1 Jerome L. and Dawn Greene Infectious Disease Laboratory, Mailman School of Public Health, Columbia University, New York, NY 10032, USA
2 Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK
Correspondence
Thomas Briese
thomas.briese{at}columbia.edu
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AY380581.
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MAIN TEXT |
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The International Committee on Taxonomy of Viruses considers Guaroa virus to be a species distinct from the species California encephalitis virus (CEV) and Bunyamwera virus (BUNV) within the genus Orthobunyavirus of the family Bunyaviridae (http://www.ncbi.nlm.nih.gov/ICTVdb/Ictv/index.htm); some investigators have suggested that GROV should not be included in either the California serogroup (CAL) or the Bunyamwera serogroup (BUN) (Whitman & Shope, 1962; Calisher & Maness, 1970
; Wellings et al., 1971
; Hunt & Calisher, 1979
; Klimas et al., 1981
). Serological assays have shown some link of GROV to both serogroups. In complement fixation (CF) assays, serological cross-reactivity was observed with BUN, but not CAL, members. In contrast, in haemagglutinationinhibition (HI) and neutralization (NT) assays, cross-reactivity was evident with CAL, but not BUN, members (Groot et al., 1959
; Casals & Whitman, 1960
; Whitman & Shope, 1962
; Tauraso, 1969
). Results similar to those of CF assays were obtained in immunodiffusion, showing no cross-reactivity between GROV and CAL viruses, but weak cross-reaction with BUNV and Tensaw virus (Calisher & Maness, 1970
; Wellings et al., 1970
). Immunoelectrophoresis, however, indicated common determinants between GROV and CAL viruses (Wellings et al., 1971
). Reaction in CF assays is determined by N, whereas reaction in NT/HI assays is determined by the glycoproteins (Lindsey et al., 1977
; Gentsch et al., 1980
; González-Scarano et al., 1982
; Kingsford & Hill, 1983
; Ludwig et al., 1991
). Discordant serological reaction may therefore indicate different phylogenetic relationships for GROV N (S segment) and the glycoproteins (M segment). S-segment sequencing suggested a closer relationship to BUN than to CAL viruses; it has thus been hypothesized that GROV may be a reassortant virus (Dunn et al., 1994
). Here, we report the GROV M-segment sequence and its analysis in comparison to other M-segment sequences.
GROV RNA was reverse-transcribed by using Superscript II (Invitrogen) and amplified by PCR (Saiki et al., 1985) using primers (1·6 µM; Table 1
), dNTPs (200 µM), MgCl2 (Table 1
) and BIO-X-ACT polymerase (Bioline) in a PTC-200 thermocycler (MJ Research) for 45 cycles of 1 min at 92 °C, 1 min at 4553 °C and 12·5 min at 68 °C (Table 1
). Products were cloned and sequenced (Sanger et al., 1977
); analysis using the Wisconsin GCG package (Accelrys) indicated one ORF of 4254 nt (1418 aa) for the assembled sequence (GenBank accession no. AY380581).
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The N-terminal portion of the next protein (nt 14454273; 108 kDa) is surprisingly divergent from other GC proteins. The C-terminal moiety, beginning about 150 aa after a conserved potential trypsin cleavage site (tttt, Fig. 1; Fazakerley et al., 1988
), is again conserved when compared to other M-segment sequences. The 3' non-coding region shows little conservation and is longer than those of the other M segments.
Five potential glycosylation sites were identified (+++; Fig. 1), including an N-terminal site in GC that is conserved among gC sequences of the cal viruses and is found in approximately the same position in grov (n492); an n-terminal glycosylation site that is conserved in gC sequences of sequenced BUN viruses was not found in the majority of GROV clones analysed. Among 10 clones, six carried AATGACAtA for N616DI (---; Fig. 1
), whereas four carried AATGACAcA, encoding the potential glycosylation site N616DT.
Analysis by TopPred2 (http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html; Claros & von Heijne, 1994) predicts six major transmembrane regions. Two are predicted for GN: the signal peptide (aa 626) and a second region that is compatible with a stop-transfer signal/membrane anchor (aa 210230), which would result in a cytoplasmic location for a strictly conserved downstream KTY motif, a stretch of mostly hydrophobic residues that includes two proline residues, and the protease cleavage motif. Three transmembrane regions are predicted for NSm (aa 315335, 368388 and 455475); the first, located 10 aa downstream of the GN C-terminus, is compatible with a third internal signal sequence, with cleavage predicted after G337 by SignalP. Signalase cleavage close to a cytoplasmic protease site may resemble the proposed situation at the C/prM cleavage site of flaviviruses (Stocks & Lobigs, 1998
; Amberg & Rice, 1999
). One transmembrane region (aa 13721392), a potential membrane anchor (Fazakerley et al., 1988
; Pekosz et al., 1995
), is predicted for GC. The overall topology appears to be well-conserved, as indicated by conservation of the same cysteine residues as in the polyproteins of all other BUN and CAL viruses (Fig. 1
) (Lees et al., 1986
; Grady et al., 1987
; Pardigon et al., 1988
). Conservation of sequence motifs with respect to CAL but not BUN virus sequences is noted for K149, Q163 and P299 in GN, P347 and N455/F456 in NSm, and H591QH, G601EKCNSA607, E958, K1036, G1243 and K1411/K1412 in GC.
