity. TGTCAATCC (3’) for PBl, and AAThis was assayed by plaque reduction in AGCAGGTCAA (3’) for PB2]. Viral RNA chick embryo fibroblast monolayers with was resolved on a 3% polyacrylamide gel the drug incorporated into the overlay mecontaining 7 M urea and 0.25% bis-acrydium at concentrations of 0.01 to 10 pg/ml lylcystamine in 0.1 X Loening’s buffer as in 0.5 log,, increments. Typically, there was described (Bean et ah, 1980). Electrophoresis was for 16 hr at 42” with a constant a 50% reduction in plaques at O.Ol-0.03% voltage of 180 V. RNA was detected with amantadine for sensitive isolates while resistant isolates required at least loo-fold the Bio-Rad Silver Stain. higher concentrations for a 50% reduction. RESULTS The amantadine resistance was also assayed in viva as described (Webster et al., Does the Avirulent H5N2 Virus Possessthe 1985). Genes Necessary for Virulence? Animals-Definitions of pathogenicity One of the central questions concerning and mean death time. Adult white leghorn chickens (>30 weeks) were used in these the acquisition of virulence by the H5N2 studies. A highly pathogenic virus is de- influenza virus is which gene segments fined as one that results in not less than other than the hemagglutinin contained 50% mortality within 8 days in at least changes that were relevant. To answer this eight healthy adult chickens inoculated question, reassortants were made which orally with bacteria-free infectious allanpossessed the hemagglutinin gene of the toic fluid. virulent virus and the seven other genes Sick chickens were defined as birds that from the avirulent parent. Since the hemagglutinin and neuraminidase of the virwere unable to stand, and that twisted ulent and avirulent viruses are antigenitheir necks indicating a central nervous system disorder. Chick embryo mean death tally almost identical, it was necessary to time is the time required to kill 50% of lotransfer the hemagglutinin and neurday-old embryos incubated at 35”. aminidase genesto “carrier” viruses (Fig. I). Nucleic acid sequencing. Nine oligonuThis was achieved by preparing reassorcleotide primers complementary to the vi- tants between uv-treated virulent Ck/1370 (H5N2) and A/Seal/Mass/l/80 (H’7N7). rion HA gene segment of Ck/l (H5N2) (Kawaoka et aZ., 1984) were synthesized on an The mixed virus yield from this infection Applied Biosystems model 380A DNA syn- was selected with monospecific antisera to thesizer by the solid-phase phosphoramiH7 and N2 to give the reassortant containdite method. Nucleic acid sequences were ing Ck/1370 HA and Seal NA (H5N7). determined by reverse transcription of viSimilarly, the avirulent Ck/l (H5N2) virus rion RNA in the presence of primers and was reassorted with Mem/Bel (H3Nl) to dideoxynucleotides as described (Air, 1979; produce the H3N2 reassortant. These two reassortant viruses (H5N7 and H3N2) were Naeve et al., 1984). The reaction products were resolved in 8% polyacrylamide-7 M used as parents to produce the reassortant urea thin gels in TBE buffer (90 mM Trispossessing the HA of the virulent virus borate, pH 8.0; 1 mM EDTA) as described (Ck/1370) and the NA of the avirulent virus (Maxam and Gilbert, 1977). (Ck/l) (H5N2). This virus was referred to Genotyping of reassortant viruses. The as reassortant 10 (RlO). The genotypes of the reassortant viruses genotype of the reassortant viruses was determined by a combination of sequence were determined by a combination of sequence analysis and electrophoresis on analysis and electrophoresis on 3% polyacrylamide gels. polyacrylamide gels. To discriminate beThe partial sequences of PA, PBl, and tween the P genes of the virulent and avirulent H5N2 virus, it was necessary to do PB2 genes were obtained by the dideoxpartial sequencing of these genes (Fig. 2). ynucleotide chain termination method In this way, each gene could be discrimidescribedabovewith the primers[GCAGGnated unambiguously. The gene composiTACTGAT (3’) for PA, ATTTGAATGGA-
168
WEBSTER, U.V.
AND
BEAN
treated
Chick/Penn/1370/83
(HSNZ)
Seal/Mass/l/80
Ck/Penn/l370
KAWAOKA,
[Virl
Chick/Penn/l/83
Mem/1/71-Be1142
(H7N7)
cH)
seal
(H5N2.j
Mem (8)
cNj
X
(H5N7)
Ck/Penn/l370
(8)
Ck/Penn/l
[Avir]
(H3Nl)
Ck/Penn/l/83
(N)
(H3N2)
(N) = RIO
(H5N2) FIG.
1. Reassortment
FIG. 2. Sequence analysis for virus reassortants. Genotyping sequencing the PBl genes using Only A reactions were analyzed sortant viruses. Mem:Mem/l/‘71 Mass/3740/65 (H6N2).
between
virulent
and avirulent
genotyping influenza of the reassortants by the dideoxy method. on this gel. 3-ll:Reas(H3N2). Ty/Mass:Ty/
Chick/Penn/83
influenza
viruses.
tion of the reassortant viruses is shown in Table 1 and an example of polyacrylamide analysis is shown in Fig. 3. The reassortant virus possessing the virulent HA and the avirulent NA (RlO) obtained five of its gene segments from the avirulent Ck/l (Table 1); the M and NS genes were derived from the Seal influenza virus and the HA from the virulent parent. This virus was as virulent as the parental virulent strain; it killed chick embryos within 2 days and caused 100% mortality in experimentally infected chickens. It could be argued that the M and NS genes from the A/Seal/Mass/l/80 influenza virus may have contributed to virulence. To rule out this possibility, a reassortant was prepared between uv-treated RlO and R5 (Table 1) and between non-uvtreated RlO and R5. R5 possesses the hemagglutinin gene from A/Mem/l/71 (H3N2) and all of the other seven genes from the avirulent virus. The reassortants produced (R12 and R13) contained the HA gene from the virulent parent and all other genes from the avirulent parent. The reassortants (R12 and R13) were as virulent as the parental strain in chick embryos and adult chickens. Isolation of two different reassortants with the same phenotype reduces the likelihood of phenotypic variation. These studies indicate that the avirulent
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OF
INFLUENZA
TABLE GENESEGMENTSOFCHICK/PENN/~~ Gene Reassortant R10 R5 Rll R12 R13
Note. Genes = Seal/Mass/l/80
PA1
PB2
INFLUENZAVIRUSASSOCIATEDWITHVIRULENCE assignment
HA
NP
NA
M
NS
A
VAA Mem
A
A
Seal A
Seal A
A = Avirulent (H7N7); Mem
169
1
PBl
AAA A A AAVAAVVV AAAVAAAA AAAVAAAA from
VIRUS
Chick/Penn/l/83 (H5N2); = Mem/l/?l (H3N2).
