Department of Virology, Faculty of Veterinary Medicine, University of Liège, Bd de Colonster 20, Bat B43b, B-4000 Liège, Belgium 1
Author for correspondence: Etienne Thiry.Fax +32 4 366 42 61. e-mail Etienne.Thiry{at}ulg.ac.be
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Abstract |
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Introduction |
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The genomes of BHV-5 and BHV-1 have the typical arrangement of herpesvirus group D genomes, consisting of a linear double-stranded DNA molecule of about 140 kb subdivided into a unique long segment (U L, approx. 105 kb) and a short segment (US, approx. 10 kb) flanked by internal and terminal repeats (IRs and TRs, approx. 11 kb each). At the genomic level, BHV-5 and BHV-1 exhibit 85% homology (Engels et al., 1986 ). However, both species can be differentiated by restriction enzyme analysis of genomic DNA, by type-specific antibodies or by type- specific hybridization probes (Metzler et al., 1986
; Engels et al., 1986
; Friedli & Metzler, 1987
). The BHV-1 genome sequence revealed ten genes that could encode glycoproteins (Schwyzer & Ackermann, 1996
); these were characterized and designated by homology with herpes simplex virus type 1 (HSV-1) gB (Whitbeck et al., 1988
) as follows: gC (Fitzpatrick et al., 1988
); gD (Tikoo et al., 1990
); gH (Baranowski et al., 1995
); gG (Keil et al., 1996
); gE (Rijsewijk et al., 1992
); gI (Rijsewijk et al. , 1995
); gL (Khattar et al., 1996
); gK (Khadr et al., 1996
); and gM (Wu et al. , 1998
). Up to now, only three BHV-5 genes, encoding glycoproteins gC (Chowdhury, 1995
), gD (Abdelmagid et al., 1995
) and gG (Engelhardt & Keil, 1996
), have been identified and their products characterized, gC being the most divergent between BHV-5 and BHV-1 (Engels et al., 1986
; Collins et al., 1993
; Chowdhury, 1995
). However, it might be assumed that BHV-5 encodes a similar number of glycoproteins as BHV-1.
The BHV-1 gH gene has been sequenced (Meyer et al., 1991 ) and BHV-1 gH has been identified and described as a 108 kDa structural component of the virus, expressed as a beta- gamma protein (Baranowski et al., 1995
). BHV-1 gH forms a complex with glycoprotein gL, which is necessary for the proper processing and transport of gH but not gL (Khattar et al., 1996
). In addition, BHV-1 gHgL complex is also required for induction of neutralizing antibody response and anchoring of gL to the plasma membrane (Khattar et al., 1996
). Characterization of BHV-1, pseudorabies virus (PrV) and HSV-1 gH null mutants has indicated that gH is an essential viral protein involved in both fusion between virion and cellular membranes during virus entry and in cell-to-cell spread of the virus (Forrester et al., 1992
; Peeters et al., 1992
; van Drunen Littel-van den Hurk et al., 1996
; Meyer et al. , 1998
). In addition, the absence of gH prevented penetration and propagation of PrV in the nervous system of adult mice after intranasal inoculation, suggesting that transneuronal spread in vivo and direct cell-to-cell spread in cell culture are governed by similar mechanisms (Babic et al., 1996
). In a similar way, gH is a target for studying the comparative neuropathogenicity of BHV-5 and BHV-1.
The present study reports the identification and characterization of the BHV-5 gH gene and gene products. The BHV-5 gH gene was sequenced and its mRNA characterized. The antigenic structure and the biochemical properties of BHV-5 gH were analysed and compared to those of BHV-1, in order to further elucidate their respective roles in the neuropathogenicity of these viruses.
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Methods |
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MAbs.
MAbs directed against BHV-1 gH (6, 8, 10, 14, 18, 20, 23, 25, 60, 61, 64, 67, 71, 83, 92, 95, 123, 126, 129, 136 and 153) were derived previously (Baranowski et al., 1993 ). Among them, MAbs 60, 61, 83, 92, 95, 126 and 153 were shown to possess neutralizing activity (Baranowski et al., 1993
). MAb 2915 directed against BHV-5 glycoprotein gC was kindly provided by M. Engels (Faculty of Veterinary Medicine, University of Zürich, Switzerland).
