Identification of naturally occurring monoclonal antibody escape ...
Journal o f General Virology (1994), 75, 609-614.
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Identification of naturally occurring monoclonal antibody escape variants of louping ill virus George F. Gao, t Mohammed H. Hussain, 2 Hugh W. Reid 2 and Ernest A. Gould 1. 1 hzstitute o f Virology and Environmental Microbiology, Mansfield Road, Oxford OX1 3 S R and 2 Moredun Research Institute, 408 Gilmerton Road, Edinburgh E H 1 7 7JH, U.K.
Louping ill virus isolates from Great Britain, Ireland and Norway were compared antigenically by indirect immunofluorescence, haemagglutination-inhibition and neutralization tests using a panel of five envelopespecific and five non-structural protein NSl-specific monoclonal antibodies raised against louping ill virus. The viruses were grouped according to their reactivities with the antibodies. Group 1, members of which were isolated between 1931 and 1987, consisted of 13 viruses that reacted with all antibodies, whereas group 2, members of which were isolated after 1980, consisted of five viruses that were positive with only eight of the 10 monoclonal antibodies. The two monoclonal antibodies that did not react with the group 2 viruses are known to be neutralizing antibodies and the amino acids that they recognize in the viral envelope protein have been
identified. We therefore refer to the group 2 viruses as naturally occurring monoclonal antibody escape variants. When compared with group 1 viruses, the escape variants showed reduced virulence for mice in terms of the time taken to kill and/or the proportion that died, following intraperitoneal inoculation. The nucleotide and deduced amino acid sequences of the envelope gene of one escape variant were compared with those of several group 1 viruses. A single amino acid substitution at residue 308 was detected in the envelope protein of the escape variant which corresponds precisely to the position in experimentally selected attenuated monoclonal antibody escape mutants. The importance and potential implications of these naturally occurring variants in louping ill epizootiology and vaccine-based control are discussed.
Introduction
which plays a major functional role in these viruses, contains approximately 500 amino acids. In order to understand the pathogenesis of viral infection at the molecular level, attempts have been made to identify and characterize viral genes that are required for virus virulence as assayed by the clinical manifestations and pathology of the disease. Although the basis of the encephalitic process has not yet been defined, experimentally derived MAb-resistant escape mutants have shown that single amino acid codon changes in the viral E protein can alter the mouse neurovirulence of Japanese encephalitis virus, TBE virus and LI virus (Holzmann et al., 1990; Cecilia & Gould, 1991; Hasegawa et al., 1992; Jiang et al., 1993). Similarly, single amino acid substitutions that affect the biological characteristics of other viruses have been reported (Parry et al., 1990; Strauss et al., 1991 ; Hern~indez et al., 1992; Woodward et al., 1991). There are no equivalent reports identifying the presence of naturally occurring attenuated MAb escape variants of TBE group viruses in the environment. LI virus was the first tick-borne flavivirus to be isolated (Pool et al., 1930). It is transmitted by the sheep tick Ixodes ri¢inus. This species is also the vector for the
Louping ill (LI) virus, a tick-transmitted member of the genus Flavivirus in the family Flaviviridae (Porterfield, 1980; Westaway et al., 1985; Francki et al., 1991), is an important pathogen for sheep and red grouse (Lagopus scoticus). LI virus occasionally infects humans. Comparative antigenic studies using polyclonal or monoclonal antibodies (MAbs) and genomic sequencing of several tick-borne flaviviruses have shown that LI virus is a distinct member in the tick-borne encephalitis (TBE) serocomplex (Stephenson et al., 1984; Gould et al., 1985a; Mandl et al., 1988, 1991; Pletnev et al., 1990; Shiu et al., 1991 ; Venugopal et al., 1992; Gritsun et al., 1993). In common with all other flaviviruses, LI virus contains a positive-stranded RNA genome which encodes three structural proteins (Shiu et al., 1991) and seven non-structural proteins (K. Venugopal, personal communication). The flavivirus envelope (E) protein,
The nucleotide sequence data reported in this paper have been submitted to the GenBank database and assigned the accession number X69975. 0001-1946 © 1994 SGM
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G. F. Gao and others
European subtype of TBE virus. So far, no strain diversity has been identified for LI or TBE virus (Heinz & Kunz, 1982; Reid, 1988; Holzmann et al., 1992). It has been reported that the E protein of LI viruses within and outside of the British Isles (Gao et at., 1993) as well as the Japanese isolate of Negishi virus (Venugopal et al., 1992) are closely related as shown by nucleotide sequence and antibody analyses. In this paper, we have extended this work by comparing 18 LI virus isolates obtained in the U.K. and Norway for their reactivities with E-specific and non-structural protein NSl-specific MAbs. Our results show evidence of naturally occurring MAbresistant variants in the environment with reduced virulence for mice.