H5N2 influenza virus that was circulating in chickens in April 1983 contained all of the gene segments necessary for virulence except the hemagglutinin. The reciprocal antigenic hybrid (Rll-Table 1) possessing
V = Virulent
Chicken killing
embryo (days) 2 >5 3.3 2 1.8
Chick/Penn/1370/83
Adult
chicken killing ah3 O/8 O/8 8/8
8/8 (H5N2);
Seal
the HA from the avirulent virus and NA, PBl, M, and NS genes from the virulent virus was avirulent in chickens and chick embryos. This emphasizes the importance of the HA gene in virulence, and indicates that the other genes involved in virulence were already present in the avirulent virus that appeared in April 1983. Do Other InJEuenxa Viruses Possessa Gene ConsteElation that Will Support Vimlence?
PI-P2 P3 HA NP NA
NS FIG. 3. Genotyping of the influenza ants on polyacrylamide gels. The viral trophoresed as given under Materials and detected by silver staining.
virus reassortRNA was elecand Methods
To determine if other influenza viruses contain gene constellations that would be virulent if they contained the virulent H5 gene, a number of human and mammalian influenza viruses were used to prepare reassortants. These are given in Table 2. The reassortant (R4) possessing six gene segments from A/Seal/Mass/l/80 (H’7N7) and the PBl and HA genes from the virulent H5N2 virus is virulent. This virus killed chick embryos within 1.8 days and caused central nervous system signs of disease in seven of eight chickens inoculated and mortality in half of the chickens. The reassortant (R9) that contains the PBl and HA gene from the virulent H5 virus and a mixture of other genes (Table 2) was also virulent in chick embryos and in chickens. Reassortants lacking the HA gene from the virulent Ck/1370 virus (Rl, Table 2; or Rll, Table 1) are avirulent in chickens and in chick embryos even though they possess all or most of the other genes required for virulence. These studies show that other viruses
WEBSTER,
170
KAWAOKA, TABLE
GENE ASSIGNMENT
AND VIRULENCE Gene
Reassortant Rl R4 R9
Mem
BEAN
2
OF REASSORTANT
INFLUENZA
assignment
PA
PB2
PBl
HA
NP
NA
M
NS
Ty* Seal Seal
Ty Seal Seal
V V V
Ty V V
V Seal Mem
v Seal Be1
v Seal V
v Seal V
Note. V = Chick/Penn/1370/83 (H7N7);
AND
= Memphis/l/71
(H5N2) (Vir); *Ty = Turkey/Mass/3740/65 (H3N2); Be1 = Belamy/ (HlNl).
VIRUSES
Chick embryo killing (days) >5 1.8 1.5 (H6N2);
Adult
chicken
Sick
Killed
O/8 7/s 6/8
O/8 418 418
Seal = Seal/Mass/l/80
otides described previously (Kawaoka et al., 1984). Sequence analysis of the amantadine-resistant Ck/1370 virus revealed that there was an amino acid change at residue 23 of HA1 from Lys in the virulent Ck/ 1370 virus to Thr in the revertant. The HA1 polypeptide from the revertant virus showed a reduction in mobility as compared with the HA1 from the parent Ck/ Penn/1370 virus (data not shown), sugWhich Mutation in the Hemagglutinin Gene gesting that the revertants regained a carbohydrate side chain. Additionally, there of H5N2 is Required for Virulence? was an amino acid sequence change at resThe virulent H5N2 virus differs from the idue 40 of HA2 from Lys to Arg. In the avirulent strain in producing plaques in three-dimensional structure of H3 influtissue culture, and the HA0 precursor is enza HA (Wilson et al, 1981), these changes cleaved into HA1 and HA2 in the absence are located in the vicinity of the cleavage of trypsin in tissue culture (Kawaoka et al., site between HA1 and HA2 (Fig. 4). 1984). There are two differences in amino The amino acid at residue 23 of the HA1 acid sequence between the HA of the virof the avirulent Ck/l influenza viruses is ulent and the avirulent viruses; one at res- the same as that in the revertant (Kawaoka idue 23 and the other at residue 78 (Ka- et al, 1984), yet the avirulent Ck/l viruses waoka and Webster, 1985). are sensitive to amantadine (data not One approach to resolving which of these shown). The amino acid change at residue two amino acid changes is critical for vir40 in HA2 may be involved in resistance of ulence is to obtain avirulent revertant vi- the revertants to amantadine. These studruses and to sequence the hemagglutinin ies indicate that the HA in the revertant gene. During the selection of amantadineviruses lost the ability to be cleaved withresistant Ck/1370 (H5N2) viruses in viva, out exogenous trypsin and that the virus such revertant viruses were isolated. The was avirulent; this was correlated with an viruses grew in chick embyros, but failed amino acid sequence change at residue 23 to plaque in tissue culture without trypsin of HAl. and HA0 was not cleaved (data not shown). These revertant viruses were avirulent in DISCUSSION adult chickens. The avirulent H5N2 influenza virus A revertant virus selected in this way was grown in chick embryos and the HA present in chickens in Pennsylvania in gene was sequenced by the dideoxy chain April 1983 possessedall of the gene segtermination method (Sanger et al., 1977; ments necessary for virulence other than the hemagglutinin. This was established by Air, 1979) using nine synthetic oligonucle-
such as A/Seal/Mass/l/80 (H7N7) are present in nature that contain a gene constellation which, in combination with the virulent hemagglutinin, has the capacity to produce a virulent virus. The reassortant possessing the Be1/40 (HlNl) neuraminidase is virulent in chickens suggesting that there is not an association between the N2 neuraminidase and virulence.