Plasmid construction.
All cloning procedures were performed using standard methods (Maniatis et al., 1982 ) and all nucleotide positions of the BHV-1 genome refer to the sequence available under EMBL accession number AJ004801. The location of the gH gene on the BHV- 5 genome and the pertinent restriction sites for cloning the BHV-5 gH gene in plasmids gH-KpnI, pBEK and pLeft are illustrated in Fig. 1
. By collinearity with the BHV-1 sequence, the BHV-5 gH gene should be contained in the 6·9 kb G fragment of the BHV-5 KpnI restriction map (Engels et al., 1986
). The presence of the BHV- 5 gH gene in this fragment was firstly confirmed by Southern blot hybridization with a BHV-1 gH probe using the colorimetric DIG High Prime DNA Labelling and Detection Starter kit I (Boehringer Mannheim) (data not shown). This 6·9 kb KpnI fragment was consecutively purified after BHV-5 genomic DNA digestion with Kpn I and cloned in the KpnI-linearized pBluescript SK(+) vector to give plasmid gH-KpnI. Comparison of the sequenced extremities of gH-KpnI with the BHV-1 genomic DNA sequence showed that the plasmid contained the entire BHV-5 gH gene. The sub-cloning strategy was performed as shown in Fig. 1
.
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mRNA isolation.
mRNAs were isolated by cell lysis with guanidine thiocyanate, followed by oligo(dT)cellulose chromatography (QuickPrep mRNA purification kit; Pharmacia Biotech). RNA concentrations were determined by spectrophotometry.
To study the course of BHV-5 gH mRNA synthesis during BHV-5 replication, MDBK cell monolayers were infected with BHV-5 at an m.o.i. of 10 p.f.u. per cell. After virus adsorption for 1 h at 37 °C, MEM with 4% horse serum was added and cells were incubated at 37 °C. mRNAs were collected at hourly intervals from 0 to 8 h post-infection (p.i.). Metabolic inhibitors were also used as previously described (Seal et al., 1992 ). Briefly, cells were treated for 2 h prior to infection and 8 h p.i. in the presence of cycloheximide (100 µg/ml) to detect immediate-early transcripts or with phosphonoacetic acid (PAA; 400 µg/ml) to detect early transcripts. Late transcripts were classified by their appearance in the absence of metabolic inhibitors.
Northern blot analysis.
Gel electrophoresis (1% agarose) of mRNA was done under 6% formaldehyde as described previously (Maniatis et al., 1982 ). Northern blot hybridization was performed on mRNAs transferred to pre-wetted nylon membranes (Boehringer Mannheim) by vacuum pressure (70 mbar, 5x SSC, 2 h). Filters with immobilized mRNAs (30 min at 120 °C) were hybridized overnight in 50% formamide buffer at 42 °C, either with specific BHV-5 gH or thymidine kinase (TK) DNA probes. All subsequent steps were performed according to the instructions of the chemiluminescent DIG High Prime DNA Labelling and Detection Starter kit II (Boehringer Mannheim). The BHV-5 gH probe was obtained by digestion of pBEK plasmid with Bam HI and SphI to generate an 852 nt fragment which was digoxigenin-labelled with a random-primed DNA labelling kit (High Prime; Boehringer Mannheim). The BHV-5 TK probe (0·5 kb) was generated using the same method by digestion of gH-KpnI plasmid with BamHI and PpuMI restriction endonucleases selected from the BHV-5 TK sequence (Smith et al., 1991
). Sizes of mRNAs were verified by using digoxigenin- labelled RNA molecular mass markers with a ladder between 1·6 and 7·4 kb (Boehringer Mannheim).
Neutralization tests.
Two-fold dilutions of ascitic fluids starting at 1 mg/ml immunoglobulins were mixed (1:1) with 100 p.f.u. BHV-1 or BHV-5 and incubated for 2 h at 37 °C. Each dilution was added in duplicate to confluent MDBK cells grown in 24-well plates and incubated for 2 h at 37 °C. Then plates were overlaid with MEM containing carboxymethylcellulose to a final concentration of 0·6%. Neutralizing activity was measured as the reciprocal of the 50% plaque-reducing end-point.
