clonal antibodies (MAbs) and eDNA probes. They showed ... (Wellemans & Leunen, 1975) and treated with actino- mycin D ..... The same conclusion could be ...
Journal of General Virology (1990), 71, 3009-3014.
3009
Printed in Great Britain
Sequence comparison between the fusion protein of human and bovine respiratory syncytial viruses Karl Walravens, 1 Richard Kettmann, 2 Alfred Collard, 3 Philippe Coppe 3. and Ars6ne Btlrny 2 1Laboratoire de Microbiologie et d'Immunologie, Facult~ Universitaire Notre Dame de la Paix, 5000 Namur, 2Laboratoire de Biologic molbculaire et de Physiologie animale, Facult~ des Sciences Agronomiques, 5800 Gembloux, 3Division d'Immunologie, Centre d'Economie Rural, rue du Carmel 1, 5406 Marloie, Belgium
The nucleotide sequence was determined for the fusion (F) protein-coding mRNA of the bovine respiratory syncytial virus (strain RB 94) and the amino acid sequence of the F protein was deduced for comparison with the sequence of human respiratory syncytial virus
subtypes A and B (RSS-2 and 18537 strains). The human and bovine RS virus F proteins (excluding the cleaved signal peptide) share 83 to 84 % homology. The greatest divergence occurred within the F2 subunit in the region preceding the cleavage activation site.
Human and bovine respiratory syncytial (RS) viruses belong to the genus Pneumovirus of the Paramyxovirus family. They are important causes of lower respiratory tract disease in infants and young calves (Stott & Taylor, 1985). Recently, several analyses on the molecular organization of the bovine RS viruses have been performed using human and bovine antisera, monoclonal antibodies (MAbs) and eDNA probes. They showed that the protein compositions of human and bovine RS viruses are very similar, with only minor differences in Mr between corresponding proteins (Cash et al., 1977; Lerch et al., 1989; Trudel et al., 1989). Electrophoretic mobilities of mRNAs of human and bovine RS viruses are comparable (Lerch et al., 1989). Human and bovine RS virus strains are also antigenically related:the fusion (F), nucleocapsid (N), matrix (M) and phospho- (P) proteins showed antigenic crossreactivity whereas the bovine RS viral G glycoprotein has major antigenic differences from its human virus counterpart (Taylor et al., 1984; Orvell et al., 1987; Lerch et al., 1989). The genome of RS virus encodes 10 genes including those for two major glycoproteins, G and F. The G glycoprotein, which has been associated with viral attachment (Levine et al., 1987) has an apparent size of approximately 90K on SDS-PAGE (Lerch et al., 1989). The F glycoprotein, which is the fusion protein (Walsh & Hruska, 1983), is synthesized as a 70K precursor molecule (F0) which is proteolytically cleaved into disulphide-linked 48K (FI) and 23K (F2) subunits (Walsh et al., 1985). By analogy to Sendai virus (Scheid & Choppin, 1974), this cleavage is a prerequisite for fusion activity and is carried out by cellular protease (s).
Two distinct antigenic subgroups of human RS virus (A and B) have been recognized (Anderson et al., 1985; Mufson et al., 1985; Gimenez et al., 1986; Orvell et al., 1987). The comparison of the F protein sequence between these two human subtypes shows 89% overall homology (Johnson & Collins, 1988), consistent with the previously described high degree of antigenic relatedness. Information derived from electrophoretic analysis, immunoprecipitation and immunoblotting, have clearly established that the F proteins of both human and bovine RS virus strains present comparable structural characteristics (Kennedy et al., 1988; Trudel et al., 1989; Lerch et al., 1989) and antigenic relatedness (Taylor et al., 1984; 0rvell et al., 1987; Lerch et al., 1989). To provide a molecular basis for these results, we present here the nucleotide and deduced amino acid sequences for the F protein of bovine RS virus strain RB 94 and compare them with human RS virus F sequence data (A and B subtypes). For this purpose, total RNA was extracted from Vero cells infected with the RB 94 strain of bovine RS virus (Wellemans & Leunen, 1975) and treated with actinomycin D (Pringle, 1985). The mRNA purified on an oligo(dT)-cellulose column had been used as a template to establish a library of cDNA clones in the phage 2gtl 1. Based on protein homologies existing between human and bovine strains of RS virus, a human RS virus Fspecific cDNA probe was used to identify by plaque hybridization the corresponding cDNA sequences of our bovine RSV strain. More specifically, we used the PstIPstI restriction fragment (586 bp) corresponding to nucleotides 62 to 648 of the human RS virus Long strain F cDNA (Lopez et al., 1988) as a 32p-labelled probe.