VIRULENCE
terminus C-terminus
OF
INFLUENZA
of HA2
of HAI
FIG. 4. The carbon tracing showing the structure A/Aichi/2/68 (H3) influenza virus hemagglutinin (Wiley et CLL, 1981). The numbers show the position the amino acid changes in the amantadine-resistant Ck/Penn/83 (H5N2) influenza virus.
of of
preparing reassortant viruses that contained the hemagglutinin gene from the virulent parent and the seven gene segments from the avirulent H5N2 parent. The gene composition of the reassortants was established by a combination of partial sequencing of the P genes and analysis of RNA migration in acrylamide gels. Studies with other influenza viruses from mammalian and avian sources showed that the A/Seal/Mass/l/80 (H7N7) influenza virus contained the necessary gene composition that, in combination with the H5 gene, was virulent in chickens. However, reassortant viruses possessing the HA from the virulent H5N2 virus and the other genes from different donor viruses [A/Turkey/Va/28215/83 (HlON8), A/ Guinea Fowl/De1/4358’7/83 (H3N8), A/ Ruddy Turnstone/l/85 (H7N3)] were avir-
VIRUS
171
ulent in chickens or chick embryos (results not shown). At this time, we do not know the minimal gene constellation required for virulence of the Ck/1370 (H5N2) virus. The studies of Rott et al. (1976,1979) with FPV and other donor viruses have shown that the minimal gene constellation required for virulence varies between each influenza virus pair studied. A practical consequence of the present studies is that there are potentially dangerous influenza viruses circulating in nature that only require a single point mutation in the HA gene to become highly virulent for chickens. Although many avirulent H5 viruses have been isolated from wild and domestic species, it is clear that this subtype is potentially more dangerous than other influenza virus subtypes. The other potentially dangerous subtype is H7, which includes FPV and other highly virulent viruses that are pathogenic for avian species and some mammalian species such as seals (Webster et aZ., 1981). Based on these studies, it appears that influenza viruses possessing H5 or H7 genes are potentially hazardous to domestic poultry and other species as well. It is therefore suggested that whenever H5 and H7 viruses are detected, they should be eradicated from domestic poultry as rapidly as possible. Which of the two amino acid changes at residues 23 or 78 of HA1 in the avirulent Ck/l (H5N2) reported by Kawaoka et al. (1985) are responsible for virulence? The availability of an amantadine-resistant H5N2 virus that requires trypsin for cleavage of HA0 into HA1 and HA2 and is avirulent in chickens provides the opportunity to determine which of the mutations is critical. Sequence analysis of a revertant virus showed that the amino acid at residue 23 changes from Lys to Thr, and SDSpolyacrylamide gel analysis suggests that the revertant HA1 is larger and possesses a carbohydrate side chain at this residue. Sequencing of the protein is in progress to establish the location and type of carbohydrate side chains on Ck/1370 HA. Isolation of an avirulent revertant Ck/ 1370 virus during selection of amantadineresistant strains may simply be fortuitous. Many of the amantadine-resistant isolates
172
WEBSTER,
KAWAOKA,
of the virulent Ck/1370 virus were virulent in chickens and chick embryos. The molecular changes and the genes involved in amantadine resistance of Ck/13’70 are under investigation. The amino acid at residue 23 in HA1 of the avirulent Ck/l influenza viruses is the same as that of the revertant, yet the avirulent Ck/l viruses are sensitive to amantadine (W. J. Bean, unpublished data). The amino acid change at residue 40 in HA2 may be involved in resistance of the revertants to amantadine. Studies by Daniels et al. (1985) have shown that amantadine-resistant mutants of FPV and X-31 have mutations in the hemagglutinin gene that affect HAl, HA2, or both. Amantadine resistance has also been associated with the matrix protein (Lubeck et al., 1978) and more recently, with the M2 protein (Dr. Allan Hay, personal communication). Studies are in progress on the other genes involved in amantadine resistance of Ck/ Penn/83 influenza viruses. These studies establish that a single critical point mutation in the hemagglutinin gene of the avirulent A/Chicken/ Pennsylvania/l/83 (H5N2) was required to produce the highly virulent Chicken/ Pennsylvania virus; the avirulent virus already possessed the other genes necessary for virulence. Our previous studies (Bean et al., 1985) also indicate that low molecular weight RNAs typical of defective interfering particles (DI) were found in the avirulent viruses isolated in April 1983, but were absent from the virulent virus. The mechanism by which the virulent virus lost the subgenomic RNAs present in the earlier virus is not known. Studies are in progress to further characterize the subgenomic RNAs and to determine their role in modulating virulence. ACKNOWLEDGMENTS This work was supported by U. S. Public Health Research Grant AI 08831, AI 52586 from the National Institute of Allergy and Infectious Disease, Cancer Center Support (CORE) Grant CA 21765, and American Lebanese Syrian Associated Charities. The authors thank Dr. Clayton W. Naeve for preparation of the oligonucleotide primers. Lisa Newberry, John Kayoma, and Hosea Clariette provided excellent
AND BEAN
technical assistance, and we thank Lisa Wilson for typing the manuscript. REFERENCES AIR, G. M. (1979). Nucleotide sequence coding for the “signal peptide” and N terminus of the hemagglutinin from an Asian (H2N2) strain of influenzavirus. Virology 97,468-472. AYMARD-HENRY, M., COLEMAN, M. T., DOWDLE, W. R., LAVER, W. G., SCHILD, G. C., and WEBSTER, R. G. (1973). Influenza virus neuraminidase-inhibition test procedures. Bull. WH.0. 48,199-202. BEAN, W. J., KAWAOKA, Y., WOOD, J. M., PEARSON, J. E., and WEBSTER, R. G. (1985). Characterization of virulent and avirulent A/Chicken/Pennsylvania/ 83 influenza A viruses: Potential role of defective interfering RNAs in nature. J. viral. 54(l), 151-160. BEAN, W. J., SRIRAM, G., and WEBSTER, R. G. (1980). Electrophoretic analysis of iodine-labeled influenza virus RNA segments. And. B&hem. 102,228232. DANIELS, R. S., DOWNIE, J. C., HAY, A. J., KNOSSOW, M., SKEHEL, J. J., WANG, M. L., and WILEY, D. C. (1985). Fusion mutants of the influenza virus hemagglutinin glyeoprotein. Cell 40,431-439. DESHPANDE, K. L., NAEVE, C. L., and WEBSTER, R. G. (1985). The neuraminidases of the virulent and avirulent A/Chicken/Pennsylvania/83 (H5N2) influenza A viruses: Sequence and antigenic analysis. Virology, 14’7, 49-60. KAWAOKA, Y., NAEVE, C. W., and WEBSTER, R. G. (1984). Is virulence of H5N2 influenza viruses in chickens associated with loss of carbohydrate from the hemagglutinin? Virology 139,303316. KAWAOKA, Y., and WEBSTER, R. G. (1985). Evolution of the A/Chicken/Pennsylvania/83 (H5N2) influenza virus. virology 146, 130-137. KOHLER, G., and MILSTEIN, C. (1976). Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion. Eur. J. Immund 6, 511519.