Methods Virus isolates and MAbs. Eighteen LI virus isolates were studied in this report. Their geographical origins, the hosts from which they were isolated and the year of isolation are listed in Table 1. Strain L1/31, which is the prototype LI virus, was used for the preparation of monoclonal and polyclonal antibodies, the antigenic specificities of which have been described previously (Venugopal et al., 1992; Gao et al., 1993). Stocks of all viruses were prepared as 10 % (w/v) suspensions of infected suckling mouse brain in PBS pH 7-2. The MAbs were selected for their capacity to bind either E or NS1 proteins of LI virus by indirect immunofluorescence (IIF) assays on LI virus-infected cell monolayers. The MAbs have all been described previously (Venugopal et al., 1992) and are designated MAb 3.1, 3.3, 4.2, 7.1 and 7.2 for E-specific MAbs and MAb 1-2, 7.3, 7.4, 7.6 and 81 for NSl-specific MAbs. All five E-specific MAbs but none of the NSl-specific MAbs neutralize the infectivity of LI virus in plaque-reduction neutralization tests. Mab 7.1 but not the other nine MAbs also shows haemagglutination inhibition (HAI) activity with LI virus. Hyperimmune antiserum against LI virus strain LI/31 was prepared by inoculation of sheep with a methanol-precipitated virus antigencontaining adjuvant (Brotherston & Boyce, 1970).
Table 1. L I virus field isolates Virus designation
Geographical origin
Host of isolation
Year of isolation
LI/31 LI/369 LI/MAI4 LI/SB526 Ll/MA27 LI/MA54 L1/MR46 L1/G L1/K L1/I L1/A LI/NOR LI/917 LI/1065 LI/261 LI/2995 LI/2996 LI/1131
Scotland Scotland Ireland Scotland Ireland Ireland Ireland Scotland Scotland Wales England Norway England England England England England Scotland
Sheep Tick* Sheep Sheep Cattle Cattle Tick* Pig Grouse Sheep Sheep Sheep Sheep Sheep Sheep Grouse Grouse Sheep
1931 1963 1967 1968 1968 1968 1971 1979 1980 1980 1980 1984 1985 1985 1987 1987 1987 1988
* Ixodes ricinus.
Plaque neutralization tests. MAbs 4"2 and 7.1 were used for neutralization tests with each LI virus strain. The tests were carried out in Linbro 12-well flat-bottomed tissue culture plates using PS cell monolayers. Serial dilutions of antibody mixed with 100 p.f.u, of virus were incubated at 37 °C for 1 h and then added to the PS cells. After incubation at 37 °C for 1 h the monolayers were washed with medium containing 0.8 % agarose, incubated at 37 °C for 3 to 5 days and then stained using naphthalene black to reveal virus plaques. H F tests. PS cell monolayers were infected with LI virus at 37 °C for 48 h, by which time approximately 25 % of the cells contained virus antigens. Antibodies were added to acetone-fixed coverslip preparations of these infected monolayers at predetermined optimal dilutions for 1 h at 37 °C, the cells on the coverslips were washed in PBS and anti-mouse immunoglobulin conjugated with fluorescein isothiocyanate was added for 1 h at 37 °C. After washing in PBS the cells were mounted in 90% glycerol-saline and fluorescent viral antigen was observed by u.v. fluorescence microscopy (Gould et al., 1985b). Haemagglutination/HAl tests. Haemagglutinins of all virus isolates that were studied were prepared from the supernatant medium of infected BHK 89 cells and concentrated using 7 % (w/v) polyethelene glycol in 0.4 M-NaC1. The haemagglutination (HA) test was carried out at pH 6-2 using gander erythrocytes (Clarke & Casals. 1958). For HAI tests, the HA antigen was adjusted to contain 4 HA units per 0.2 ml using 0.4% BSA in borate-buffered saline. MAb 7.1, as ascitic fluid, and polyclonal antiserum, were used and the tests were carried out as described previously (Clarke & Casals, 1958). The HA1 titres were expressed as log10 reciprocal of the end-point dilution giving 50 % inhibition of HA. Passive protection o f mice with M A b 4"2. The potential therapeutic value of MAb 4.2 was evaluated using a dose of 100 lal of 1:2 dilution of ascitic fluid for each mouse. Twenty male Swiss mice aged 3 weeks were injected intraperitoneally with the diluted ascitic fluid at - 6 , 0, 14, 24, 48 or 96 h before or after challenge with virulent LI virus, strain LI/SB526. The mice were challenged subcutaneously with 100 ~tl containing 2 x 102 p.f.u, of virus. A control group of 20 mice was injected with PBS, 6 h before virus challenge. Mice were observed for up to 25 days. Nucleotide sequencing and analysis. Viral E gene sequencing of LI/A virus, chosen randomly from the five antibody escape variants, was carried out as described below. A 10 % (w/v) mouse brain suspension of the plaque-purified virus was used for the extraction of viral RNA as previously described (Gao et al., 1993). The virus suspension was treated with proteinase K and extracted with phenol and ether. Firststrand cDNA was synthesized using the conserved downstream primer 5' ctcgaattcGGTAGTATGCATAGTT 3', complementary to LI virus nucleotides 2449 to 2464 and the E gene was subsequently amplified by standard PCR using the upstream primer (5' cttagatctaatATGGTCGCCGTTGTGTGGCTA 3', nucleotides 781 to 801 of the L1 virus gene sequence) and the same downstream primer used in the synthesis of the first-strand eDNA. The PCR product was cloned into the pUCll8 vector (Gao et al., 1993) and sequenced in both directions by doublestranded dideoxynucleotide sequencing (Sanger et al., 1977). The sequencing data were compared with other published E gene sequences of the LI virus isolates LI/31, LI/369, LI/SB526, LI/K and LI/NOR (Shiu et al., 1991; Venugopal et al., 1992: Gao et al., 1993). The sequence identity calculations were performed using the CLUSTAL program and the University of Wisconsin Genetics Computer Group (GCG) packages. Mouse neurovirulenee comparison o f L I viruses. Groups of 10 female Swiss mice aged 3 weeks were used for virus neurovirulence comparisons. Approximately 1 x 103 p.f.u, of virus was inoculated
Louping ill virus escape variants
611
Table 2. Grouping of LI virus field isolates by IIF test MAb Virus
3.1
3.3
4.2
7.1
7.2
1.2
7.3
7.4
7.6
8.1
Polyclonal serum
Group
+ + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + --
+ + + + + + + + + + + + + -
+ + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + +
1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2
LI/31 LI/369 LI/MA14 LI/SB526 LI/MA27 LI/MA54 LI/MR46 LI/G LI/K LI/NOR LI/2995 LI/2996 LI/ll31 LI/I LI/A LI/917 LI/1065 LI/261
intraperitoneally into each mouse and the mice were observed for up to 16 days.
