I+u.r Research, 22 (1992) 93-106 0 1992 Elsevier Science Publishers B.V. All rights reserved 016%1702/92/$05.00

93

VIRUS 00726

Sequence analysis of the equine H7 influenza virus haemagglutinin gene C.A. Gibson Ipa,R.S. Daniels l,b, J.S. Oxford ‘,’ and J.W. McCauley 2 I National Institute for Biological Standards and Control, Potters Bar, U.K. and ’ Division of Molecular Biology, AFRC Institute for Animal Health, Pirbright Laboratory, Pirbright, UK (Received 2 August 1991; revision received and accepted 23 October 1991)

Summary

The nucleotide sequences of ten haemagglutinin genes of representative H7N7 equine influenza viruses isolated between 1956 and 1977 have been determined by primer extension sequencing. Their nucleotide and deduced amino acid sequences demonstrate a high degree of homology. These equine viruses can be divided into two distinct subgroups, the prototype-like, and a group comprising the early American isolates and the remaining equine viruses. The equine H7 haemagglutinins form a quite distinct group compared to H7 haemagglutinins isolated from other species. Each of these equine H7 haemagglutinins possess a tetrabasic amino acid cleavage site separating the HA1 and HA2 domains but, in addition, all ten contain a nine amino acid insertion prior to the tetrabasic sequence. The haemagglutinin glycoproteins of all ten viruses are capable of cleavage activation in virus infected primary chicken embryo fibroblast cells.

Nucleotide sequence; RNA segment 4; HA glycoprotein; Hemagglutinin

Correspondence

Equine influenza virus;

to: J.W. McCauley, APRC Institute for Animal Health, Compton Laboratory, Compton, Newbury, Berks, RG16 ONN, U.K. a Present address: Veterinary Research Institute, Attwood, Vie., Australia. b Present address: Division of Virology, National Institute for Medical Research, London, U.K. ’ Present address: Department of Medical Microbiology, The Royal London Hospital Medical College, London, U.K.

94

Introduction Two subtypes of influenza A virus cause clinical disease in horses, the H7N7 and H3N8 subtypes, previously referred to as the equi-1 and equi-2 subtypes respectively (Sovinova et al., 1958; Waddell et al., 1963). Both subtypes circulate in horse populations throughout the world, with the possible exception of Australasia (Doll, 1961; Wilson et al., 1965; Mahaffey, 1964; Beveridge, 1965; Halterman and McQueen, 1968; Powell et al., 1974; Rose et al., 1974; Burrows, 1978; Burrows and Denyer, 19821. Although H3N8 viruses have been isolated recently (Meldrum, 1989; Cullen, 1989; Rojahn, 1989; Webster and Guo, 1991), the last recorded isolation of the H7N7 influenza virus from horses in the field was in 1978 (Turnova, 1980). However, there is serological evidence to suggest the continued circulation of these viruses in horse populations (Bull. WHO, 1987). Influenza virus subtypes infecting other mammalian and avian hosts demonstrate varying degrees of antigenic shift and drift. Antigenic studies (Powell et al., 1974; Burrows and Denyer, 1982), of H7N7 virus isolates and immune sera from horses, detected only minor antigenic drift in the haemagglutinin (HA). This contrasted sharply with studies on the Hl (Raymond et al., 1983, 1986) and H3 subtype in man (reviewed by Webster et al., 19821, and in horses (Hinshaw et al., 1983; Daniels et al., 1985; Kawaoka et al., 1989). The degree of antigenic variation within the equine influenza viruses is of interest since the current commercial equine vaccines are still produced using the two prototype strains. However, in recent years a later antigenic variant of the H3N8 subtype has been incorporated in several commercial preparations to produce a trivalent vaccine. In the present study, we have determined the nucleotide sequences of RNA segment 4 (coding for the HA glycoprotein) from a range of H7N7 equine influenza viruses, isolated over a 20 year period (1956-77). These nucleotide sequences and deduced amino acid sequences are compared to each other and we further compare the equine H7 haemagglutinin and its gene with those from other H7 influenza viruses and show that the haemagglutinin of the H7 equine viruses are members of a rare group of viruses which contain an amino acid insertion at the cleavage activation site of the haemagglutinin.

Materials and Methods The following H7N7 viruses were used in this study: A/Prague/i/56, A/Cambridge/l/63, A/Detroit/i/64, A/C/Detroit/l/64 (a vaccine manufacturer’s seed stock), A/Lexington/i/66, A/Switzerland/137/72, A/London/1416/73, A/Cambridge/i/73, A/Sao Paula/1/76, and A/Newmarket/l/77, all of which were maintained in the reference collection at NIBSC. All were grown in lo-day-old embryonated hens eggs purified by centrifugation through sucrose gradients, and the RNA was prepared using phenol-SDS extraction as described previously (Hay et al., 1977).