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OF INFLUENZA
niques for influenza diagnosis. In “Immunology Series No. 6,” pp. 51-52. U. S. Department of Health, Education, and Welfare. ROTT, R., ORLICH, M., and SCHOLTISSEK, C. (1976). Attenuation of pathogenicity of fowl plague virus by recombination with other influenza A viruses nonpathogenic for fowl: Nonexclusive dependence of pathogenicity on hemagglutinin and neuraminidase of the virus. J. Viral. 19,54-60. ROTT, R., ORLICH, M., and SCHOLTISSEK, C. (1979). Correlation of pathogenicity and gene constellation of influenza A viruses. III. Non-pathogenic recombinants derived from highly pathogenic parent strains. J. Gen. Viral. 44,471-477. SANGER, F. S., NICKLEN, S., and COULSON,A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad Sci. USA 74,5463-5467. WEBSTER, R. G. (1970). Antigenic hybrids of influenza A viruses with surface antigens to order. IJiroZogy 42,633-642.
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WEBSTER, R. G., HINSHAW, V. S., BEAN, W. J., VAN WYKE, K. L., GERACI, J. R., ST. AUBIN, D. J., and PETURSSON,G. (1981). Characterization of an influenza A virus from seals. firology 113,712-724. WEBSTER, R. G., HINSHAW, V. S., and LAVER, W. G. (1982). Selection and analysis of antigenic variants of the neuraminidase of N2 influenza virus with monoclonal antibody. IJirology 117,93-104. WEBSTER, R. G., ISACHENKO, V. A., and CARTER, M. (1974). A new avian influenza virus from feral birds in the USSR: Recombination in nature? Bull WH.0. 51,324-332. WEBSTER, R. G., KAWAOKA, Y., BEAN, W. J., BEARD, C. W., and BRUGH, M. (1985). Chemotherapy and vaccination: A possible strategy for the control of highly virulent influenza virus. J. viral. 55,173-176. WILSON, I. A., SKEHEL, J. J., and WILEY, D. C. (1981). Structure of the hemagglutinin membrane glycoprotein of influenza virus’at 3A resolution. Nature (London)
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(1986)
Molecular Changes
in A/Chicken/Pennsylvania/83
Associated ROBERT
G. WEBSTER,l
with Acquisition
YOSHIHIRO
Received
September
of Virulence
KAWAOKA,
St. Jude Children’s Research Hospital, Department Research Hospital, 332 North Lauderdale,
(H5N2) Influenza Virus WILLIAM
AND
of Viyologl/ and Molecular P.O. Box 318, Memphis,
18, 1985; accepted
November
J. BEAN,
Biology, St. Jude Tennessee 38101
Children
JR. k
14, 1985
One of the unresolved questions concerning the acquisition of virulence by the A/Chicken/ Pennsylvania/83 (H5N2) influenza virus is which gene segments other than the hemagglutinin (HA) showed changes that were relevant. To answer this question, reassortants were made possessing the hemagglutinin gene of the virulent virus and the seven other genes from the avirulent parent. Since both the virulent and avirulent H5N2 strains are antigenically almost indistinguishable, it was necessary to transfer the genes of interest to a “carrier” virus before the appropriate reassortment could be selected. The gene compositions of the reassortants was established by a combination of sequence analysis and migration on polyacrylamide gels. These analyses established that the avirulent influenza virus present in April 1983 possessed seven of the eight gene segments necessary for virulence; mutation(s) in the HA gene were required for acquisition of virulence. Other viruses such as A/Seal/Mass/l/80 (H7N7) could provide the other genes necessary for virulence. Two changes in the HA have been associated with the acquisition of virulence; these are at amino acid residues 23 and 78 (H3 numbering) (Y. Kawaoka and R. G. Webster, Virology, 146, 130-137 (1985)). Isolation of an amantadine-resistant avirulent revertant virus provided the opportunity to determine which of the two amino acid changes in HA is critical. Sequence analysis of the revertant virus revealed amino acid changes at residues 23 in HA1 and 40 in HA2 (H3 numbering). The change at residue 23 of HA1 is probably associated with reversion to avirulence, of cleavability of HA, and inability to plaque in tissue culture without trypsin; while the change at residue 40 of HA2 may be associated with the amantadine-resistant phenotype. These studies establish that a single critical point mutation in the hemagglutinin gene of the avirulent A/Chicken/Pennsylvania/l/ 83 (H5N2) was probably all that was required to produce the highly virulent Chicken/ Pennsylvania virus; the avirulent virus already possessed the other genes necessary for VirUknCe.
0 1986 Academic
Press. Inc.