0000-9764 © 1990 SGM
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3010
A~G GOG ACA ACA Met Ala q1~r Thr Glu Leu Leu Glu Leu Leu
1
GCC Ala Ile Ile
ATG AGG Met hr~ Leu Lys llis
ATG b½t Thr Set
ACC TAT GTG lle lle Set lle lie Pile lle Set q'hr q'~r Val Asn Ala Ile Thr Ala Ile Leu Ala Ala Val Thr Set Ala Ile Phe leu Thr Leu Ala val Ash Ala A'I~C ATC AGC ATC Aq~ ' I T C ATC T c r
ACA ~*r Leu Leu
CAT His Cys Tyr
A'IU ACr TI'A TGC CAA AAC ATA RB94
Ile Tilt leu Cys Gln Ash I]e Phe Ala Ser Set RSS-2 Leu Se~ Set ]8537
RB9.4 ACA GAA GAA TFF TAT CAA ~CA ACA ~ C AGP C~A ~]'I' A(~r AGA (3Gr TAC cFr AGr (3CA TI'A AGA ACT (]CA q~3G TAT AC~ A~P ~ ---~-]Glu G]u Phe Tyr G|n Set q~r Cys Set A|a Val Set Arg Gly q~,r [~eu Ser Aid Leu Arg Thr Gly Trp ~ r Thr Ser Val ESS-2 Lys 18537 • Phe
85 29
|
AAC G~G AAA TrA ATA AAG CAA GAA CTA GAA RB94 Ash Val Lys Leu lie Lys Gln Glu Leu G]u Lys Asp RSS-2 Lys Asp 18537
GTA ACA ATA GAG ~ AGC AAA ATA CAA AAA AAT Val Thr Ile Glu Leu Ser Lys Ile Gln Lys Asn Ile Asn Lys Glu Ile ASh Lys Glu Thr
253 85
AGA TAC AAC AAT GCA GTA Arg Tyr Ash Ash Ala Va ~ Lys Lys Ser Lys Lys
337 113
GAG Glu Arg Gin
421 141
~ CTA ~ ATF GC_.~q-UP (121' ATe' ~ A~r GG~ G'PA ~ (21~.;~L~C ~ (IPA CTA CAC ~ GAG ~ GAG ~ K~T ~ AT? ~ ~94 leu Leu GIv lie G|y Set A l a ~ l e Ala Ser ~1¥ Val Ala Va) Set Lys Val i~u iijs l~u Glu Gly G|u Val Ash Lys lle Lys Val -- '" lle RSS-2 Val lle 18537
505 169
AAT GC~ Ci~ CTA TCC ACA AAT AAA GCA GrA GT~ AC~F CTA qL~C AAT GGA GST A ~ GPC CPA ACT AGC AAA GTA C]'~ CAT CTA AAG RB94 Asn Ala Leu Leu Set Thr Ash Lys Ala Val Val Set Leu Set ASh Gly val Set Val Leu Thr Set Lys Val Leu Asp Leu Lys Ser RSS- 2 ]8537
589 197
AAC TAT ATA CAC AAA GAG CTT CFA C ~ AAA GY]TM AAC AAT CAT CAT T~I' CAG ATA TCC AAC ATA GCA ACT ~rG ATA C~A T]~ CAA RB94 ASh qyr Ile Asp Lys G]u Leu Leu Pro Lys Val ASh Ash ]}is Asp Cys Gln lle Ser Asn lle Ala Thr val lle Glu Phe Girl Gin Ile l,ys Gin Ser Ser Glu RSS-2 Asr| Ash Arg lie Gin G|n Ser • Arg G]u 18537
673 225
CAA AAA AAC AAT AGA T~3 TI~ GAA A'I'{~ AGG GAA TIT AGT GTA AAT Gcr GGT ATT AOC ACA Cf~C C]~ A ~ ACA TAC AT(] TI~ RB94 Gln Lys Asn ASh Arg Leu leu Glu lie Ala Arg Glu ?he Set Val ASn A]a Gly Ile ~ir ~lr Pro Leu Set ~ir Tyr Met Leu Thr Val Va[ RSS-2 Met Set T~r Va[ 18537
757 253
AOC AAT A~T ~ TI~A CTA ~CA CTA ATr AAT CAT ATG CCT ATA AOG AAT CAC CAA AAA AAG C['A AT(: TCA A~r AAT GTT CAA ATA RB94 Thr Ash Set Glu Leu Leu Set Leu Ile ASh Asp Met Pro lie Thr Asn Asp Gln Lys Lys Leu Met Set Ser Asn Val Gln Ile Ash RSS-2 18537