95

Nucleotide sequences were determined using the dideoxynucleotide chain termination procedure of Sanger et al. (19771, and reverse transcription using I~‘-~~P labelled oligonucleotide primers as previously described (Daniels et al., 1983). All primers were made on an Applied Biosystems 381A DNA-synthesiser and corresponded to bases 5-AAAGCAGGGGA-15, 151-GTTGTCAATGCA-162, 412ATGGGATTCAC-422, 585-CTGGGGAATCCACCA-599, 877-TGTGAAGGTGAATG-890, 1153-GCACAAGGAGAAGG-1166 and 1492-CACCAATGTGAC-1503 of the A/Prague/l/56 sequence. Sequence analyses employed UWGCG (Deveraux et al., 1984) and FASTA (Pearson and Lipman, 1988) software packages. Cleavage of the haemagglutinin was determined by labelling virus infected chick embryo fibroblasts in serum-free medium at 5 h post infection for 5 min with 35S-methionine and chasing with medium which contained 10 mM methionine for 30 and 60 min (described in detail by McCauley and Penn, 1990). Electrophoresis of infected cell lysates was carried out on 12.5% acrylamide gels (Laemmlli, 1970). We have used the following abbreviations for the equine viruses in the figures and text and have been assigned their respective DNA database accession numbers by EMBL: PRAG56, Prague/i/56, accession no. X62552; CAMB63, Cambridge/i/63, accession no. X62553; DETR64, Detroit/i/64, accession no. X61627; CDET64, G/Detroit/l/64, accession no. X62555; LXTN66, Lexington/l/66, X62556; SWTZ72, Switzerland/137/72, accession no. X62557; LOND73, London/1416/73, accession no. X62560; CAMB73, Cambridge/i/73, accession no. X62558; SNPL76, Sao Paula/1/76, X62559; NMKT77, Newmarket/1/77, accession no. X62554. The following virus sequences were retrieved from EMBL databases: A/FPV/Rostock/34 (Porter et al., 19791, abbreviated to ROST34; A/FPV/Chicken/Victoria/85 (Nestorowicz et al., 1987), CHVC85; A/Turkey/Oregon/71 (Orlich et al., 1990), TYOR71; and A/ Seal/ l/ 80 (Naeve and Webster, 19841, SEAL80.

Results and Discussion Nucleotide sequences of H7 haemagglutinin genes from equine viruses The nucleotide sequences of RNA segment 4, the haemagglutinin

gene, have been determined by direct sequencing of virion RNA from ten H7 equine influenza virus strains representative of major outbreaks of disease during the period from 1956 to 1977. All RNA segments 4 from the ten equine H7 isolates were 1761 nucleotides in length and potentially encode 570 amino acid heamagglutinin precursors.

Variation within H7 haemagglutinins from equine viruses isolated between 1956 and 1977 All ten viruses show a high level of nucleotide sequence homology, between 96

and 99% for each paired comparison (Fig. la) and this reflects in a similar homology in the haemagglutinin polypeptide (94-99%, Fig. lb). Using these

96 LXTN66 LOND73 Swz72 CAN873 NflKT77 SNPL76 ______________-~________l_~~~~_______l__--~~~~~---------. ___________--------98.7 99.1 99.1 99.1 98.2 : NNKT77 I 100 100 99.3 99.3 99.4 98.4 : SNPL76 : 100 99.6 99.8 98.3 : HIND73 : loo 99.7 98.8 : wz72 : 100 98.8 : CANS73 I 100 I LIT1166 I : CDETM : : DEW64 : I CANE63 I : PM656 :

NNKl77

SIP176

CAR73

LOND73

SNTZ72

CDET64

DEW84

91.0 97.8

97.0 97.8 98.2 98.2 98.2 99.2 99.7

98.1 98.1 98.2

99.1 100

PM656 _________94.5 94.1 94.9

Cm63 94.3 94.5 94.6 94.6 94.7 95.5

94-a a)

94.0

95.1

100

95.2

100

100

LXlN66

CDET64

DETRM

cm63

__________________________________~~__________~~~~~~~~ __________-_-I 1111177

I

I I I : 1 I : I :

I I I I : I I : :

SNPL76 CAM73 LOND73 SNTZ72 LXTN66 CDET64 DETR64 CANB63 PM656

100

9S.6

100

95.1 95.4 95.5 99.4

____I~~________~__~~______-~~~~~~~~~~~~

98.9

98.6

98.4

98.4

99.6 100

99.3 99.6 100

99.2 99.5 99.1 100

99.1

97.9

97.9

95.3

97.9

91.9

95.6

99.5

98.2

98.2

99.1 93.9 loo

97.9 97.7

97.9 97.7

96.0 95.6

98.4

100

98.4 99.4

100

95.4 96.1 95.4 95.4

100

PM56 95.6 95.6 96.0 96.0 95.4

b)

%.I 95.4 95.4

99.1 100

Fig. 1. (a) Nucleotide sequence homologies between each of the ten equine H7 haemagglutinin genes sequenced in this study. (b) Protein sequence homologies between the precursor haemagglutinins. Abbreviations for the viruses are described in Materials and Methods. Homologies are scored by the percentage of exact matches.

figures, the two early European isolates (PRAG56 and CAMB63) can be grouped as a very closely related pair distantly related to the later viruses. All viruses isolated from 1964 onwards exhibit a high degree of homology, greater than 98% homology amongst one another, and viruses isolated from 1973 onwards make up a group even more closely related to each other (greater than 99% homology). A comparison of the nucleotide sequences of segment 4 of PRAG56 and DETR64 is shown in Fig. 2. CAMB63 differed from the prototype PRAG56 at only ten nucleotide positions (373 A + C, 527 C + U, 584 U --) C, 696 U + C, 717 G + C, 949 G + A, 1248 C + U, 1506 U + C, 1527 A + G, 1657 U + C). These changes resulted in amino acid substitutions: 118 K + Q; 169 T + I; 188 I + T; 232 M --$ I; 310 A + T. The nucleotide sequence differences and the resulting amino acid substitutions between DETR64 and viruses isolated after 1964 are shown in Table 1, whilst Fig. 3 contains full amino acid translations for PRAG56 and DETR64. Fig, 4 maps the amino acid substitutions onto the structure of an H3 haemagglutinin monomer (Wilson et al., 1981) in the absence of an H7 haemagglutinin structure. The antigenicity of strains of human influenza virus currently circulating in the population (Hl and H3 sub-types) has been studied in detail and the locations of antibody binding sites have been proposed (Caton et al., 1982; Wiley et