INTRODUCTION
The highly virulent H5N2 influenza A virus that occurred in chickens in Pennsylvania in October 1983 first appeared in an avirulent form in April 1983 (Bean et al., 1985). This provided a unique opportunity to study the molecular changes in a virus that occur during the acquisition of virulence. Studies to date on this virus have concentrated on the molecular changes in the hemagglutinin (HA) and neuraminidase (NA) genes (Kawaoka et ah, 1984; ’ Author addressed.
to whom
requests
for reprints
should
be
165
Deshpande et al., 1985). The acquisition of virulence by A/Chicken/Pennsylvania/ 13’70183 (H5N2) [Ck/1370] has been associated with ability to plaque in tissue cultures and cleavage of HA into HA1 and HA2 in the absence of trypsin and reduction in molecular weight of HA1 commensurate with loss of a carbohydrate side chain (Kawaoka et al., 1984). Sequence analysis of the hemagglutinin genes of the avirulent and virulent viruses demonstrated four amino acid differences between the two viruses. Subsequent analysis of the HA genes from several virulent and avirulent H5N2 viruses showed that there were two amino acid differences be0042-6822/86 Copyright All rights
$3.00
0 1986 by Academic Press, Inc. of reproduction in any form reserved.
166
WEBSTER,
KAWAOKA,
tween the virulent and avirulent H5N2 viruses, at residues 23 and 78 (H3 numbering, Wilson et ah, 1981) in the hemagglutinin molecule (Kawaoka and Webster, 1985). One of these sequence changes (residue 23) is in the middle of the stalk and results in the loss in the virulent virus of a potential carbohydrate side chain in the vicinity of the cleavage site between HA1 and HA2, while the change at 78 is in an antigenic region (site E). The studies to date have not resolved which of the two sequence changes is critical for acquisition of virulence. Comparison of the deduced amino acid sequences of the neuraminidase genes from the virulent and avirulent strains show four amino acid changes; one in the transmembrane segment, one in the stalk, and two in the head (Deshpande et al., 1985). The NA of both the virulent and avirulent H5N2 viruses possessed a short (stubby) stalk with a 20-amino acid deletion as compared with other influenza viruses containing N2. The aim of the present study is to determine which gene segments of the A/ Chicken/Pennsylvania/l/83 (H5N2)[Ck/l] in addition to the HA are required for virulence and which of the amino acid substitutions in HA is essential for virulence. We will establish that the avirulent H5N2 influenza virus present in April 1983 possessed all of the gene segments necessary for virulence, and that a single point mutation in the hemagglutinin gene was probably all that was required for the acquisition of virulence. The present studies show that the neuraminidase from either the virulent or avirulent parent in association with the HA from the virulent virus is fully virulent. There is no evidence that the changes in NA were associated with acquisition of virulence. MATERIALS
AND
METHODS
Viruses and viral RNA. The avirulent Ck/l (H5N2) virus isolated from the index case in April 1983 (Kawaoka et al,, 1984) and the virulent Ck/13’70 virus isolated in October 1983, were used in these studies. The other influenza viruses used were from the repository at St. Jude Children’s Re-
AND
BEAN
search Hospital and are given in the tables. Viruses were grown in U-day-old embryonated chicken eggs and were purified by differential sedimentation through 25-70% sucrose gradient in a Beckman SW 28 rotor. Virion RNA was isolated by treatment of purified virus with proteinase K and sodium dodecyl sulfate, followed by extraction with phenol:chloroform (1:l) as described previously (Bean et aZ., 1980). The viruses used in this study were handled in a P3 containment laboratory that was approved for such use by the United States Department of Agriculture. Specific antibodies. Antisera specific for the isolated HA and NA antigens of the reference strains of influenza A viruses were prepared in goats (Webster et al., 1974). Monoclonal antibodies to the HA and NA of many of the viruses used in this study were prepared by the method of Kohler and Milstein (1976) as described by Webster et aZ. (1982). Serological tests. HA titrations and hemagglutination inhibition tests were performed in microtiter plates with receptordestroying enzyme-treated sera (Palmer et al., 1975). NA titrations and NA inhibition tests were done by the procedure of Aymard-Henry et al. (1973). Reaasortant viruses. Reassortant influenza viruses were prepared in chicken embryos as described (Webster, 1970). When necessary the virulent Ck/1370 (H5N2) virus was partially inactivated with uv light (>99% reduction in infectivity). This minimized the contribution of genes from the uv inactivated parent. Reassortants were selected in embryonated eggs using either monospecific antisera or monoclonal antibodies specific for the HA or NA of the virus.