Immunoprecipitation and SDSPAGE.
Preparation of BHV-5 radiolabelled cell lysates was performed as described previously (Baranowski et al., 1993 ). Briefly, an MDBK monolayer was overlaid with methionine-free medium 2 h before infection with BHV-5 (m.o.i. of 10). At 2 h p.i., 20 µCi [35S]methionine per ml medium was added to the cultures.
The expression of glycoprotein gH was studied during each step of BHV-5 protein synthesis using cycloheximide and actinomycin D for alpha protein labelling and PAA for beta protein labelling (Baranowski et al., 1995 ). In this method, infected cells were harvested at 8 h p.i.
Pulsechase experiments were performed to study the course of glycoprotein gH synthesis during BHV-5 replication in MDBK cells. Proteins were radiolabelled at hourly intervals from 0 to 7 h p.i. Cells were first incubated in methionine-free MEM for 30 min before 1 h labelling with 100 µCi/ml Trans35S- label (ICN Flow) and then harvested after 30 min incubation with unlabelled methionine. Protein radiolabelling in the presence of glycosylation inhibitors was described by van Drunen Littel-van den Hurk & Babiuk (1985) . Infected cells treated with tunicamycin and monensin were harvested at 8 h p.i. Precipitates digested by endoglycosidase F/N-glycosidase F (endo-F; Boehringer Mannheim) and endoglycosidase H (endo-H; Boehringer Mannheim) were obtained from BHV-5-infected cells at 20 h p.i. The immunoprecipitation method (RIP assay) was previously described by Baranowski et al. (1995)
.
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Results |
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Nucleotide comparisons between the overall BHV-5 and corresponding BHV-1 SphI/SalI fragments revealed 84·6% homology. When comparisons were performed between BHV-5 and BHV-1 gH ORFs, the gaps comprise essentially single or multiple triplets except for two BHV-5-specific segments located at nt 732756 and 19231953 on the BHV-5 sequence. The highest divergence between BHV-5 and BHV-1 (49% homology) was found in the last 170 nt of the SphI/SalI fragment located downstream from the BHV-5 gH poly(A) signal. In BHV-5, this short sequence contains an A+T-rich region characterized by 14 repetitions of the TAT sequence which was not found in BHV-1. In addition, high G+C repeat fragments followed this A+T-rich region.
BHV-5 gH amino acid sequence analysis and comparison
The BHV-5 gH ORF (nt 2402787) encodes a predicted protein of 849 aa, with a deduced molecular mass of 89·01 kDa. The only start codon of the gH ORF (nt 240) is contained in a consensus sequence of translation initiation (A/GNNATGG; Kozak, 1986 ) with one substitution at +4 reading 5' AGGATGC 3'. The BHV- 5 gH protein has several features that are characteristic of a membrane protein. Based on empirical rules for predicting signal sequences (von Heinje, 1986
), aa 123 have the position, length, hydrophobicity and consensus cleavage site characteristic of a signal sequence. ChouFasman analyses (Chou & Fasman, 1978
) predicted a ß-turn structure preceding the respective glycine residue (position 23), which is characteristic before or after a signal peptidase recognition site (Kreil, 1981
). Thus, after cleavage of the proposed signal sequence, the non-glycosylated gH should comprise 826 aa and exhibit a molecular mass of 86·8 kDa. Similarly, one hydrophobic region in an
-helical conformation near the C terminus of the protein from position 810830 may serve as a transmembrane anchor sequence leading to a small cytoplasmic domain of only 19 aa. The proposed BHV-5 gH ORF surface domain contains six potential N-glycosylation sites and 11 cysteine residues (10 of them located in the extracellular domain) which are completely conserved in the BHV-1 gH sequence. However, the secondary structure predictions calculated by the ChouFasman method indicate that the BHV-5 and BHV-1 amino acid backbones may be folded differently, specifically between aa 150350.