97

al., 1981; Wiley and Skehel, 1987) although much less detailed knowledge is available for the binding of neutralising antibody to sites in the H7 haemagglutinin. There are five antigenic sites defined for the H3 haemagglutinin (A to E) and amino acid sequence changes can be found within the H7 equine haemagglutinin at sites B, D and E. Antigenic site B is located at the top of the haemagglutinin trimer and the amino acid substitutions seen in the equine H7 haemagglutinins at positions 164, 168, 169, 198, 202 and 208 (numbered for the H7 HA as shown in Fig. 3 and Table 1) can be mapped to this site. Mutations at residues 182, 183, 211, 220, 223 and 253 are part of site D; and the mutations at 67 and 96 map to site E. The degree of variation seen in these ten equine viruses is much lower than that seen in human H3 viruses isolated over a similar time period (Wiley and Skehel, 1987) and in most cases the changes seen in the equine H7 viruses reflect the more distant relationship between the 1956 and 1963 pair and the later viruses. Viruses isolated after 1964 exhibit little variation at the antigenic sites: the only variations observed are at 168 (site B), 182 (site D), 202 (site B), 208 (site B), 211 (site D>, 220 (site D) and 253 (site D). These amino acid substitutions are found in only one (position 252 and 168), two (position 88) or three of the viruses (position 182). Carbohydrate side chains can potentially modulate the biological activity of a glycoprotein. In both the Hl and H3 haemagglutinins the addition of a carbohydrate moiety can block antibody binding at a site to which, in the absence of carbohydrate, it would be capable of binding (Wiley and Skehel, 1987). Possible examples of viruses which use this mechanism to avoid antigenic surveillance in human hosts have been documented for both H3 and Hl subtype haemagglutinins (Wiley and Skehel, 1987; Caton et al., 1982). All viruses maintain the two potential carbohydrate attachment sites in HA, at positions 46 and 249 and those in HA, (positions 431 and 503 of the precursor HA), all of which are maintained and utilized in the prototype H7 avian haemagglutinin (Keil et al., 1985). A potential carbohydrate attachment site in antigenic site B (at position 167) is lost in all viruses except the prototype equine strain (PRAG56), whilst LOND73 has lost the site at position 30. Two new sites appear: one at position 253 is very close to the conserved site at position 249 and is only present in three viruses (DETR64, CDET64 and NMKT771, and the other is at position 335, close to the HA cleavage site and which is maintained in all viruses isolated from 1964 onwards. The latter two glycosylation sites might modulate antigenicity. However, since the potential site at position 335 is so close to the haemagglutinin cleavage site (which is not thought to be an antigenic site), we consider that it is unlikely to influence antigenicity. The glycosylation sites at residue 30 is, like 335, close to the HA,/HA, proteolytic processing site. The presence of carbohydrate close to the haemagglutinin cleavage site in H5 haemagglutinins can dramatically influence virus pathogenicity and haemagglutinin cleavage in tissue culture (Deshpande et al., 1987). We examined the cleavage of the haemagglutinin in tissue culture by pulse-chase experiments, the results are shown in Fig. 5. Cleavage of all the equine haemagglutinins was detected irrespective of these two potential glycosylation sites, so should these sites prove to be used, glycosylation proximal to the cleavage site seems not