84] 281
GTC AGG CAA CAG A ~ TAT TCC AT? ATG TCA GI~ Val Arg Gln Gln Set Tyr Ser Ile Met Set Val lle lie
925 309
ATA GAC AOC COC ]~GT TGG /~:~ CTA CAC A(]C TCg OCA TPA %~C AOC Acr CAT AAT AAA GAA GGG TCA AAC ATC TGC TrA ACT AGG RB94 lle Asp Thr Pro Cys Trp Lys Leu His Thr Set Pro Leu Cys ]1|r Thr Asp Ash Lys Glu G]y Ser Ash Ile Cys Leu ~1*r Arg RSS-2 Ash Tier lle • 18537
TI~ Ieu Phe Tyr
ATA CAT TAT lle {{is Tyr ~etlAsn MetlAsn
C,YA T(/r AAC AGr AC]' CAT Val Cys Ash Ser q%rlAs p Lys G]y Lys • GIy
qUA Ser A|a Thr
]69 57
I
¢-fG GAA T[U CAG q~?A CI'P A'L~ ~ AAT GAA (3~G G(3C TCC TCC Val Glu Leu Gln S t Leu Met Gln Asn G]u Pro Ala Set Set Thr Leu Ser Thr Thr Asn ~r Leu Thr Ala Asn
AAA AGA AAC Lys Arg Ash ThrlLeu I Thr I lle I
~ ACA AAA Set ThrlLys Ash Thr I
AAG Lys Ash Ash
TIT Phe Thr Leu
Af/l'AGA GCA AAA AGA GGG ATA (3CA RB94 Set Arg Ala Lys Arg Gly Ile Pro Asn Arg Glu Leu RSS-2 Ash Arg Glu Ala ]8537
TAT GGG C]'A ATG GGC AAA AAG AGA AAA AGG ACA TIT T?A GGA T~C RB94 ~fr Gly ThrlLeu[eU ~et Gly Lys Lys Arg Lys Arg A r g ~ Asn Val Set RSS-2 As,l Val Serl lle Ser e ]8537
GI~ AAA GAA GAG ~ ATG Gcr TAT GTr GTA CAA TI~A OCT AT? TAT GGA GIT RB94 Val Lys G|u Glu Val Met Ala Tyr Val Val Gin Leu Pro I]e Tyr Gly VaI Leu RSS-2 lle Leu 18537 lle Leu
]009 ACA CAT CGr ¢ ~ G TAT ~ r c~:r m T ~Z~ ~ ~nr ¢ I ~ I v r 1 ' r r T r c c c r CAG GCA GAG ACA ~ F AAA &TA CAA TCA AAT AGA RB94 337 Thr Asp Arg Gly Trp Tyr Cys Asp Ash A]a Gly Set Val Set Phe Pile Pro Gln Ala Glu Thr Cys Lys Va[ Gill Ser ASh Arg l~u PSS-2 Asp • ]8537 CCT ArT 1093 GTG T~C TGT CAC ACA ATG AAC AGT TrA ACT ~ 365 Val Phe Cys Asp Thr Met Asrl Set Ieu Thr Leu Pro Thr Ser Ser
CAT GFr AAC TTA TGC AAC ACT GAC ATA Trc AAT GCA AAG TAT CAC RB94 Asp Val Ash Leu Cys ASh T~]r Asp Ile Phe Asn A]a Lys Tyr Asp Ile Pro RSS-2 Glu • Ser 18537 Glu Set
1177 ~ AAA ATA ATG ACA ~ P AAA AC|' C~%C ATA AGT AGC q~F GTA ATA AC? ~CA ATA GGA c c r ATF GI'A TCA TGC TAT GGG AAG ACA RB94 393 Cys Lys lle Met q1~r Ser l,ys Thr Asp lie Ser Se[ Set VoI lie q~ir Set I|e Gly Ala Ile V~l Set Cys Tyr Gly Lys ~llr l~u RSS-2 Val Leu • ] 8537 ACA GOC TCT AAT AAA AAT C~F GGA ATC ATA AAG ACT TIT TCC AAT OGG 'Ib~PGAT TAT 6"rA TCA AAC AGA (r~ 6"IT CAT RB94 Lys Cys Thr Ala Set Ash Lys Ash Arg Gly lle Ile Lys Thr Phe Set Ash Gly Cys Asp Tyr Val Set Asn Arg Gly Val Asp Asp Lys RSS-2 Lys 18537
1261 AAA ~
421