Results
Grouping and antigenic characterization of 18 LI virus field isolates The 18 LI virus strains were tested by IIF for their ability to bind each of the 10 MAbs defined in Methods. The results in Table 2 show that the viruses formed two groups. In group 1, 13 LI virus isolates were recognized by all 10 MAbs. These group 1 viruses were isolated between 1931 and 1987 from either Scotland, England, Ireland or Norway. The other five viruses (group 2) reacted with all the MAbs except MAb 4.2 and MAb 7.1. These five viruses were isolated after 1980 from either England or Wales. Experimentally produced MAb 4-2
Table 3. Passive protection of mice against L I virus infection using MAb 4.2 Time of injecting MAb 4.2
No. of survivors
(h)
challenged
- 6
20/20 18/19 17/20 9/16 14/20 12/18 7/18
0 + 14 + 24 +48 + 96
Control* * Injection with PBS.
/no.
Interpretation of Fisher's exact test Very significant Very significant Significant Not significant Not significant Not significant
escape mutants also failed to react with MAb 7"1 (Jiang et al., 1993), indicating that the epitopes recognized by these two MAbs are very closely associated. Further quantitative analysis of the antigenicities of each of the 18 viruses by neutralization, HA and HAI revealed no other significant antigenic divergence among the virus isolates. However, the five variants did produce smaller plaques than the non-variant viruses. This characteristic was also observed with the MAb-resistant mutants reported by Jiang et al. (1993).
Passive protection of mice us#Tg MAb 4"2 Mice inoculated intraperitoneally with MAb 4.2 were challenged with LI virus (strain LI/SB526) at different intervals either before or after antibody administration as described in Methods. The results show that MAb 4"2 protected mice against challenge with virulent LI virus (Table 3). Significant protection was achieved when the antibody was administered either before or up to 14 h after virus challenge but not at 24, 48 or 96 h postinfection. Similar protective and therapeutic effects were also observed when MAb 7"1 was analysed in the same manner (data not shown).
Analysis of the mouse neurovirulence of escape variants Two representative LI viruses (LI/31 and LI/MA54) and five naturally occurring variants that escape neutralization by MAb 4.2 (LI/917, LI/261, LI/1065, LI/A and LI/I) were compared in mouse neurovirulence assays as described in Methods. The results of a typical experiment (Fig. 1) show that all the variants were less virulent than
612
G. F. Gao and others
100
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.am.,-, - m , , . , .urn.,,,. n "
. m&'" sn.'
~::::~.... "~ 50
/'/ m" ±
±
Jr.
i
0
1 2
3 4
5
6
7
8 9 10 11 12 13 1'4 1'5 16
Fig. 1. Examination of the virulence for mice of LI/31 and LI/MA54 virus compared with five different escape variants. • . . . . i , LI/MA54; O, LI/31 ; C), LI/917; [~, LI/261 : • • , LI/1065; &, LI/A; /~, LI/I strains.
41 81 83
II
230238266 308
II
i
i
405 431 482 483
I
I[
:G,;~
Sites of all amino j acid changes in all sequenced LI viruses LI/369 LI/369 Escape mutant LI/A (Natural escape variant) LI/K
:~
,±
LI/NOR II
I
[
II
II I
I
I
LI/SB526
I
LI/31
Fig. 2. Comparison of the amino acid substitutions in the E genes of selected, sequenced strains and experimentally derived MAb escape mutants of LI virus. All strains are compared with LI/369 virus (Shiu et al., 1991). The positions of amino acid substitutions are indicated by vertical bars. The top box shows the positions of all known substitutions.
LI/31 and LI/MA54 viruses. LI/917 virus was the least attenuated but could be differentiated from LI/31 and LI/MA54 viruses by the longer time taken to cause fatal infection. Determination of the nucleotide and deduced amino acid sequence of the E gO,coprotein gene of L I / A virus LI/A virus was chosen as the representative strain of naturally occurring variants for nucleotide sequencing. A schematic representation of the E gene amino acid sequence of the LI/A virus aligned with other published sequences, including the experimentally selected MAb 4.2 escape mutant of LI/369 virus (Jiang et al., 1993), is shown in Fig. 2. Although four amino acid codon changes were seen in the E gene of LI/A virus compared with LI/369 virus, three of them occur in all of the sequenced E genes of LI virus isolates that are virulent
for mice. The fourth nucleotide change, corresponding to an amino acid substitution at position 308, resulted in a non-conservative amino acid change. This corresponded with the amino acid substitution seen in experimentally selected MAb 4.2 escape mutants (Jiang et al., 1993) and is therefore responsible for the failure of this variant to bind MAb 4.2. The substitution of aspartate to asparagine at amino acid residue 308 produced a potential glycosylation site in the experimentally derived escape mutants. However, the substitution of aspartate to glutamic acid in the naturally occurring escape mutant did not lead to either a potential glycosylation site or a change in charge.