98

I

100

DETRbl PM656

XXXXXXXXXX

XXXXXXXXXA

AAffiAACACU

CAMWCUM

UAUUA6CC#

c 6 UWUAUWAC

WC66CAUlK

W6CA6AUAA

AAUCUGECUA

66ACAucAu6

101

2OD

DETRM

C

C

PRA6Sb

CU6U6UCUAA

6 U66AACCAAA

GUMACACCC

WACUGAMA

A

A66AAUA6AA

6WWCAAuS

CMCAGAAAC

MWSAACAA

C UCCUCCCCM

C UW6ACCAAU

6AMCAUUM

6AAAMUACU

C

ACAMCAUCC

CUAA6AUCuG

201

300

DETRb4 PM656

6 CUCAAAA66A

A AAACA6ACU6

A WSACCUUS6

UCAAU6U66A

WACUA666A

CC6UUAW66

A66UAAWAC

AUUU6WAUC

CA66cAAAW

U6ACMU6AA

WClRJ6MW

CUCU6CUAAU

CAGAMAUCC

66ffi6AAWA

301

400

DETRb4 PUffi

A

6

WMUA6W6

A

6

AAAMA666A

C

401

SO0

DElRb4

A

A

PM656

AAAA66A6AA

UAU&&UlUC

ACMMMCC6

6116MAMAC

CAAU66A616

ACUA6C6CAU

6UA6AA66UC

AA6MJlJCC

UWJAU6CA6

A6AU6AAM16

501 DETRb4 PRA656

600 C

A

6CUUCUAUCC

6U

AMACAGACA

C

AUSMCAW

A

UCCACAMUG

6

ACAAA6UtCU

ACAA6AACAC

A6

UAA6AA66UA

CCffiClJClJM

UACUCU6666

AAUCCACCAC

601

100

DEVIL4 PM656

6

C6 CMCUGAACA

UCA66AUCAA

6ACUA6AUUA

A UAffi6AA6U6

6

66AAUAAAUU

U GAUAACAGUU

C

U66A6CUCCA

AAUACCAACA

AuCUUUU6lK

CCAAAUCCU6

701

800

DETRb4 PM656

6

A

6ACCMACC

c

6CAAAMAAU

U

66UCAAUCA6

6AffiAAW6A

CUWCACffiD

U

A

CUMUGCUA6

AUCCCAMIGA

UACKUCACU

u WCAGUUWA

AU6666CCVU

601

900

DEIRM PM656

C

6 UAUA6CACCU

6ACCSC6CCA

6UUUKiJAA6

A66UAMUCU

CUA66AAUUC

YA!4UABCAA

CW6CCCUW

CMAACAWA

AUAWA6ffiC

AAAU6UUCCU

AAUA GAffiCUCCUG

c CACUIAAACA

U ACUAACUCAU

AM6U6AU6C

U

ACAACWGAC

AAUAAW6u6

AAUt6PAAA

UGCCCUGAU

ACWMAGCA

CACAUGCGCA

G AAAAAA6A66

AA66U6AAU6

CUAUCAUAW

901

1000

DETRb4 PM656

C 66A6SUACUA

U

6

A

A

A

6AA6ASCWA

AU6CUA6CM

1001 DETRb4 PRA656

1100

CA66AAU6AA

UiAlAUUU66U

6CAAUA6CA6

GAUUCAUUGA 1200

1101 DETRb4 PM656

U AAAUSW66

6AA66AUUAA

UA6AC66AU6

C 6UAUG6AUAU

A

6A AA6CAUCA6A

AUGCACAAGG

AGAA666ACU

GCU6CA6ACU

ACAAAAGUAC

ACAAUCUGCU 1300

I201 DETRb4 PRA656

G

U AUCAACCAAA

A

UAKC66AAA

AUUGAACAGA

U CUAAUA6AAA

AMCCAACCA

G

GCAAUUCGAA

CUAAUAGAUA

AIBAGUUCAA

U6AAAUA6AA

1400

1301 A

DETRb4 PM656

6CAAU6UUAU

UAAClh36ACU

A6A6AUUCUA

UCAUCGMGU

6 AU66UCAUAU

AAUGCA6AAU

UCCUCGUAGC

AW66ffiAAU

CMCACACUA

U

PM656

U6ACUCA6AA

6

U AU6AACAAAC

UAUAU6AM.A

66UAA6AA61

6CAU66CCA6

CUlUA6AAAC

AACACAUAUG

WACAAASAU

6 AUAAUACUUU

CAACU616A6

AAAAffiCU6A

66AA6AU66U

AAU66CU6W

UUGAAAUAUU

CCACCAMIGU

ACCAUAAAAA

AUACA6AAAA

6A6SCAAUAC

AMACMUJ

UCAGAWGAC

GCAWAAAGU

UUCUUGCCAU

A U6CAAu666U

C CUU6UWUCA

C UAUSUAUMA

1600

1501 DElRb4

U 6ACAAU6AUU

1700

1601 DElRb4 PRA656

C UEMCA6u66

ffiUWA6CW

C6666CAUCA

U6UUUtUUAU 1761

1701 DETRb4 PRA656

UUGAUUUAAC IS00

1401 DETRb4

PRA656

AAACMAWG

AAAU66AAAC

AUK66ffiCA

CUAUW6UAU

AUAAGUWGA

AAAAACACCC

UU6UUUCUAX

X

Fig. 2. Nucleotide sequences of RNA segment 4 of A/equine/Prague/56 and A/equine/Detroit/64. Regions of RNA sequence that were not determined are marked x. Only nucleotide differences are shown for DETR64.

99 TABLE 1 Differences between hemagglutinins of A/Detroit/64

Wucleotide

l

no. md base in woetroi t/64 33 50 95 I& 104 108 116 153 254 321 325 330 355 373 405 459 510 524 566 579 612 615 626 639 643 645 652 6aO 696 711 717 777 779 763 a50 a58 YCQ 948 975 1002 1041 1065 1095 1107 1140 1201 1275 1339 136a 1443 1446 1491 1542 1560 1590 1596 1620 1621 1672 1679

and later equine H7 influenza A virus isolates

COET 64

Race LXTU 66

oubotitutimr Sun 72

in Lam 73

Later viruses CMR SWPL 73 76

A C

A i C

”

c

u

U

G U

G

c A U U

C

C ” u A A C G C A A C G G G C u G U A u A A ” G * C A ” C U ” G A A A G C G

U

NmKl 77

U IJ u

U U

U U

u u C

C

C

C

G

G

G

A G C

C

C G G

G

U A

A A G C

*

A

G C

G C

A

A

C

G C ”

A A IJ C A C

A

G C

C

C

C

C

C

u C C A C

” C C

” C C

U C C

C

C

C

A

*

A

C G A U A

C G A U A

C G A U A

A G C G A u 1

C G G C ”

C G U C C C A C G A u A G

G

A

A

G

G A

1

G G

G C C

U

A C A U G A

U G u

A G

A G U

G

G

A G ”

A G U

A G U

vr=J ui th 4 10 25 i7 28 29 32 44 78 100 102 103 112 ii8 128 146 163 168 162 186 197 198 202 206 208

C

”

A

Reid StbRtitutiom

Amh

A G u

211 220 225 230 232 252 253 254 277 279 296 309 318 327 340 34a 358 362 373 394 418 440 449 474 475 490 507 513 523 525 533 534 551 553

WDctroi

t/64 G T-I H-R A A-E A-V S T-I ” I-T E N-D D O-N G-K K A S G-E

R-K’ L A R-K S N-O N I-V G-R

(17) 120)

1241 170) 194) 1104) 1110)

1159) 1173)

1193) 1199) 1202) (211)

N

P 1-M V N-T F D-K 0 T R V 1 0 K F G

1223) 12451 1269)

I I-V

E I-V V G 1 F 0 K D V D-E V-I 1-A G-V

145) (91)

1184) 11851 (202) iio4;

Nucleotide sequence differences between RNA segment 4 of A/equine/Detroit/64 and that of viruses subsequently isolated. The resulting amino acid substitutions are also given. The signal peptide of the haemagglutinin, HAl, the connecting peptide and HA2 domains are separated by lines. The numbering for the protein sequence is for the baemagglutinin precursor polypeptide, numbers in parentheses refer to equivalent positions of amino acids in the H3 (X31) HA structure (Wilson et al., 1981).