]345 ACT GI'A TCY GTr GGT AAC ACA CTA TAT TAT GTA A,NP bAG CI'A GAG GGG AAA GCA C ~ TAT ATA AAG ~ CAA CCA ATT AT? AAT RB94 449 Thr Val Set Val Gly Ash Thr Leu 'l~r 5~r Val Ash Lys Ieu G]u Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro I]e Ile ASh Gin Ser Vat RSS-2 Asn Val 18537 ]429 TAC TAT GAT CCA CTA GI'A TIP CCT 5cr CAT GAG TiT GAP GCA TCA AqT CCC CAA GrA AAC GCA AAA ATA AAC CAA AGC CrG Gcr RB94 477 Tyr Tyr Asp Pr~ Leu Val Phe Pro Set Asp Glu Phe Asp Ala Ser lie Ala Gin Val Ash A|a I,ys lleiAsn Gin SerlLeu Ala Phe Set Glu l I RSS-2 Set Glu 18537
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1513 ~ ATA (3GT OGA TCT CAT GAG qTA CTF CAC AG'F GI'A CAT G~A GGA AAA T(3C ACC ACA AAT G£A GTA ATP ACT ACT ATr ATC ATA RB94 505 Phe Ile Arg Arg Set Asp Glu I~u [mu His Set Val Asp Val G]y Lys Set Thr Thr Asn Val Va3 lie Thr Thr lle lie lle Lys Ash Ash A]a lle Met RSS-2 ASI! ASll Thr I l e Met 18537 1597 GTG ATA GTA GTA ~ 533 Val lle VaI Val Val lle I|e Ile
ATA q'PA Aq~3 T~A ATA GCr GFA GC.~ TFA CI~ TIT TAC AC,V AAG ACC AGG AG~ ACT fL-T ATC ATG TI'A GGA RB94 Ile Leu Met Leu Ile Ala Val GI~ leu leu Phe T~r Set Lys Thr Arg Set Thr Pro Ile Met Leu Gly Leu Set Ieu Cys Ala Val Thr Set RSS-2 Leu Ser Ile Leu Cys Ala Lys Asn Val Thr Set 18537
1681 AAG GAT CAG 6TF AGF GGT A~U AAC AAT CIT 561 Lys Asp Gln Leu Set G[y Ile ASh Asn Leu Ile Ile
TCC TIT AGT AAA TGA AAT (]CA TAA ~ T Ser Phe Set Lys *** Ala Asn *** Ala ***
TrA CAA TCT AAA 06T AAG AAT CAT AAA RB94 RSS-2 18537
1765 qL~T CAT GAG CTA AAT TFA CTA ATA CAT TCA AAA G'IT CFA q~C GOC AAG ACC TGC AIT TI'I' ATC AGG TCT TAC ATA AGC TAA CCT RB94 1849 TAC A~3 CTA CAC qFA GCT CCA qb~lI TAA TAG 'ITA TAT R1394
Fig. 1. Nucleotideand deducedamino acid sequencesof the cDNA clone RB 94 F-11 correspondingto the bovine RS virus (Strain RB 94) F protein, comparedto that of the F protein of the human RS virus subtypesA and B (strain RSS-2,Baybutt& Pringle, 1987;strain 18537, Johnson & Collins, 1988).The amino acid changes in the two human RS virus proteinsare indicated belowthe protein sequence of the RB 94 F protein. The arrow locates the activation cleavage site between F2 and F1. The presumptive signal peptide and membrane anchoragedomain are underlined.The hydrophobicsequenceat the N terminusofF1 is denoted by a doubleline. Potential glycosylationsites are boxed, and cysteine residues common to the three proteins are indicated by solid triangles. Numbers at the beginning of each line refer to nucleotide or amino acid positions in the RB 94 F gene or protein.