Isolation of amuntadine-resistant avirulent revertant viruses. Two adult white leghorn chickens were infected by dropping 0.1 ml of virus containing lo4 EID5,, of virulent Chick/Penn virus into the nasal cleft. Two days after infection, the birds were put in contact with two uninfected birds and all were given 0.01% amantadine in their drinking water. Tracheal and cloaca1 swabs were taken daily, inoculated into embryonated eggs, and the virus yields
VIRULENCE
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INFLUENZA
VIRUS
167
were tested for amantadine sensitivity. TGTCAATCC (3’) for PBl, and AAThis was assayed by plaque reduction in AGCAGGTCAA (3’) for PB2]. Viral RNA chick embryo fibroblast monolayers with was resolved on a 3% polyacrylamide gel the drug incorporated into the overlay mecontaining 7 M urea and 0.25% bis-acrydium at concentrations of 0.01 to 10 pg/ml lylcystamine in 0.1 X Loening’s buffer as in 0.5 log,, increments. Typically, there was described (Bean et ah, 1980). Electrophoresis was for 16 hr at 42” with a constant a 50% reduction in plaques at O.Ol-0.03% voltage of 180 V. RNA was detected with amantadine for sensitive isolates while resistant isolates required at least loo-fold the Bio-Rad Silver Stain. higher concentrations for a 50% reduction. RESULTS The amantadine resistance was also assayed in viva as described (Webster et al., Does the Avirulent H5N2 Virus Possessthe 1985). Genes Necessary for Virulence? Animals-Definitions of pathogenicity One of the central questions concerning and mean death time. Adult white leghorn chickens (>30 weeks) were used in these the acquisition of virulence by the H5N2 studies. A highly pathogenic virus is de- influenza virus is which gene segments fined as one that results in not less than other than the hemagglutinin contained 50% mortality within 8 days in at least changes that were relevant. To answer this eight healthy adult chickens inoculated question, reassortants were made which orally with bacteria-free infectious allanpossessed the hemagglutinin gene of the toic fluid. virulent virus and the seven other genes Sick chickens were defined as birds that from the avirulent parent. Since the hemagglutinin and neuraminidase of the virwere unable to stand, and that twisted ulent and avirulent viruses are antigenitheir necks indicating a central nervous system disorder. Chick embryo mean death tally almost identical, it was necessary to time is the time required to kill 50% of lotransfer the hemagglutinin and neurday-old embryos incubated at 35”. aminidase genesto “carrier” viruses (Fig. I). Nucleic acid sequencing. Nine oligonuThis was achieved by preparing reassorcleotide primers complementary to the vi- tants between uv-treated virulent Ck/1370 (H5N2) and A/Seal/Mass/l/80 (H’7N7). rion HA gene segment of Ck/l (H5N2) (Kawaoka et aZ., 1984) were synthesized on an The mixed virus yield from this infection Applied Biosystems model 380A DNA syn- was selected with monospecific antisera to thesizer by the solid-phase phosphoramiH7 and N2 to give the reassortant containdite method. Nucleic acid sequences were ing Ck/1370 HA and Seal NA (H5N7). determined by reverse transcription of viSimilarly, the avirulent Ck/l (H5N2) virus rion RNA in the presence of primers and was reassorted with Mem/Bel (H3Nl) to dideoxynucleotides as described (Air, 1979; produce the H3N2 reassortant. These two reassortant viruses (H5N7 and H3N2) were Naeve et al., 1984). The reaction products were resolved in 8% polyacrylamide-7 M used as parents to produce the reassortant urea thin gels in TBE buffer (90 mM Trispossessing the HA of the virulent virus borate, pH 8.0; 1 mM EDTA) as described (Ck/1370) and the NA of the avirulent virus (Maxam and Gilbert, 1977). (Ck/l) (H5N2). This virus was referred to Genotyping of reassortant viruses. The as reassortant 10 (RlO). The genotypes of the reassortant viruses genotype of the reassortant viruses was determined by a combination of sequence were determined by a combination of sequence analysis and electrophoresis on analysis and electrophoresis on 3% polyacrylamide gels. polyacrylamide gels. To discriminate beThe partial sequences of PA, PBl, and tween the P genes of the virulent and avirulent H5N2 virus, it was necessary to do PB2 genes were obtained by the dideoxpartial sequencing of these genes (Fig. 2). ynucleotide chain termination method In this way, each gene could be discrimidescribedabovewith the primers[GCAGGnated unambiguously. The gene composiTACTGAT (3’) for PA, ATTTGAATGGA-
168
WEBSTER, U.V.
AND
BEAN
treated
Chick/Penn/1370/83
(HSNZ)
Seal/Mass/l/80
Ck/Penn/l370
KAWAOKA,
[Virl
Chick/Penn/l/83
Mem/1/71-Be1142
(H7N7)
cH)
seal
(H5N2.j
Mem (8)
cNj
X
(H5N7)
Ck/Penn/l370
(8)
Ck/Penn/l
[Avir]
(H3Nl)
Ck/Penn/l/83
(N)
(H3N2)
(N) = RIO
(H5N2) FIG.
1. Reassortment
FIG. 2. Sequence analysis for virus reassortants. Genotyping sequencing the PBl genes using Only A reactions were analyzed sortant viruses. Mem:Mem/l/‘71 Mass/3740/65 (H6N2).
between
virulent
and avirulent
genotyping influenza of the reassortants by the dideoxy method. on this gel. 3-ll:Reas(H3N2). Ty/Mass:Ty/
Chick/Penn/83
influenza
viruses.
tion of the reassortant viruses is shown in Table 1 and an example of polyacrylamide analysis is shown in Fig. 3. The reassortant virus possessing the virulent HA and the avirulent NA (RlO) obtained five of its gene segments from the avirulent Ck/l (Table 1); the M and NS genes were derived from the Seal influenza virus and the HA from the virulent parent. This virus was as virulent as the parental virulent strain; it killed chick embryos within 2 days and caused 100% mortality in experimentally infected chickens. It could be argued that the M and NS genes from the A/Seal/Mass/l/80 influenza virus may have contributed to virulence. To rule out this possibility, a reassortant was prepared between uv-treated RlO and R5 (Table 1) and between non-uvtreated RlO and R5. R5 possesses the hemagglutinin gene from A/Mem/l/71 (H3N2) and all of the other seven genes from the avirulent virus. The reassortants produced (R12 and R13) contained the HA gene from the virulent parent and all other genes from the avirulent parent. The reassortants (R12 and R13) were as virulent as the parental strain in chick embryos and adult chickens. Isolation of two different reassortants with the same phenotype reduces the likelihood of phenotypic variation. These studies indicate that the avirulent
VIRULENCE
OF
INFLUENZA
TABLE GENESEGMENTSOFCHICK/PENN/~~ Gene Reassortant R10 R5 Rll R12 R13
Note. Genes = Seal/Mass/l/80
PA1
PB2
INFLUENZAVIRUSASSOCIATEDWITHVIRULENCE assignment
HA
NP
NA
M
NS
A
VAA Mem
A
A
Seal A
Seal A
A = Avirulent (H7N7); Mem
169
1
PBl
AAA A A AAVAAVVV AAAVAAAA AAAVAAAA from
VIRUS
Chick/Penn/l/83 (H5N2); = Mem/l/?l (H3N2).
H5N2 influenza virus that was circulating in chickens in April 1983 contained all of the gene segments necessary for virulence except the hemagglutinin. The reciprocal antigenic hybrid (Rll-Table 1) possessing
V = Virulent
Chicken killing
embryo (days) 2 >5 3.3 2 1.8
Chick/Penn/1370/83
Adult
chicken killing ah3 O/8 O/8 8/8
8/8 (H5N2);
Seal
the HA from the avirulent virus and NA, PBl, M, and NS genes from the virulent virus was avirulent in chickens and chick embryos. This emphasizes the importance of the HA gene in virulence, and indicates that the other genes involved in virulence were already present in the avirulent virus that appeared in April 1983. Do Other InJEuenxa Viruses Possessa Gene ConsteElation that Will Support Vimlence?