100 I

ROST34 CHVC85 TVOR71 SEAL80 DETR64 PRAS56

VF 1 11 WA WA #TIMLILAT

v VIPTN V AWN CVLIKAKS CVLIEAKS LC SAFFVVRADK

A A A ICLSHHAVSN

RT R RR1 R 1 KAl IA 6 6 TO RPT TA 6 TO RPT K I VEQTNIPKIC SKSKQTVDLS

R v II v

N N NR NR STKVSTLTEK

SIEVVNATET

IT IIT 1 1 I C’CS11STVIS

PPOCDRFLEF

100 D I TD t ESD I ES I I SAMLIVERRE 200

101 ROST34 CHVCSS TVOR71 SEALS0 DETRCI PRAS56

ROST34 CHVCSS TVOR71 SEALS0 DETR64 PRAS56

V V V SNDICVPSKF

V A0 V A0 1 SE 1 SQ D Q DNEETLRKIl

201 K K K

6 DT E N 11 6 DS SVDS RKSSSIKKEN

SH 6 N E 6

TRLVSSSNKL

ST1 SA TSA TSA A SFVPNPSPRP

ITVSSSKYOR

SI SI 61 SI

6

1 V A 1

NSFTVTSVRT

16 SA 6

NSETSACRRS

RSSFVAEIIKN

I IN 1 1

V V V I PflMS8SSRID

E

N AS RRES V N AA RNE V NS AA R P RSK V NS AA RPRNK V RE N V LLSSTDNSTF PWKSVKNT KKVPALIIWS

N VFK

V 1 1

V FHNLIILDPND

N

N TVTFSFNSAF

A I E E

F

IAPDRASFLR

A

A IHHSSSTTEO

V VA EV PV 1 VPSSNFS V VPSSDFS

SKSLSIOSDA

ELDNNCESEC

300 TR 1 VS VS

S N

VHISSTIISN

301 ROST34 WCS5 TVOR71 SEALS0 DETR64 PRAM6

400 V v

E

1 1

PSXXXX v I XXXX RPL R N KTXX TV v PL R TV N KTXX NST LPFRNIMSRA ISKCPRVVKO KSLIILATSHK NVPEAPAHKO

V V

XXXKK XXXKK E XXXXXXXX XXXXXXXX LTHHIIRKKRS

LFSAIASFIE

FR FR FR FR R VSVKHONAQ6 ESTAADVKST

NWESLIDSN

D D D D QSAINOITSK

401 ROST34 CHVCSS TVOR71 SEALS0 DETR64 PRAS56

TV T v D D

AS

1

KT

11 DL 1 1

1 t!T MT

0 0

A A A A

I( I! n II

R

K K K

1 CT 1 1

K K K K

500 D D DO Dil E

I LNRLIEKTWP

BFELIDNEFN

EIEKOISNVI

NNTRDSIIEV

E II E II A 61 AKSL

P P P P

V v

IISVNAEFLVA

VENOHTIDLT

DSEANKLVEK

VRRQLRENAE

EDSNSCFEIF

HOCDNDCtlAS

570

501 ROSl34 CHVCSS TVOR71 SEALS0 DETR64 PRAS56

R R R R

S ST 78 I0 IRNNTVDHKK

VRKEAIUNRI

UK

OIDAVKLSSS

1

1 1

V IIV

1 1 V VKDIILNFSF

SASCFLFLAI

T A AMSLVFICIK

NSRRCTICI

Fig. 3. The alignment of the H7 subtype haemagglutinin polypeptides from PRAG 56 and DETR64 compared with other published H7 haemagglutinin sequences from turkeys: TYOR71 (Orlich et al., 1990), chickens: ROST34 and CHVC76 (Porter et al., 1979; Nestarowicz et al., 1988) and seals: SEAL77 (Naeve and Webster, 1983). Padding characters have been inserted into the non-equine virus haemagglutinins to maximise the alignment and are marked x.

to influence cleavage in vitro. Similarly, there has been no evidence reported for any attenuation of virulence of those strains that possess additional potential carbohydrate addition sites through the modulation of carbohydrate addition. Host-cell selection plays a role in the variation seen in laboratory isolates of influenza viruses as evinced by the observations that virus grown in hen’s eggs

101

Fig, 4. A map of the of amino acid substitutions found in the equine H7 haemagglutinin of viruses isolated from 1956 to 1977. Amino acid substitutions are mapped onto the homologous position on the peptide backbone of the H3 subtype haemagglutinin (Wilson et al., 1981). A single monomer is shown with substitutions in HA, and HA,. The arrow indicates the receptor binding site and a black square marks the C-terminus of HA, which is extended in all equine H7 HAS. Antigenic sites (A + E) determined from the genetic drift of the human infhrenza H3 haemagglutinin, are shown.