Among positive plaques identified, some recombinant phages were likely to contain a complete copy of the F m R N A as the size of their inserts (1.9 kb) corresponded to the full-length copy of the F c D N A clones of human RS virus (Collins et al., i984). The larger clone called RB 94 F-11 was chosen for nucleotide sequencing by the dideoxynucleotide chain termination method (Sanger et al., 1977). The nucleotide sequence of the c D N A clone RB 94 F11 orientated 5' to 3' and its deduced amino acid sequence are represented in Fig. 1. The c D N A nucleotide sequence begins with the 5' A T G initiation signal of translation and ends in the consensus sequence A G T T A T A T 3' at position 1885. The F mRNA-coding sequence of strain RB 94 has 7 5 ~ identity with both human RS virus strains RSS-2 (subtype A strain; Baybutt & Pringle, 1987) and 18537 (subtype B strain; Johnson & Collins, 1988) F m R N A sequences (Table 1). As shown for the comparison of nucleotide sequences between human subtypes A and B (Johnson & Collins, 1988), we observed also that coding regions of the m R N A were more conserved than the 3' non-translated regions (data not shown). However, at the 3' end of it, the sequence A G T T A N A T 3' is perfectly conserved between the two human RS virus subtypes (Johnson & Collins, 1988) and our bovine strain (Fig. 1). The nucleotide sequence encoding the F protein was predicted to be an open reading frame (ORF) identical in length (nucleotides 1 to 1723), with the O R F described for the human RS virus strains RSS-2 and 18537. The F protein sequence of the three RS virus strains (human subtype A strain RSS-2, human subtype B strain 18537 and bovine strain RB 94) have been aligned in Fig. 1.
The amino acid sequences of these proteins showed 80 ~o overall homology between RB 94 and RSS-2 and 81 between RB 94 and 18537 (Fig. 1 and Table 1). This identity between the bovine and both human subtypes is less than that reported for the F proteins of subtypes A and B (90~o overall homology between the RSS-2 and 18537 strains; Table 1). This suggests that the bovine strains of RS virus constitute a well defined entity different from human strains as previously suggested on account of antigenic differences in the major glycoprotein G between human and bovine RS viruses (Taylor et al., 1984; Orvell etal., 1987; Lerch etal., 1989), and the unequal abilities of these strains to infect cells of different species in vitro (Paccaud & Jacquier, 1970; Matsumoto et al., 1974). The bovine strain RB 94 F1 subunit had 8 8 ~ and 8 9 ~ amino acid sequence identity with the human strains RSS-2 and 18537, whereas the F2 subunit homology was somewhat lower ( 6 7 ~ and 6 8 ~ ; Table 1, Fig. 1). This is also in agreement with the amino acid sequence comparison between human subtypes A and B (Johnson & Collins, 1988). It seems that a greater selective pressure (in terms of conservation of the fusion activity) was operative on the F1 region. Such a result was expected since one fusion-inhibiting epitope has been localized in the F1 subunit (Walsh et al., 1986). Moreover, two well defined neutralization epitopes, located in the F1 subunit around amino acid residues 221 (lie) to 232 (Glu) and 262 (Asn) to 268 (Asn) respectively are perfectly conserved between RSS-2 and RB 94 strains (Trudel et al., 1987; Lopez et al., 1990). The neutralization epitope described by Lopez et al. (1990) is also conserved in the human subtype B prototype strain
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Table 1. Percentage of nucleotide and amino acid identities between human R S virus subtype A strain RSS-2, human R S virus subtype B strain 18537 and bovine R S virus strain R B 94 RB 94/RSS-2
RB 94/18537
RSS-2/18537
Domain of the
fusion protein
NSH (%)*
AASH (%)t
NSH (%)
Signal peptide FI F2~ F1 + F2~ Overall
36 77 73 76 75§
4 88 67 83 80
39 77 71 76 75§
AASH (%)
NSH (%)
ASH (%)
12
61
48
89
84
93
68 84 81
81 83 82§
86 92 90
* NSH, Nucleotidesequence identity(%). t AASH, Aminoacid sequence identity(%). :~Exclusiveof the predicted signal peptide (mature protein). § Exclusiveof the 3' terminusnon-codingnucleotidesequence.