Discussion Two neutralizing MAbs, which map at or near to amino acid position 308 in the LI virus E protein (Jiang et al., 1993), were able to identify five out of 18 LI virus field isolates that are naturally occurring MAb escape variants. Three other E-specific and five NSl-specific MAbs, on the other hand, recognized epitopes in all the virus strains, demonstrating the antigenic similarity and stability of these viruses. It is not yet known whether the occurrence of escape variants, with reduced virulence, in the environment has any significant effects on LI virus epidemiology but the frequency with which these naturally occurring variants arise seems very high. The possibility that they could be laboratory artefacts of the isolation procedure, as observed with influenza and mumps viruses, seems low since identical procedures were used to isolate the 13 non-variant viruses. The finding of attenuated LI virus variants may at least in part explain the apparent differences in severity of LI epidemics in some regions of the U.K. Epizootiological data appear to support this idea since in some epizootic areas the severity of LI disease may be on the decline (Reid, 1988). This concept is also supported by evidence from other viral diseases. Recently, Snyder et al. (1992) reported that naturally occurring escape variants could define the epizootiology of infectious bursal disease virus (IBDV) in the U.S.A. Live IBDV vaccine containing only standard IBDV strains is used in broiler flocks, but isolation of classic IBDV in the eastern U.S.A. occurred less frequently than in other areas in which standard IBDV strains were predominant. It is believed that the wide use of vaccine in the eastern U.S.A. is responsible for the predominance of IBDV escape variants. Moreover, the IBDV variants were isolated after 1985 and in our study all the LI virus escape variants were isolated after 1980. This could mean that the introduction of vaccine into an area may result in the selection of antibody-resistant variants with accompanying altered
Louping ill virus escape variants
virulence characteristics. Several different preparations of LI virus vaccine have been used since the 1930s, although their use has recently declined. By analogy with the periodic variation of influenza viruses, it will be interesting, in future studies of field isolates, to see whether other variants appear. On the other hand, there is evidence from studies with experimentally selected escape mutants of Sindbis virus and poliovirus (Stec et al., 1986; Ketterlinus et al., 1993), that reversion of mutants to wild-type can occur. Whether or not the appearance of antibody-resistant variants represents a temporary state in LI virus epizootiology has yet to be determined. It is interesting to note that all the antibodyresistant variants identified by us were from south of the Scottish border. It is not yet known if this observation has significance, but it could reflect either different vaccination strategies or different ecological pressures on the southern isolates. Nucleotide and amino acid sequencing of the E gene of one variant virus (LI/A) identified a mutation at amino acid position 308, responsible for substitution of aspartate by glutamate. This substitution site corresponds with that of experimentally selected antibody-resistant escape mutants derived using neutralizing MAb 4.2 and reported previously (Jiang et al., 1993). The reduced neurovirulence for mice of the naturally occurring variants, as compared with non-variant LI viruses, is also compatible with the results reported previously for MAb 4.2-derived escape mutants. Thus, the antigenic site containing amino acid 308 clearly has significance in determining the pathogenic characteristics of LI virus, at least for mice. The mouse neurovirulence assays with the derived escape variants showed that they were not all attenuated to the same extent and that this is probably due to different amino acid substitutions at or near to position 308 as noted previously (Jiang et al., 1993). Alternatively, substitutions in genes other than the E gene could contribute to the variable virulence characteristics. It is also of interest that the amino acid substitution sites for both experimentally selected mutants and the naturally occurring variants of LI virus are the same, but that different amino acids have been substituted. However, the substituted amino acids did not cause changes in charge, demonstrating that factors other than charge can influence the presentation of this epitope in the E protein. This contrasts with the evidence observed for Sindbis virus antibody escape mutants since in this case the charged nature of the substituted amino acid was important for interaction with antibody (Strauss et al., 1991). Future vaccine development strategies will need to take into account the effects of using antigenic preparations that might induce selective immunological pressures on LI virus.
613
The authors thank Mrs W. R. Jiang, Dr K. Venugopal and Dr T. S. Gritsun (NERC Institute of Virology and Environmental Microbiology) and H. T. Zhang (Institute of Molecular Medicine, Imperial Cancer Research Fund) for helpful discussions during preparation of this manuscript. G. F. Gao is a scholar supported by the Sino-British Friendship Scholarship Scheme of the British Council and the Chinese Government.
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occurring-neutralizing monoclonal antibody escape variants define the epidemiology of infectious bursal disease viruses in the United States. Archives of Virology 127, 89-101. STEC, D.S., WADDELL, A., SCHMALJOHN, C.S., COLE, G.A. & SCHMALJOHN, A.L. (1986). Antibody-selected variation and reversion in Sindbis virus neutralization epitopes. Journal of Virology 57, 715 720. STEPgENSON,J. R., LEE,J. M. & WILTON-SMITH,P. D. (1984). Antigenic variation among members of the tick-borne encephalitis complex. Journal of General Virology 65, 81-89. STRAUSS, E.G., STEC, D.S., SCHMALJOHN,A.L. & STRAUSS,J.H. (1991). Identification of antigenically important domains in the glycoproteins of Sindbis virus by analysis of antibody escape variants. Journal of Virology 65, 4654-4664. VENUGOPAL,K., BUCKLEY,A., REID, H. W. & GOULD, E. A. (1992). Nucleotide sequence of the envelope glycoprotein of Negishi virus shows very close homology to louping ill virus. Virology 190, 515-521. WESTAWAY,E. G., BRINTON,M. A., GAIDAMOVICH,S. YU., HORZINEK, M. C., IGARASHI,A., K)~.~RIAINEN,L., Lvov, D. K., PORTEREIELD, J. S., RUSSELL,P. K. & TRENT, D.W. (1985). Flaviviridae. bitervirology 24, 183-192. WOODWARD, Z.M., MILLER, B.R., BEATY, B.J., TRENT, D.W. & ROEHRIG, J.Z. (1991). A single amino acid change in the E2 glycoprotein of Venezuelan equine encephalitis virus affects replication and dissemination in Aedes aegypti mosquitoes. Journal of General Virology 72, 2431-2435.