-HA1

Fig. 5. Polyactylamide gel electrophoresis of [ %]methionine labelled cells infected with ten strains of equine influenza H7 subtype virus. Infection of 96-well multiwell dishes with infectious allantoic fluid was carried out and cells were incubated with [35S]methionine (100 &i/ml) for 5 min at 5 h after infection. After removal of label, cells were incubated in medium which contained 10 mmol/l methionine for 0, 30 or 60 min as indicated. Uncleaved haemagglutinin (HA) and the cleavage products (HA, and HA,) are indicated. a, PRAG56; b, CAMB63; c, DETR64; d, CDET64; e, LXTN66; f, SWTZ72; g, LOND73; h, CAMB73; i, SNPL76; j, NMKT77. Abbreviations used the the viruses are described in Materials and Methods.

show antigenic differences from those isolated in mammalian tissue culture @child et al., 1983). Residues at the tip of the trimer and close to the sialic acid receptor binding site have been shown to be determinants of such host-cell selection of influenza viruses and have been seen in Hl, H3 and Type B influenza viruses (Robertson et al., 1985, 1987; Katz et al., 1987; Katz and Webster, 1988). In many cases, individual viruses show alternate selected mutations upon adaptation to growth in eggs (Robertson et al., 1987). We are not in a position to specify whether there has been selection of the equine H7 viruses studied here due to growth in eggs nor whether host-cell selection plays a role in the variation seen in the haemagglutinin. However, we do see changes at antigenic site B which is frequently implicated in host-cell selection. It is therefore a strong possibility that certain of the mutations itemised above are associated with selection of virus to grow in embryonated hen’s eggs. Comparison of equine H7 haemagglutinin sequences with other H7 haemagglutinins

The predicted

amino acid sequences

of precursor

H7 haemagglutinins

from

103 SEA177 PRAfB6 ctlWl5 TYORfl R&Z34 _______________________~~~~~~~~~~~~~~~~-~~--~---- ___________ : Ro!x# : 109 93.5 75.3 76.8 18.4 :cwcR5: 100 75.1 74.8 77.0 : TYOR71 : 100 92.5 74.1 : SEAL77 : 100 74.2 loo f PRffi56 :

Ross4

CHVCRS

lYtJR71

___________________-____I___-----”---------------100 90.1 85.6 : RffiT34 : 100 83.1 : mvcRs : 100 i TYORll : : SEAM : : PRRRs6 :

REAM0

PRM6

86.0 83.5 %.4 100

04.7 E;

. R2.t to0

a)

b)

Fig. 6. Nucleotide (a) and amino-acid (b) sequence homologies between H7 haemagglutinin genes and polypeptides of H7 viruses described in Fig. 3 and the prototype equine H7 influenza virus-PRAG54.

viruses isolated from chickens (Porter et al., 1979; Nestorowicz et al., 19871, turkeys (Orlich et al., 19901, seals (Naeve and Webster, 1984) and of the equine H7 strains, PRAG56 and DETR64, are compared in Fig. 3. Fig. 6 tabulates the nucleotide and amino acid sequence identities among these viruses. The overall sequence homology, shown in Fig. 6, of U-85% amino acid identity and 74-78% nucleotide identity between the other H7 viruses and the prototype equine strain (PRAG56) suggests that the HA of equine H7 viruses represent a unique class of HA and does not show any close relationship to any of the avian strains sequenced to date. This is unlike the haemagglutinin of the seal influenza virus, in which the haemagglutinin gene is closely related to an avian influenza virus of North American origin. A unique feature of all ten equine H7 viruses compared to all other influenza viruses is an insertion of 9 amino acids at the carboxyl terminus of HA,, close to the HA,/HA, cleavage site. At the cleavage site equine H7 HAS have four basic amino acid residues, comparable with the pathogenic avian H7 strains (ROST34, CHVCS5, and others (McCauley, 1987)) and with the pathogenic H5 avian strains (Kawaoka et al., 1987; Kawaoka and Webster, 1988). The insertion of nine amino acids at the cleavage site is analogous to one described by Khatchikian et al. (1989) where following extensive passage of a low-pathogenici~ avian H7 virus in tissue culture in the absence of trypsin, a virus was isolated which contained an insertion of 18 amino acids at the HAr/HA, cleavage site. This insertion resulted from a recombination event in which a 54 nucleotide tract from 285 ribosomal RNA was inserted into the HA gene (Khatchikian et al., 1989; Orlich et al., 1990). As a result of this insertion haemagglutinin of this ‘recombinant’ virus became capable of cleavage activation by the endogenous processing proteases in the cell. As discussed above, a11ten of the equine H7 haemagglutinins we have examined are capable of cleavage activation in chick embryo fibroblast cultures (Fig. 51, but at this stage we cannot infer whether it is due to the four basic amino acids at the cleavage site (which are sufficient for cleavage of the HA in some H5 haemagglu-

104

tinins (Kawaoka and Webster, 1988)), to the insertion of additional amino acids preceding the cleavage site basic residues, or both. We have been unable to detect significant homology between the nucleotide sequence inserted into the equine segment 4 RNA and any entry in the nucleotide databases when searched in either sense. Neither has any homology been found between its predicted amino acid translation and sequences in the protein sequence databases. Conclusions