Comparisons with the published sequence of the BHV-1 Cooper strain (Meyer et al., 1991 ) indicated that the BHV-5 gH ORF is 7 aa longer with a molecular mass of 89·01 kDa compared to 88·37 kDa for BHV-1 gH. The BHV-5 and BHV-1 gH amino acid sequences showed 86·4% identity and 87·7% similarity. Alignment of the two sequences revealed that the third C- terminal part of the protein is well conserved (Fig. 2
) except for one segment of 9 aa (563573) which is completely different between the two viruses. Sequence alignment showed that this fragment is not conserved among herpesviruses. The most divergence was found in the N-terminal region, consisting essentially of single or double amino acid substitutions except for one specific region located at aa 166188 (Fig. 2
). This divergent domain is characterized by an addition of 8 aa for BHV-5 which are not present in the BHV-1 sequence. The first amino acids (166 and 167) of this domain are responsible for two hydrophilic peaks separated by one hydrophobic fragment of 3 aa in BHV- 5 compared to one broad hydrophilic peak for BHV-1.
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Northern blot analysis
Northern blot analysis with a BHV-5 gH probe revealed two transcripts of 3·03·1 and 4·34·4 kb in mRNAs isolated 2 h p.i. which were always present 8 h p.i. (Fig. 3 ). At 2 and 3 h p.i., the 3·03·1 and 4·34·4 kb transcripts exhibited roughly the same intensity; at later times, the 3·03·1 kb transcript was much more prominent (Fig. 3
). The two transcripts were detected after synthesis in the presence of PAA (Fig. 4
). In contrast, no transcripts were isolated from infected cells in the presence of cycloheximide. Upon PAA treatment, a smear due to partial mRNA degradation was observed for the two transcripts which migrate faster than without treatment (Fig. 4
). In addition, hybridization of BHV-5 mRNA blots with the BHV-5 TK-specific probe only recognized the 4·34·4 kb mRNA (Fig. 4
).
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In addition, radiolabelled cells were infected by BHV-5 in the presence of tunicamycin or monensin (Fig. 5a). No bands were detected from cells treated with tunicamycin using either the five most reactive BHV-1 MAbs or the seven additional MAbs which were able to detect BHV-5 gH by RIP assay. According to Baranowski et al. (1995)
, only MAbs 10 and 67 recognize gH from tunicamycin-treated BHV-1 infected cells. In this study, gH was not recognized by MAb 10. MAb 67 only immunoprecipitated BHV-5 gH without tunicamycin treatment. The efficacy of tunicamycin was checked by using MAb 2915 raised against BHV-5 glycoprotein gC, a gamma-2 protein. BHV-5 gC was detected as an 87 kDa protein which shifted to a 64 kDa band after tunicamycin treatment. In the presence of monensin, all selected MAbs were able to precipitate only the 102 kDa band when infected cells were harvested at 8 h p.i. (Fig. 5a
). Moreover, after endo-H digestion of BHV-5 infected cells treated with monensin, a molecular mass shift from 102 kDa to approximately 92 kDa was observed (Fig. 5c
).
The expression of glycoprotein gH was studied during each step of BHV-5 protein synthesis. Glycoprotein gH was not immunoprecipitated from infected cells treated with actinomycin D and cycloheximide suggesting that this protein is not expressed during expression of alpha protein (Fig. 6). The treatment of BHV-5 infected cells with PAA did not influence immunoprecipitation of gH. Consequently, the gH synthesis is not dependent on DNA replication. The efficacy of PAA was checked by using MAb 2915 raised against BHV-5 glycoprotein gC, a gamma-2 protein. After PAA treatment, the 87 kDa band corresponding to gC was not detected.
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Discussion |
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Up to now, only three BHV-5 genes encoding glycoproteins gC (Chowdhury, 1995 ), gD (Abdelmagid et al., 1995
) and gG (Engelhardt & Keil, 1996
) have been identified and their products characterized. Amino acid comparisons showed that gH is the most conserved between BHV-5 and BHV-1 (86·4% identity). The primary structure of the putative transmembrane anchor domain is highly conserved, as are the positions of all cysteine residues and glycosylation sites found in BHV-5 gH. However, the secondary structure prediction indicated that BHV-5 and BHV-1 gH may be folded differently between aa 150350. In addition, two different domains located at aa 166174 and 563573 of the BHV-5 sequence clearly differentiate BHV-5 and BHV- 1 gH amino acid sequences. The first divergent domain, located in the divergent secondary structure predictive region, is characterized by a sequence of 8 aa for BHV-5, which is not present in the BHV-1 sequence. Comparisons of amino acid sequences of BHV-5 gH over this region with those of other alphaherpesviruses suggest that the 8 aa lacking in BHV- 1 gH are the result of a deletion during evolution. Considering this point, the BHV-5 gH glycoprotein, in contrast to BHV-1 gH, is clearly similar to that of other mammalian alphaherpesviruses.