PI-P2 P3 HA NP NA
NS FIG. 3. Genotyping of the influenza ants on polyacrylamide gels. The viral trophoresed as given under Materials and detected by silver staining.
virus reassortRNA was elecand Methods
To determine if other influenza viruses contain gene constellations that would be virulent if they contained the virulent H5 gene, a number of human and mammalian influenza viruses were used to prepare reassortants. These are given in Table 2. The reassortant (R4) possessing six gene segments from A/Seal/Mass/l/80 (H’7N7) and the PBl and HA genes from the virulent H5N2 virus is virulent. This virus killed chick embryos within 1.8 days and caused central nervous system signs of disease in seven of eight chickens inoculated and mortality in half of the chickens. The reassortant (R9) that contains the PBl and HA gene from the virulent H5 virus and a mixture of other genes (Table 2) was also virulent in chick embryos and in chickens. Reassortants lacking the HA gene from the virulent Ck/1370 virus (Rl, Table 2; or Rll, Table 1) are avirulent in chickens and in chick embryos even though they possess all or most of the other genes required for virulence. These studies show that other viruses
WEBSTER,
170
KAWAOKA, TABLE
GENE ASSIGNMENT
AND VIRULENCE Gene
Reassortant Rl R4 R9
Mem
BEAN
2
OF REASSORTANT
INFLUENZA
assignment
PA
PB2
PBl
HA
NP
NA
M
NS
Ty* Seal Seal
Ty Seal Seal
V V V
Ty V V
V Seal Mem
v Seal Be1
v Seal V
v Seal V
Note. V = Chick/Penn/1370/83 (H7N7);
AND
= Memphis/l/71
(H5N2) (Vir); *Ty = Turkey/Mass/3740/65 (H3N2); Be1 = Belamy/ (HlNl).
VIRUSES
Chick embryo killing (days) >5 1.8 1.5 (H6N2);
Adult
chicken
Sick
Killed
O/8 7/s 6/8
O/8 418 418
Seal = Seal/Mass/l/80
otides described previously (Kawaoka et al., 1984). Sequence analysis of the amantadine-resistant Ck/1370 virus revealed that there was an amino acid change at residue 23 of HA1 from Lys in the virulent Ck/ 1370 virus to Thr in the revertant. The HA1 polypeptide from the revertant virus showed a reduction in mobility as compared with the HA1 from the parent Ck/ Penn/1370 virus (data not shown), sugWhich Mutation in the Hemagglutinin Gene gesting that the revertants regained a carbohydrate side chain. Additionally, there of H5N2 is Required for Virulence? was an amino acid sequence change at resThe virulent H5N2 virus differs from the idue 40 of HA2 from Lys to Arg. In the avirulent strain in producing plaques in three-dimensional structure of H3 influtissue culture, and the HA0 precursor is enza HA (Wilson et al, 1981), these changes cleaved into HA1 and HA2 in the absence are located in the vicinity of the cleavage of trypsin in tissue culture (Kawaoka et al., site between HA1 and HA2 (Fig. 4). 1984). There are two differences in amino The amino acid at residue 23 of the HA1 acid sequence between the HA of the virof the avirulent Ck/l influenza viruses is ulent and the avirulent viruses; one at res- the same as that in the revertant (Kawaoka idue 23 and the other at residue 78 (Ka- et al, 1984), yet the avirulent Ck/l viruses waoka and Webster, 1985). are sensitive to amantadine (data not One approach to resolving which of these shown). The amino acid change at residue two amino acid changes is critical for vir40 in HA2 may be involved in resistance of ulence is to obtain avirulent revertant vi- the revertants to amantadine. These studruses and to sequence the hemagglutinin ies indicate that the HA in the revertant gene. During the selection of amantadineviruses lost the ability to be cleaved withresistant Ck/1370 (H5N2) viruses in viva, out exogenous trypsin and that the virus such revertant viruses were isolated. The was avirulent; this was correlated with an viruses grew in chick embyros, but failed amino acid sequence change at residue 23 to plaque in tissue culture without trypsin of HAl. and HA0 was not cleaved (data not shown). These revertant viruses were avirulent in DISCUSSION adult chickens. The avirulent H5N2 influenza virus A revertant virus selected in this way was grown in chick embryos and the HA present in chickens in Pennsylvania in gene was sequenced by the dideoxy chain April 1983 possessedall of the gene segtermination method (Sanger et al., 1977; ments necessary for virulence other than the hemagglutinin. This was established by Air, 1979) using nine synthetic oligonucle-
such as A/Seal/Mass/l/80 (H7N7) are present in nature that contain a gene constellation which, in combination with the virulent hemagglutinin, has the capacity to produce a virulent virus. The reassortant possessing the Be1/40 (HlNl) neuraminidase is virulent in chickens suggesting that there is not an association between the N2 neuraminidase and virulence.
VIRULENCE
terminus C-terminus
OF
INFLUENZA
of HA2
of HAI
FIG. 4. The carbon tracing showing the structure A/Aichi/2/68 (H3) influenza virus hemagglutinin (Wiley et CLL, 1981). The numbers show the position the amino acid changes in the amantadine-resistant Ck/Penn/83 (H5N2) influenza virus.