(Received 7 July 1993; Accepted 1 November 1993)
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Identification of naturally occurring monoclonal antibody escape variants of louping ill virus George F. Gao, t Mohammed H. Hussain, 2 Hugh W. Reid 2 and Ernest A. Gould 1. 1 hzstitute o f Virology and Environmental Microbiology, Mansfield Road, Oxford OX1 3 S R and 2 Moredun Research Institute, 408 Gilmerton Road, Edinburgh E H 1 7 7JH, U.K.
Louping ill virus isolates from Great Britain, Ireland and Norway were compared antigenically by indirect immunofluorescence, haemagglutination-inhibition and neutralization tests using a panel of five envelopespecific and five non-structural protein NSl-specific monoclonal antibodies raised against louping ill virus. The viruses were grouped according to their reactivities with the antibodies. Group 1, members of which were isolated between 1931 and 1987, consisted of 13 viruses that reacted with all antibodies, whereas group 2, members of which were isolated after 1980, consisted of five viruses that were positive with only eight of the 10 monoclonal antibodies. The two monoclonal antibodies that did not react with the group 2 viruses are known to be neutralizing antibodies and the amino acids that they recognize in the viral envelope protein have been
identified. We therefore refer to the group 2 viruses as naturally occurring monoclonal antibody escape variants. When compared with group 1 viruses, the escape variants showed reduced virulence for mice in terms of the time taken to kill and/or the proportion that died, following intraperitoneal inoculation. The nucleotide and deduced amino acid sequences of the envelope gene of one escape variant were compared with those of several group 1 viruses. A single amino acid substitution at residue 308 was detected in the envelope protein of the escape variant which corresponds precisely to the position in experimentally selected attenuated monoclonal antibody escape mutants. The importance and potential implications of these naturally occurring variants in louping ill epizootiology and vaccine-based control are discussed.
Introduction
which plays a major functional role in these viruses, contains approximately 500 amino acids. In order to understand the pathogenesis of viral infection at the molecular level, attempts have been made to identify and characterize viral genes that are required for virus virulence as assayed by the clinical manifestations and pathology of the disease. Although the basis of the encephalitic process has not yet been defined, experimentally derived MAb-resistant escape mutants have shown that single amino acid codon changes in the viral E protein can alter the mouse neurovirulence of Japanese encephalitis virus, TBE virus and LI virus (Holzmann et al., 1990; Cecilia & Gould, 1991; Hasegawa et al., 1992; Jiang et al., 1993). Similarly, single amino acid substitutions that affect the biological characteristics of other viruses have been reported (Parry et al., 1990; Strauss et al., 1991 ; Hern~indez et al., 1992; Woodward et al., 1991). There are no equivalent reports identifying the presence of naturally occurring attenuated MAb escape variants of TBE group viruses in the environment. LI virus was the first tick-borne flavivirus to be isolated (Pool et al., 1930). It is transmitted by the sheep tick Ixodes ri¢inus. This species is also the vector for the
Louping ill (LI) virus, a tick-transmitted member of the genus Flavivirus in the family Flaviviridae (Porterfield, 1980; Westaway et al., 1985; Francki et al., 1991), is an important pathogen for sheep and red grouse (Lagopus scoticus). LI virus occasionally infects humans. Comparative antigenic studies using polyclonal or monoclonal antibodies (MAbs) and genomic sequencing of several tick-borne flaviviruses have shown that LI virus is a distinct member in the tick-borne encephalitis (TBE) serocomplex (Stephenson et al., 1984; Gould et al., 1985a; Mandl et al., 1988, 1991; Pletnev et al., 1990; Shiu et al., 1991 ; Venugopal et al., 1992; Gritsun et al., 1993). In common with all other flaviviruses, LI virus contains a positive-stranded RNA genome which encodes three structural proteins (Shiu et al., 1991) and seven non-structural proteins (K. Venugopal, personal communication). The flavivirus envelope (E) protein,
The nucleotide sequence data reported in this paper have been submitted to the GenBank database and assigned the accession number X69975. 0001-1946 © 1994 SGM
610
G. F. Gao and others
European subtype of TBE virus. So far, no strain diversity has been identified for LI or TBE virus (Heinz & Kunz, 1982; Reid, 1988; Holzmann et al., 1992). It has been reported that the E protein of LI viruses within and outside of the British Isles (Gao et at., 1993) as well as the Japanese isolate of Negishi virus (Venugopal et al., 1992) are closely related as shown by nucleotide sequence and antibody analyses. In this paper, we have extended this work by comparing 18 LI virus isolates obtained in the U.K. and Norway for their reactivities with E-specific and non-structural protein NSl-specific MAbs. Our results show evidence of naturally occurring MAbresistant variants in the environment with reduced virulence for mice.