In the light of the results presented here, we submit that the haemagglutinin of H7 equine influenza viruses shows only limited variation, and that the HA gene of early (pre-1964) strains of virus are quite distinct from the more recent (1964 and subsequent) strains of virus. These conclusions conform with the proposal that the internal genes of 1957 equine virus strains were replaced by others prior to 1973, a deduction based on sequence analysis of the internal genes of a wide range of influenza viruses including two of the viruses studied here, PRAG56 and LOND73 (Gorman et al., 1990a,b; Okasaki et al., 1989). The equine H7 viruses seem unique amongst natural influenza virus isolates in the possession of an insertion at the haemagglutinin cleavage site. We further suggest that, should vaccination against H7 influenza viruses prove again necessary, a virus from the post-1964 group might prove a more effective vaccine than would A/equine/Prague/56, which is still in use by some vaccine manufacturers. Acknowledgements

This work was supported by the Racehorse and Betting Levy Board, through a studentship awarded to CAG and NIAID A120591 (R.S.D.). Some of the data derived from this work were presented at the Fifth International Conference on Equine Infectious Diseases, and are published in the proceedings, Equine Infectious Diseases V: proceedings of the Fifth International Conference, Ed. D.G. Powell, (19881, University of Kentucky Press, pp. 51-59. We thank Paul Thomas for critical reading of the manuscript. References Beveridge, W.I.B. (1965) Some topical comments on influenza in horses. Vet. Rec. 77, 427. Bulletin of WHO (1987) Molecular epidemiology of influenza viruses: memorandum from WHO. Bull. WHO, 65, 161-165. Burrows, R. (1978) Equine influenza viruses: field and experimental observations of infection and vaccination. Proc. Annu. Gonv. Am. Assoc. Equine Pratt. 24, 37-48. Burrows, R., Denyer, M., Goodridge, D. and Hamilton, F. (1981) Field and laboratory studies of equine influenza viruses isolated in 1979. Vet. Rec. 109, 353-356. Burrows, R. and Denyer, M. (1982) Antigenic properties of some equine influenza viruses. Arch. Virol. 73, 15-24.

105 Caton, A.J., Brownlee, G.G., Yewdell, J.W. and Gerhard, W. (1982) The antigenic structure of the influenza virus A/PR/8/34 hemagglutinin (Hl subtype) Cell 31, 417-427. Cullen, R.G. (1989) Equine influenza in Ireland. Bull. Off. Int. Epiz. 101, 700. Daniels, R.S., Douglas, A.R., Gonsalves-Scamo, F., Palu, G., Skehel, J.J., Brown, E., Knossow, M., Wilson, I.A. and Wiley, D.C. (1983) Antigenic structure of influenza virus haemagglutinin. In: The Origin of Pandemic Influenza Viruses. Laver, W.G. (Ed.). Elsevier, Amsterdam, pp. 9-18. Daniels, R.S., Skehel, J.J. and Wiley, DC. (1985) Amino acid sequences of haemagglutinins of influenza viruses of the H3 subtype isolated from horses. J. Gen. Virol. 66, 457-464. Deshpande, K.L., Fried, V.A., Ando, M. and Webster, R.G. (1987) Glycosylation affects cleavage of an H5N2 influenza virus hemagglutinin and regulates virulence. Proc. Natl. Acad. Sci. U.S.A. 84, 36-40. Devereux, J., Haeberli, P. and Smithies, 0. (1984) Nucleic Acids Res. 12, 387-395. Doll, E.R. (1961) Influenza of horses. Am. Rev. Resp. Dis. 83, 48-53. Gorman, O.T., Bean, W.J., Kawoaka, Y. and Webster, R.G. (1990a) Evolution of the nucleoprotein gene of influenza A virus. J. Virol. 64, 1487-1497. Gorman, O.T., Donis, R.O., Kawoaka, Y. and Webster, R.G. (199Ob) Evolution of influenza A virus PB2 genes: implications for evolution of the ribonucleoprotein complex and origin of human influenza A virus. J. Viral. 64, 4863-4902. Halterman, L.G. and McQueen, J.L. (1968) A serological study of equine influenza. JAVMA 153, 1069-1073. Hay, A.J., Lomniczi, B., Bellamy, A.R. and Skehel, J.J. (1977) Transcription of the influenza virus genome. Virology 83,337-355. Hinshaw, VS., Naeve, C.W., Webster, R.G., Douglas, A., Skehel, J.J. and Bryans, J. (1983) Analysis of antigenic variation in equine 2 influenza viruses. Bull. WHO. 61, 153-158. Katz, J.M., Naeve, C.W. and Webster, R.G. (1987) Host cell-mediated variation in H3N2 influenza viruses. Virology 156, 386-395. Katz, J.M. and Webster, R.G. (1988) Antigenic and structural characterization of multiple subpopulations of H3N2 influenza virus from an individual. Virology 165, 446-456. Kawoaka, Y. and Webster, R.G. (1988) Sequence requirements for cleavage activation of influenza virus hemagglutinin expressed in mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 85, 324-328. Kawoaka, Y., Nestorowicz, A., Alexander, D.J. and Webster, R.G. (1987) Molecular analyses of the hemagglutinin genes of H5 influenza viruses: origin of a virulent turkey strain. Virology 158, 218-227. Kawoaka, Y., Bean, W.J. and Webster, R.G. (1989) Evolution of the hemagglutinin of equine H3 influenza viruses. Virology 169, 283-292. Keil, W., Geyer, R., Dabrowski, J., Dabrowski, U., Niemann, H., Stirm, S. and Klenk, H.-D. (1985) Carbohydrates of influenza virus. Structural elucidation of the individual glycans of the FPV hemagglutinin by two-dimensional ‘H NMR and methylation analysis. EMBO J. 4, 2711-2720. Khatchikian, D., Orlich, M. and Rott, R. (1989) Increased viral pathogenicity after insertion of a 28s ribosomal RNA sequence into the haemagglutinin gene of an influenza virus. Nature 340, 156-157. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of bacteriophage T4. Nature 227, 680-685. Mahaffey, L.W. (1964) Equine influenza, coughing epidemics are due to different types of influenza virus. Br. Racehorse Oct., 470-472. McCauley, J.W. (1987) Influenza virus glycoproteins and their role in disease. In W.C. Russell and J.W. Almond (Eds.) Molecular Basis of Virus Disease, Symp. Sot. Gen. Microbial. 40, 21-50. McCauley, J.W. and Penn, C.R. (1990) The critical cut-off temperature of avian influenza viruses. Virus Res. 17, 191-198. Meldrum, K.C. (1989) Equine influenza in Great Britain. Bull. Off. Int. Epiz. 101, 700. Naeve, C.W. and Webster, R.G. (1983) Sequence of the haemagglutinin gene from influenza virus A/seal/Mass/i/80. Virology 129, 298-308. Nestorowicz, A., Kawaoka, Y., Bean, W.J. and Webster, R.G. (1987) Molecular analysis of the hemagglutinin genes of Australian H7N7 influenza viruses: role of passerine birds in maintenance of transmission? Virology 160, 411-418. Okazaki, K., Kawaoka, Y. and Webster, R.G. (1989) Evolutionary pathways of the PA gene of influenza A viruses. Virology 172, 601-608.