of of
preparing reassortant viruses that contained the hemagglutinin gene from the virulent parent and the seven gene segments from the avirulent H5N2 parent. The gene composition of the reassortants was established by a combination of partial sequencing of the P genes and analysis of RNA migration in acrylamide gels. Studies with other influenza viruses from mammalian and avian sources showed that the A/Seal/Mass/l/80 (H7N7) influenza virus contained the necessary gene composition that, in combination with the H5 gene, was virulent in chickens. However, reassortant viruses possessing the HA from the virulent H5N2 virus and the other genes from different donor viruses [A/Turkey/Va/28215/83 (HlON8), A/ Guinea Fowl/De1/4358’7/83 (H3N8), A/ Ruddy Turnstone/l/85 (H7N3)] were avir-
VIRUS
171
ulent in chickens or chick embryos (results not shown). At this time, we do not know the minimal gene constellation required for virulence of the Ck/1370 (H5N2) virus. The studies of Rott et al. (1976,1979) with FPV and other donor viruses have shown that the minimal gene constellation required for virulence varies between each influenza virus pair studied. A practical consequence of the present studies is that there are potentially dangerous influenza viruses circulating in nature that only require a single point mutation in the HA gene to become highly virulent for chickens. Although many avirulent H5 viruses have been isolated from wild and domestic species, it is clear that this subtype is potentially more dangerous than other influenza virus subtypes. The other potentially dangerous subtype is H7, which includes FPV and other highly virulent viruses that are pathogenic for avian species and some mammalian species such as seals (Webster et aZ., 1981). Based on these studies, it appears that influenza viruses possessing H5 or H7 genes are potentially hazardous to domestic poultry and other species as well. It is therefore suggested that whenever H5 and H7 viruses are detected, they should be eradicated from domestic poultry as rapidly as possible. Which of the two amino acid changes at residues 23 or 78 of HA1 in the avirulent Ck/l (H5N2) reported by Kawaoka et al. (1985) are responsible for virulence? The availability of an amantadine-resistant H5N2 virus that requires trypsin for cleavage of HA0 into HA1 and HA2 and is avirulent in chickens provides the opportunity to determine which of the mutations is critical. Sequence analysis of a revertant virus showed that the amino acid at residue 23 changes from Lys to Thr, and SDSpolyacrylamide gel analysis suggests that the revertant HA1 is larger and possesses a carbohydrate side chain at this residue. Sequencing of the protein is in progress to establish the location and type of carbohydrate side chains on Ck/1370 HA. Isolation of an avirulent revertant Ck/ 1370 virus during selection of amantadineresistant strains may simply be fortuitous. Many of the amantadine-resistant isolates
172
WEBSTER,
KAWAOKA,
of the virulent Ck/1370 virus were virulent in chickens and chick embryos. The molecular changes and the genes involved in amantadine resistance of Ck/13’70 are under investigation. The amino acid at residue 23 in HA1 of the avirulent Ck/l influenza viruses is the same as that of the revertant, yet the avirulent Ck/l viruses are sensitive to amantadine (W. J. Bean, unpublished data). The amino acid change at residue 40 in HA2 may be involved in resistance of the revertants to amantadine. Studies by Daniels et al. (1985) have shown that amantadine-resistant mutants of FPV and X-31 have mutations in the hemagglutinin gene that affect HAl, HA2, or both. Amantadine resistance has also been associated with the matrix protein (Lubeck et al., 1978) and more recently, with the M2 protein (Dr. Allan Hay, personal communication). Studies are in progress on the other genes involved in amantadine resistance of Ck/ Penn/83 influenza viruses. These studies establish that a single critical point mutation in the hemagglutinin gene of the avirulent A/Chicken/ Pennsylvania/l/83 (H5N2) was required to produce the highly virulent Chicken/ Pennsylvania virus; the avirulent virus already possessed the other genes necessary for virulence. Our previous studies (Bean et al., 1985) also indicate that low molecular weight RNAs typical of defective interfering particles (DI) were found in the avirulent viruses isolated in April 1983, but were absent from the virulent virus. The mechanism by which the virulent virus lost the subgenomic RNAs present in the earlier virus is not known. Studies are in progress to further characterize the subgenomic RNAs and to determine their role in modulating virulence. ACKNOWLEDGMENTS This work was supported by U. S. Public Health Research Grant AI 08831, AI 52586 from the National Institute of Allergy and Infectious Disease, Cancer Center Support (CORE) Grant CA 21765, and American Lebanese Syrian Associated Charities. The authors thank Dr. Clayton W. Naeve for preparation of the oligonucleotide primers. Lisa Newberry, John Kayoma, and Hosea Clariette provided excellent
AND BEAN
technical assistance, and we thank Lisa Wilson for typing the manuscript. REFERENCES AIR, G. M. (1979). Nucleotide sequence coding for the “signal peptide” and N terminus of the hemagglutinin from an Asian (H2N2) strain of influenzavirus. Virology 97,468-472. AYMARD-HENRY, M., COLEMAN, M. T., DOWDLE, W. R., LAVER, W. G., SCHILD, G. C., and WEBSTER, R. G. (1973). Influenza virus neuraminidase-inhibition test procedures. Bull. WH.0. 48,199-202. BEAN, W. J., KAWAOKA, Y., WOOD, J. M., PEARSON, J. E., and WEBSTER, R. G. (1985). Characterization of virulent and avirulent A/Chicken/Pennsylvania/ 83 influenza A viruses: Potential role of defective interfering RNAs in nature. J. viral. 54(l), 151-160. BEAN, W. J., SRIRAM, G., and WEBSTER, R. G. (1980). Electrophoretic analysis of iodine-labeled influenza virus RNA segments. And. B&hem. 102,228232. DANIELS, R. S., DOWNIE, J. C., HAY, A. J., KNOSSOW, M., SKEHEL, J. J., WANG, M. L., and WILEY, D. C. (1985). Fusion mutants of the influenza virus hemagglutinin glyeoprotein. Cell 40,431-439. DESHPANDE, K. L., NAEVE, C. L., and WEBSTER, R. G. (1985). The neuraminidases of the virulent and avirulent A/Chicken/Pennsylvania/83 (H5N2) influenza A viruses: Sequence and antigenic analysis. Virology, 14’7, 49-60. KAWAOKA, Y., NAEVE, C. W., and WEBSTER, R. G. (1984). Is virulence of H5N2 influenza viruses in chickens associated with loss of carbohydrate from the hemagglutinin? Virology 139,303316. KAWAOKA, Y., and WEBSTER, R. G. (1985). Evolution of the A/Chicken/Pennsylvania/83 (H5N2) influenza virus. virology 146, 130-137. KOHLER, G., and MILSTEIN, C. (1976). Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion. Eur. J. Immund 6, 511519.
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VIRULENCE
OF INFLUENZA
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