Methods Virus isolates and MAbs. Eighteen LI virus isolates were studied in this report. Their geographical origins, the hosts from which they were isolated and the year of isolation are listed in Table 1. Strain L1/31, which is the prototype LI virus, was used for the preparation of monoclonal and polyclonal antibodies, the antigenic specificities of which have been described previously (Venugopal et al., 1992; Gao et al., 1993). Stocks of all viruses were prepared as 10 % (w/v) suspensions of infected suckling mouse brain in PBS pH 7-2. The MAbs were selected for their capacity to bind either E or NS1 proteins of LI virus by indirect immunofluorescence (IIF) assays on LI virus-infected cell monolayers. The MAbs have all been described previously (Venugopal et al., 1992) and are designated MAb 3.1, 3.3, 4.2, 7.1 and 7.2 for E-specific MAbs and MAb 1-2, 7.3, 7.4, 7.6 and 81 for NSl-specific MAbs. All five E-specific MAbs but none of the NSl-specific MAbs neutralize the infectivity of LI virus in plaque-reduction neutralization tests. Mab 7.1 but not the other nine MAbs also shows haemagglutination inhibition (HAI) activity with LI virus. Hyperimmune antiserum against LI virus strain LI/31 was prepared by inoculation of sheep with a methanol-precipitated virus antigencontaining adjuvant (Brotherston & Boyce, 1970).
Table 1. L I virus field isolates Virus designation
Geographical origin
Host of isolation
Year of isolation
LI/31 LI/369 LI/MAI4 LI/SB526 Ll/MA27 LI/MA54 L1/MR46 L1/G L1/K L1/I L1/A LI/NOR LI/917 LI/1065 LI/261 LI/2995 LI/2996 LI/1131
Scotland Scotland Ireland Scotland Ireland Ireland Ireland Scotland Scotland Wales England Norway England England England England England Scotland
Sheep Tick* Sheep Sheep Cattle Cattle Tick* Pig Grouse Sheep Sheep Sheep Sheep Sheep Sheep Grouse Grouse Sheep
1931 1963 1967 1968 1968 1968 1971 1979 1980 1980 1980 1984 1985 1985 1987 1987 1987 1988
* Ixodes ricinus.
Plaque neutralization tests. MAbs 4"2 and 7.1 were used for neutralization tests with each LI virus strain. The tests were carried out in Linbro 12-well flat-bottomed tissue culture plates using PS cell monolayers. Serial dilutions of antibody mixed with 100 p.f.u, of virus were incubated at 37 °C for 1 h and then added to the PS cells. After incubation at 37 °C for 1 h the monolayers were washed with medium containing 0.8 % agarose, incubated at 37 °C for 3 to 5 days and then stained using naphthalene black to reveal virus plaques. H F tests. PS cell monolayers were infected with LI virus at 37 °C for 48 h, by which time approximately 25 % of the cells contained virus antigens. Antibodies were added to acetone-fixed coverslip preparations of these infected monolayers at predetermined optimal dilutions for 1 h at 37 °C, the cells on the coverslips were washed in PBS and anti-mouse immunoglobulin conjugated with fluorescein isothiocyanate was added for 1 h at 37 °C. After washing in PBS the cells were mounted in 90% glycerol-saline and fluorescent viral antigen was observed by u.v. fluorescence microscopy (Gould et al., 1985b). Haemagglutination/HAl tests. Haemagglutinins of all virus isolates that were studied were prepared from the supernatant medium of infected BHK 89 cells and concentrated using 7 % (w/v) polyethelene glycol in 0.4 M-NaC1. The haemagglutination (HA) test was carried out at pH 6-2 using gander erythrocytes (Clarke & Casals. 1958). For HAI tests, the HA antigen was adjusted to contain 4 HA units per 0.2 ml using 0.4% BSA in borate-buffered saline. MAb 7.1, as ascitic fluid, and polyclonal antiserum, were used and the tests were carried out as described previously (Clarke & Casals, 1958). The HA1 titres were expressed as log10 reciprocal of the end-point dilution giving 50 % inhibition of HA. Passive protection o f mice with M A b 4"2. The potential therapeutic value of MAb 4.2 was evaluated using a dose of 100 lal of 1:2 dilution of ascitic fluid for each mouse. Twenty male Swiss mice aged 3 weeks were injected intraperitoneally with the diluted ascitic fluid at - 6 , 0, 14, 24, 48 or 96 h before or after challenge with virulent LI virus, strain LI/SB526. The mice were challenged subcutaneously with 100 ~tl containing 2 x 102 p.f.u, of virus. A control group of 20 mice was injected with PBS, 6 h before virus challenge. Mice were observed for up to 25 days. Nucleotide sequencing and analysis. Viral E gene sequencing of LI/A virus, chosen randomly from the five antibody escape variants, was carried out as described below. A 10 % (w/v) mouse brain suspension of the plaque-purified virus was used for the extraction of viral RNA as previously described (Gao et al., 1993). The virus suspension was treated with proteinase K and extracted with phenol and ether. Firststrand cDNA was synthesized using the conserved downstream primer 5' ctcgaattcGGTAGTATGCATAGTT 3', complementary to LI virus nucleotides 2449 to 2464 and the E gene was subsequently amplified by standard PCR using the upstream primer (5' cttagatctaatATGGTCGCCGTTGTGTGGCTA 3', nucleotides 781 to 801 of the L1 virus gene sequence) and the same downstream primer used in the synthesis of the first-strand eDNA. The PCR product was cloned into the pUCll8 vector (Gao et al., 1993) and sequenced in both directions by doublestranded dideoxynucleotide sequencing (Sanger et al., 1977). The sequencing data were compared with other published E gene sequences of the LI virus isolates LI/31, LI/369, LI/SB526, LI/K and LI/NOR (Shiu et al., 1991; Venugopal et al., 1992: Gao et al., 1993). The sequence identity calculations were performed using the CLUSTAL program and the University of Wisconsin Genetics Computer Group (GCG) packages. Mouse neurovirulenee comparison o f L I viruses. Groups of 10 female Swiss mice aged 3 weeks were used for virus neurovirulence comparisons. Approximately 1 x 103 p.f.u, of virus was inoculated
Louping ill virus escape variants
611
Table 2. Grouping of LI virus field isolates by IIF test MAb Virus
3.1
3.3
4.2
7.1
7.2
1.2
7.3
7.4
7.6
8.1
Polyclonal serum
Group
+ + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + --
+ + + + + + + + + + + + + -
+ + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + +
1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2
LI/31 LI/369 LI/MA14 LI/SB526 LI/MA27 LI/MA54 LI/MR46 LI/G LI/K LI/NOR LI/2995 LI/2996 LI/ll31 LI/I LI/A LI/917 LI/1065 LI/261
intraperitoneally into each mouse and the mice were observed for up to 16 days.