Nucleotide analysis revealed one specific BHV-5 domain located just after the polyadenylation signal of the gH transcript. This BHV-5 sequence contains an A+T-rich region which was directly followed by high G+C repeat fragments. It was previously reported that this region, between UL21 and UL22 (gH) of PrV, EHV-1 and EHV-4, encompasses an origin of replication containing repeated sequences around a A+T-rich stretch of DNA (Klupp & Mettenleiter, 1991 ; Nicolson et al., 1990
; Baumann et al., 1989
). Alphaherpesvirus origins are characterized by the presence of a 9 nt conserved sequence element CGTTCGCAC, proximal to the A+T- rich region. No consensus BHV-5 origin sequence could be found after the gH gene despite the fact that this region contains A+T-rich sequences. However, since repeat elements in other herpesvirus genomes are known to be unstable when cloned, sequencing needs to be performed on BHV-5 genomic DNA to determine the possible location of an origin of replication between UL21 and UL22.
The BHV-5 gH ORF is contained within two early-late transcripts of 3·03·1 and 4·34·4 kb detected from 2 h p.i. Sequence analysis revealed no polyadenylation signal after the TK stop codon. Based on these observations, it was suggested that the 3·03·1 kb transcript constitutes the gH mRNA and that the 4·34·4 kb mRNA most likely represents a structurally bicistronic transcript which encompasses the TK ORF and terminates at the gH polyadenylation signal. Confirmation was obtained by hybridization of BHV-5 mRNA blots with a BHV-5 TK probe which only recognized the 4·34·4 kb mRNA. Similar results were obtained for PrV, BHV-1 and MDV/HVT (Klupp & Mettenleiter, 1991 ; Bello et al., 1992
; Scott et al., 1993
) but not for HSV-1, VZV, EHV- 1 or EHV-4 (Davison & Scott, 1986
; McGeoch & Davison, 1986
; Nicolson et al., 1990
; Telford et al., 1992
), suggesting distinct transcriptional regulation in these alphaherpesviruses.
By RIP assay, MAbs directed against BHV-1 gH detected specifically a 108 kDa glycoprotein in both virus particles and infected cells. This glycoprotein was then designated BHV-5 gH. The expression of BHV-5 gH during each step of protein synthesis showed that BHV-5 gH could be classified as a beta-gamma regulated protein. In the pulsechase experiments, BHV-1 (Jura strain) and BHV-5 (N569 strain) gH were immunoprecipitated as early as 3 h p.i. and were still detected 7 h p.i. Previous studies (van Drunen Littel-van den Hurk et al., 1986 ; Baranowski et al., 1995
) detected BHV-1 Cooper strain gH at 2 h p.i. This difference in time could be due to the sensitivity of the RIP assay. Nevertheless, the results are quite similar due to the fact that samples were collected at hourly intervals. Moreover, the expression of gH starting at 3 h p.i. correlated with the expression kinetics of BHV-5 gH transcripts which were detected as early as 2 h p.i.
The acquisition of resistance to endo-H corresponds to the intracellular transport time required for the high mannose glycosylated peptides to move from the rough endoplasmic reticulum to elements of the Golgi, where processing reactions convert them to endo-H-resistant complex chains (Kornfeld & Kornfeld, 1985 ; Tarentino et al., 1989
). In the present study, radiolabelled BHV-5 gH was resistant to endo-H but an apparent molecular mass shift from 108 to 92 kDa was observed after endo-F digestion. This molecular mass reduction with endo-F is consistent with all six potential N -linked glycosylation sites being used, assuming 3000 Da for each site.