106

Orlich, M., Khatchikan, D., Teigler, A. and Rott, R. (1990) Structural variation occurring in the hemagglutinin of influenza virus A/Turkey/Oregon/71 during adaptation to different cell types. Virology 176, 531-538. Pearson, W.R. and Lipman, D.J. (1988) Improved tools for biological sequence comparison. Proc. Natl. Acad. Sci. U.S.A. 85, 2444-2448. Porter, A.G., Barber, C., Carey, N.H., Hallewell, R.A., Threlfall, G. and Emtage, J.S. (1979) Complete nucleotide sequence of an influenza virus haemagglutinin gene from cloned DNA. Nature 282, 471-477. Powell, D.G., Thompson, G.R., Spooner, P., Plowright, W., Burrows, R. and Schild, G.C. (1974) The outbreak of influenza in England, April/May 1973. Vet. Rec. 94, 282-287. Raymond, F.L., Caton, A.J., Cox, N.J., Kendal, A.P. and Brownlee, G.G. (19831 Antigenicity and evolution amongst recent influenza viruses of the HlNl subtype. Nucl. Acids Res. 11, 7191-7203. Raymond, F.L., Caton, A.J., Cox, N.J., Kendal, A.P. and Brownlee, G.G. (1986) The antigenicity and evolution of influenza Hl haemagglutinin from 1950-1956 and 1977-1985; two pathways from one gene. Virology 148, 275-287. Robertson, J.S., Naeve, C.W., Webster, R.G., Bootman, J.S., Newman, R. and Schild, G.C. (1985) Alterations in the hemagglutinin associated with the adaptation of influenza B virus to growth in eggs. Virology 143, 166-174. Robertson, J.S., Bootman, J.S., Newman, R., Oxford, J.S., Daniels, R.S., Webster, R.G. and Schild, G.C. (19871 Structural changes in the haemagglutinin which accompany egg adaptation of an influenza A (HlNl) virus. Virology 160, 31-37. Rojahn, L. (1989) Equine influenza in the Federal Republic of Germany. Bull. Off. Int. Epiz. 101, 701. Rose, M.A., Hopes, R., Rossdale, P.D. and Beveridge, W.I.B. (1974) Virus infections of horses at Newmarket, 1972 and 1973. Vet. Rec. 95, 484-488. Sanger, F., Nicklen, S. and Coulsen, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proc. Nat. Acad. Sci. U.S.A. 74, 5463-5467. Schild, G.C., Oxford, J.S., de Jong, J.C. and Webster, R.G. (1983) Evidence for host-cell selection of influenza virus antigenic variants. Nature 303, 706-709. Sovinova, O., Tumova, B., Pouska, F. and Nemec, J. (1958) Isolation of a virus causing respiratory diseases in horses. Acta Virol. 1, 52-61. Tumova, B. (1980) Equine influenza - a segment in influenza virus ecology. Camp. Immun. Microbial. Infect. Dis. 3, 45-59. Waddell, G.H. Teigland, M.B. and Sigel, M.M. (1963) A new influenza virus associated with equine respiratory disease. JAVMA 143, 587. Webster, R.G. and Guo Y. (1991) New influenza virus in horses. Nature 351, 527. Webster, R.G., Laver, W.G., Air, G.M. and Schild, G.C. (1982) Molecular mechanisms of variation in influenza viruses. Nature 296, 115-121. Wiley, D.C. and Skehel, J.J. (1987) The structure and function of the hemagglutinin membrane glycoprotein of influenza virus. Annu. Rev. Biochem. 56, 365-394. Wiley, D. C, Wilson, I.A., and Skehel, J.J. (1981) Structural identification of the antibody-binding sites of Hong-Kong influenza haemagglutinin and their involvement in antigenic variation. Nature 289, 373-378. Wilson, I.A., Skehel, J.J. and Wiley, DC. (1981) Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3A resolution. Nature 289, 366-373. Wilson, J.C., Bryans, J.T., Doll, E.R. and Tudor, L. (1965) Isolation of a newly identified equine respiratory virus. Cornell Vet. 55, 42.5-431.

Note added in proof

It has recently been shown (Banbura et al. Virology 184, 469-471) that virus reassortants which contain the haemagglutinin gene from A/ Equine/ London/ 1416/73 in an avian influenza background were lethal for chickens.