Results
Grouping and antigenic characterization of 18 LI virus field isolates The 18 LI virus strains were tested by IIF for their ability to bind each of the 10 MAbs defined in Methods. The results in Table 2 show that the viruses formed two groups. In group 1, 13 LI virus isolates were recognized by all 10 MAbs. These group 1 viruses were isolated between 1931 and 1987 from either Scotland, England, Ireland or Norway. The other five viruses (group 2) reacted with all the MAbs except MAb 4.2 and MAb 7.1. These five viruses were isolated after 1980 from either England or Wales. Experimentally produced MAb 4-2
Table 3. Passive protection of mice against L I virus infection using MAb 4.2 Time of injecting MAb 4.2
No. of survivors
(h)
challenged
- 6
20/20 18/19 17/20 9/16 14/20 12/18 7/18
0 + 14 + 24 +48 + 96
Control* * Injection with PBS.
/no.
Interpretation of Fisher's exact test Very significant Very significant Significant Not significant Not significant Not significant
escape mutants also failed to react with MAb 7"1 (Jiang et al., 1993), indicating that the epitopes recognized by these two MAbs are very closely associated. Further quantitative analysis of the antigenicities of each of the 18 viruses by neutralization, HA and HAI revealed no other significant antigenic divergence among the virus isolates. However, the five variants did produce smaller plaques than the non-variant viruses. This characteristic was also observed with the MAb-resistant mutants reported by Jiang et al. (1993).
Passive protection of mice us#Tg MAb 4"2 Mice inoculated intraperitoneally with MAb 4.2 were challenged with LI virus (strain LI/SB526) at different intervals either before or after antibody administration as described in Methods. The results show that MAb 4"2 protected mice against challenge with virulent LI virus (Table 3). Significant protection was achieved when the antibody was administered either before or up to 14 h after virus challenge but not at 24, 48 or 96 h postinfection. Similar protective and therapeutic effects were also observed when MAb 7"1 was analysed in the same manner (data not shown).
Analysis of the mouse neurovirulence of escape variants Two representative LI viruses (LI/31 and LI/MA54) and five naturally occurring variants that escape neutralization by MAb 4.2 (LI/917, LI/261, LI/1065, LI/A and LI/I) were compared in mouse neurovirulence assays as described in Methods. The results of a typical experiment (Fig. 1) show that all the variants were less virulent than
612
G. F. Gao and others
100
. . . . . . . . . . . . . . . •. . ' m ' " '
.am.,-, - m , , . , .urn.,,,. n "
. m&'" sn.'
~::::~.... "~ 50
/'/ m" ±
±
Jr.
i
0
1 2
3 4
5
6
7
8 9 10 11 12 13 1'4 1'5 16
Fig. 1. Examination of the virulence for mice of LI/31 and LI/MA54 virus compared with five different escape variants. • . . . . i , LI/MA54; O, LI/31 ; C), LI/917; [~, LI/261 : • • , LI/1065; &, LI/A; /~, LI/I strains.
41 81 83
II
230238266 308
II
i
i
405 431 482 483
I
I[
:G,;~
Sites of all amino j acid changes in all sequenced LI viruses LI/369 LI/369 Escape mutant LI/A (Natural escape variant) LI/K
:~
,±
LI/NOR II
I
[
II
II I
I
I
LI/SB526
I
LI/31
Fig. 2. Comparison of the amino acid substitutions in the E genes of selected, sequenced strains and experimentally derived MAb escape mutants of LI virus. All strains are compared with LI/369 virus (Shiu et al., 1991). The positions of amino acid substitutions are indicated by vertical bars. The top box shows the positions of all known substitutions.