Pairwise, sliding-window distance analysis (SimPlot; http://sray.med.som.jhmi.edu/RaySoft/SimPlot/; Lole et al., 1999) between GROV and BUN and CAL viruses indicated an almost equidistant position of GROV, with lowest distance scores in the GN region (approx. position 1300; Fig. 2a
) and highest scores obtained in the NSm sequence (approx. position 300500) and in the N-terminal portion of GC (approx. position 5001400). Serogroup-specific differences appear to be most pronounced in three regions (approx. positions 100200, 550650 and 12001275), where a separation between sequences of BUNV, CVV and GERV and CEV, Melao virus (MELV) and Trivittatus virus (TVTV) is observed. GROV appears to be less distant from BUNV/GERV/CVV than from CEV/MELV/TVTV in all regions except for the second part of the second region (at the N-terminus of GC toward N616), where CEV/MELV/TVTV are less distant from GROV than BUNV/GERV; however, CVV remains the most closely related sequence throughout. In a reconstructed phylogenetic tree, the GROV M-segment sequence is placed in a closer relationship to sequences of BUN than of CAL viruses (62 % bootstrap support; Fig. 2b
). Phylogenetic relationships of each individual ORF are similar to that of the entire sequence (data not shown).
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The structural determinants of GROV's unique serological reaction pattern are obscure. GN is not a major target of neutralizing antibodies that interfere with infection of mammalian cells (Ludwig et al., 1989; Cheng et al., 2000
) and the few amino acids that are conserved in GN between grov and cal viruses do not correlate with identified epitopes of gN (/; Fig. 1
; Cheng et al., 2000
). Therefore, GN is unlikely to form major determinants of the reaction of GROV in NT/HI assays. Likewise, NSm is unlikely to be involved. Epitopes detected in NT/HI assays have been mapped to the N-terminal portion of GC, mainly in relation to the trypsin site of LACV/SSHV (González-Scarano et al., 1982
; Kingsford et al., 1983
; Najjar et al., 1985
; Kingsford & Boucquey, 1990
). However, their relation to primary amino acid sequence is not defined and only in one case has a particular amino acid that is involved in neutralization been identified (residue 29 of LACV GC; #, Fig. 1
; Bupp & González-Scarano, 1998
). The divergence of this region in comparison to available BUN virus sequences may explain the lack of cross-reaction between GROV and these viruses in NT/HI assays. Although also divergent when compared to sequences of CAL viruses, this region does contain motifs that are conserved with respect to CAL, but not BUN, viruses (H591QH and G601EKCNSA607) and two glycosylation sites. N492, which is conserved amongst CAL but not BUN virus sequences, is present, whereas N616, which is conserved amongst BUN but not CAL virus sequences, is only present in a minority of GROV clones. N492 flanks the first putative antigenic domain that was proposed by Brockus & Grimstad (2001)
and both conserved amino acid motifs and N616 are located in their second putative antigenic domain. It is conceivable that these positions contribute to the serological reaction of GROV. The mutation at the glycosylation site N616DI/T is intriguing, given that M-segment sequence has been associated with plaque size (Iroegbu & Pringle, 1981
) and the observation that GROV can generate both large and small plaque morphologies, of which only the small variant elicited antibodies that were cross-reactive with CEV and TAHV (Tauraso, 1969
).
In summary, our analysis of the GROV M-segment sequence indicates a relative phylogenetic relationship that is comparable to that reported for the GROV S-segment sequence (Fig. 2b; Dunn et al., 1994
), and does not provide evidence for genome-segment reassortment. Instead, in a sequence that is almost equidistant to published CAL and BUN virus sequences, isolated determinants in the N-terminal portion of GC were identified that potentially relate to the unique serological reaction pattern of GROV and are more compatible with GROV forming a bridge between both serogroups, as originally proposed by Whitman & Shope (1962)
.
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ACKNOWLEDGEMENTS |
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Received 20 March 2004;
accepted 21 June 2004.