18537, but not the other epitope where two amino acid substitutions were found at positions 226 (Met for Lys) and 228 (Ser for Asn) (Johnson & Collins, 1988; Fig. 1). Nevertheless, these two regions of the F protein seem to have high biological importance because of the extensive conservation of neutralizing epitopes. In addition, Scopes et al. (1990) have described an epitope (amino acids 483 to 488) recognized by human sera with high titres of neutralizing antibodies. We observe here that this epitope is perfectly conserved between human and bovine RS viruses (Fig. 1). This result, in conjunction with the high level of conservation of the two neutralizing epitopes so far mapped on the F protein (Trudel et al., 1987; Lopez et al., 1990), is very important in view of tire development of a bovine RS virus vaccine. Fourteen cysteine residues were conserved on the F protein between the three strains compared here (Fig. 1), indicating the importance of these residues to the overall structure and function of the mature protein. One cysteine residue present in the RB 94 strain at position 25 but present neither in the RSS-2 strain nor in the 18537 strain, is presumed to be the cleavage site of the signal peptide (Austen, 1979) and is not present in the mature protein. Another cysteine residue is present in both RSS2 and 18537 strains at the carboxy terminus of the anchor region (amino acid 550) and is described as an attachment site for palmitate in human RS virus (Arumugham et al., 1989). This cysteine is replaced in the RB 94 strain by a serine residue which is also described as a potential acceptor site for palmitoylation (Schmidt & Lambrecht, 1985). Of the four potential acceptor sites of N-linked glycosylation presented in the F protein sequence of our bovine strain, three (at positions 27, 70 and 500) are well conserved in the F protein sequence of human subtypes A and B (Fig. 1). The fourth acceptor site is located at
position 120 in a highly variable domain (Fig. 1). Two Nglycosylation sites at positions 116 and 127 have been described in the F protein sequences of strains of human subtypes A and B; they are not observed in our sequence (Fig. 1). The fourth acceptor site, mentioned above, is present in RSS-2, Long, Edinburgh and 18537 human RS virus strains (Baybutt & Pringle, 1987; Lopez et al., 1988; Scopes et al., 1990; Johnson & Collins, 1988) but not in the A2 strain (Collins et al., 1984). This indicates that high variability in amino acid content and potential glycosylation sites in the carboxy terminus of the F2 subunit (residues 100 to 129) exists within human strains as well as between human and bovine RS viruses. The high hydrophilicity of that region in both the RSS-2 and RB 94 strains strongly suggests surface localization. This area might form strain-specific epitopes (human/bovine) as suggested by Johnson & Collins (1988) for human strains. Further studies with MAbs, polyclonal sera, F protein mutants, etc. should help verify this hypothesis. In contrast, the protease cleavage region (residues 130 to 136; Collins et al., 1984) is highly conserved. Therefore, it seems likely that this confined domain plays an important biological role as for the F protein of Newcastle disease virus (Le et al., 1988). The signal peptide (residues 1 to 25) which would be predicted to be cleaved at cysteine residue 25 (Austen, 1979) exhibited extensive sequence divergence between human and bovine RS virus strains (Table 1, Fig. 1), although its hydrophobic nature was maintained. Such variability in the signal peptide sequence of the F protein has been previously reported between human subtypes A and B (Table 1; Johnson & Collins, 1988). These observations suggest that signal functions depend on features of hydrophobicity and local secondary structures rather than on specific sequence. The same conclusion could be extended to the membrane anchor region (Fig. 1).
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The N terminus of the F1 subunit (residues 137 to 154) which is thought to have a direct role in membrane fusion is well conserved between bovine and human RS viruses as observed for other Paramyxoviridae (Morrison, 1988). Only two conservative amino acid substitutions (Val to Ile and lie to Val at positions 8 and 16 respectively; see Fig. 1) have been observed between both human subtype and RB 94 strains. Comparing the two major surface glycoprotein, G and F, the fusion protein was described as the major neutralization antigen (Olmsted et al., 1986; Johnson et al., 1987). In addition, the high degree of sequence conservation of the F proteins between both human subgroups and between human and bovine RS virus indicate that the F protein should be a good candidate for a human and also bovine subunit vaccine. The amino acid sequence of the F protein of the bovine RS virus strain RB 94 is of a particular interest for the correlation of genetic variation with changes in antigenic properties. In addition, the conjunction of sequence and MAb crossreaction data may serve to define protein domains of biological importance in order to understand the fusion mechanisms. We gratefully acknowledge Dr J. A. Melero (Centro National de Microbiologia, Virologia e Immunologia Sanitarias, Majadahonda, 28220 Madrid, Spain) for providing the plasmid SLF 88. R. K. and K. W. are respectively Maitre de recherches of the Fond National de la Recherche Scientifique and fellow of the Institut pour l'encouragement de la Recherche Scientifique dans l'Industrie et l'Agriculture.
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(Received 25 June 1990; Accepted 10 September 1990)