LI/31 and LI/MA54 viruses. LI/917 virus was the least attenuated but could be differentiated from LI/31 and LI/MA54 viruses by the longer time taken to cause fatal infection. Determination of the nucleotide and deduced amino acid sequence of the E gO,coprotein gene of L I / A virus LI/A virus was chosen as the representative strain of naturally occurring variants for nucleotide sequencing. A schematic representation of the E gene amino acid sequence of the LI/A virus aligned with other published sequences, including the experimentally selected MAb 4.2 escape mutant of LI/369 virus (Jiang et al., 1993), is shown in Fig. 2. Although four amino acid codon changes were seen in the E gene of LI/A virus compared with LI/369 virus, three of them occur in all of the sequenced E genes of LI virus isolates that are virulent
for mice. The fourth nucleotide change, corresponding to an amino acid substitution at position 308, resulted in a non-conservative amino acid change. This corresponded with the amino acid substitution seen in experimentally selected MAb 4.2 escape mutants (Jiang et al., 1993) and is therefore responsible for the failure of this variant to bind MAb 4.2. The substitution of aspartate to asparagine at amino acid residue 308 produced a potential glycosylation site in the experimentally derived escape mutants. However, the substitution of aspartate to glutamic acid in the naturally occurring escape mutant did not lead to either a potential glycosylation site or a change in charge.
Discussion Two neutralizing MAbs, which map at or near to amino acid position 308 in the LI virus E protein (Jiang et al., 1993), were able to identify five out of 18 LI virus field isolates that are naturally occurring MAb escape variants. Three other E-specific and five NSl-specific MAbs, on the other hand, recognized epitopes in all the virus strains, demonstrating the antigenic similarity and stability of these viruses. It is not yet known whether the occurrence of escape variants, with reduced virulence, in the environment has any significant effects on LI virus epidemiology but the frequency with which these naturally occurring variants arise seems very high. The possibility that they could be laboratory artefacts of the isolation procedure, as observed with influenza and mumps viruses, seems low since identical procedures were used to isolate the 13 non-variant viruses. The finding of attenuated LI virus variants may at least in part explain the apparent differences in severity of LI epidemics in some regions of the U.K. Epizootiological data appear to support this idea since in some epizootic areas the severity of LI disease may be on the decline (Reid, 1988). This concept is also supported by evidence from other viral diseases. Recently, Snyder et al. (1992) reported that naturally occurring escape variants could define the epizootiology of infectious bursal disease virus (IBDV) in the U.S.A. Live IBDV vaccine containing only standard IBDV strains is used in broiler flocks, but isolation of classic IBDV in the eastern U.S.A. occurred less frequently than in other areas in which standard IBDV strains were predominant. It is believed that the wide use of vaccine in the eastern U.S.A. is responsible for the predominance of IBDV escape variants. Moreover, the IBDV variants were isolated after 1985 and in our study all the LI virus escape variants were isolated after 1980. This could mean that the introduction of vaccine into an area may result in the selection of antibody-resistant variants with accompanying altered
Louping ill virus escape variants
virulence characteristics. Several different preparations of LI virus vaccine have been used since the 1930s, although their use has recently declined. By analogy with the periodic variation of influenza viruses, it will be interesting, in future studies of field isolates, to see whether other variants appear. On the other hand, there is evidence from studies with experimentally selected escape mutants of Sindbis virus and poliovirus (Stec et al., 1986; Ketterlinus et al., 1993), that reversion of mutants to wild-type can occur. Whether or not the appearance of antibody-resistant variants represents a temporary state in LI virus epizootiology has yet to be determined. It is interesting to note that all the antibodyresistant variants identified by us were from south of the Scottish border. It is not yet known if this observation has significance, but it could reflect either different vaccination strategies or different ecological pressures on the southern isolates. Nucleotide and amino acid sequencing of the E gene of one variant virus (LI/A) identified a mutation at amino acid position 308, responsible for substitution of aspartate by glutamate. This substitution site corresponds with that of experimentally selected antibody-resistant escape mutants derived using neutralizing MAb 4.2 and reported previously (Jiang et al., 1993). The reduced neurovirulence for mice of the naturally occurring variants, as compared with non-variant LI viruses, is also compatible with the results reported previously for MAb 4.2-derived escape mutants. Thus, the antigenic site containing amino acid 308 clearly has significance in determining the pathogenic characteristics of LI virus, at least for mice. The mouse neurovirulence assays with the derived escape variants showed that they were not all attenuated to the same extent and that this is probably due to different amino acid substitutions at or near to position 308 as noted previously (Jiang et al., 1993). Alternatively, substitutions in genes other than the E gene could contribute to the variable virulence characteristics. It is also of interest that the amino acid substitution sites for both experimentally selected mutants and the naturally occurring variants of LI virus are the same, but that different amino acids have been substituted. However, the substituted amino acids did not cause changes in charge, demonstrating that factors other than charge can influence the presentation of this epitope in the E protein. This contrasts with the evidence observed for Sindbis virus antibody escape mutants since in this case the charged nature of the substituted amino acid was important for interaction with antibody (Strauss et al., 1991). Future vaccine development strategies will need to take into account the effects of using antigenic preparations that might induce selective immunological pressures on LI virus.
613
The authors thank Mrs W. R. Jiang, Dr K. Venugopal and Dr T. S. Gritsun (NERC Institute of Virology and Environmental Microbiology) and H. T. Zhang (Institute of Molecular Medicine, Imperial Cancer Research Fund) for helpful discussions during preparation of this manuscript. G. F. Gao is a scholar supported by the Sino-British Friendship Scholarship Scheme of the British Council and the Chinese Government.
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(Received 7 July 1993; Accepted 1 November 1993)