Interestingly, when BHV-5 gH was detected at 8 h p.i. or pulse- labelled between 38 h p.i., a second specific band of 102 kDa was recognized by MAbs using RIP assay. This band showed the same electrophoretic mobility as BHV-5 gH expressed under monensin treatment. Moreover, BHV-5 gH expressed under monensin treatment appeared to be sensitive to endo-F and endo-H digestions with a molecular mass shift from 102 kDa to 92 kDa. These results suggest that BHV-5 gH is processed in MDBK cells by N- glycosylation from an endo-H-sensitive precursor of 102 kDa to an endo-H-resistant form of 108 kDa, containing complex and hybrid type oligosaccharides. Similar results were described for BHV-1 gH in Georgia bovine kidney or MDBK cells (van Drunen Littel-van den Hurk & Babiuk, 1986 ; Baranowski et al., 1995
). The MAbs used in this study were unable to precipitate BHV-1 or BHV-5 gH when radiolabelling was performed in the presence of tunicamycin. This suggests that the presence of N-linked precursors on BHV-1 and BHV-5 gH is crucial for epitope conformation and recognition by all of the MAbs used, although processing in the Golgi apparatus is dispensable. Another possibility would be that gH without carbohydrate is unstable and is consequently degraded when cell extracts are made.
Complex formation between glycoproteins gH and gL has been described in several alphaherpesviruses, including BHV-1 (Khattar et al. , 1996 ). This association is essential for gH transport, glycosylation and epitope formation (Khattar et al., 1996
). The BHV-1 MAbs used in this study were previously shown to be unable to detect a BHV-1 gHgL complex (Baranowski et al., 1995
). Similar results were obtained with a potential BHV-5 gHgL complex because no band corresponding to hypothetical BHV-5 gL could be immunoprecipitated with the BHV-1 gH MAbs using either [35S]methionine, [35S]cysteine, [3H]leucine and/or [3H]glucosamine (data not shown).
Differences in the antigenic structure of the major glycoproteins gB, gC and gD of BHV-1 and BHV-5 were previously demonstrated with a panel of MAbs prepared against BHV-1 or BHV-5 glycoproteins (Metzler et al., 1986 ; Friedli & Metzler, 1987
; Collins et al., 1993
). Of the three glycoproteins, gC showed the main antigenic differences between the two viruses, while gB and gD are closely related antigenically. In this study, we report differences in the antigenic structure of gH between BHV-1 and BHV-5. A panel of 21 MAbs directed against BHV-1 gH (Baranowski et al., 1993
) were tested for reactivity to lysates of BHV-5- infected cells. Twelve out of the 21 MAbs were able to specifically recognize BHV-5 gH by RIP assay, indicating the same antigenic relationship to BHV-1 as for gB and gD. The differences observed in the viral glycoproteins involve several biological functions. Glycoprotein gC has a predominant role in attachment of BHV-1 to cells (Okazaki et al., 1991
, 1994
; Liang et al., 1991
), gB and gD participate in attachment and penetration (Liang et al., 1991
), while gH only plays a role in the penetration of virus into target cells (Meyer et al., 1998
). In addition, gH is involved in the neuroinvasiveness of PrV (Babic et al., 1996
). Some of the functional epitopes on these BHV-1 glycoproteins are absent from those of BHV-5. For BHV-1 gH, at least two antigenic domains were shown to be involved in penetration (Baranowski, 1996
). From our virus neutralization data, one of these domains, binding with MAbs 61, 92 and 95, differed between BHV- 1 and BHV-5. MAbs 61 and 92 did not immunoprecipitate BHV-5 gH suggesting that amino acid differences within the epitopes could prevent DNA binding. MAb 95 binds to BHV-1 and BHV-5. Binding of MAb 95 to BHV-1 gH interferes with its function, resulting in virus neutralization. In contrast, due to amino acid differences, binding of MAb 95 to BHV-5 gH may not interfere with its function. These antigenic differences between BHV-5 and BHV-1 gH, as well as sequence data comparisons which essentially focused on divergence between BHV-5 and BHV-1 for one amino acid domain located between aa 150250, may possibly relate to in vivo differences in virus tropism.
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Acknowledgments |
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Footnotes |
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References |
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Received 10 May 1999;
accepted 26 July 1999.