Antibody-dependent enhancement of Murray Valley encephalitis virus virulence in mice

M. J. Wallace1, D. W. Smith2, A. K. Broom1, J. S. Mackenzie3, R. A. Hall3, G. R. Shellam1 and P. C. McMinn1,4

1 Discipline of Microbiology, School of Biomedical and Chemical Sciences, The University of Western Australia, WA, Australia
2 Division of Microbiology and Infectious Diseases, PathCentre, Perth, WA, Australia
3 Department of Microbiology and Parasitology, School of Microbial and Molecular Sciences, The University of Queensland, QLD, Australia
4 Division of Virology, Telethon Institute for Child Health Research, 100 Roberts Road, Subiaco, WA 6008, Australia

Correspondence
Peter McMinn (at address 4)
peterm{at}ichr.uwa.edu.au


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Enhancement of flavivirus infection in vitro in the presence of subneutralizing concentrations of homologous or heterologous antiserum has been well described. However, the importance of this phenomenon in the enhancement of flavivirus infection in vivo has not been established. In order to study antibody-mediated enhancement of flavivirus infection in vivo, we investigated the effect of passive immunization of mice with Japanese encephalitis virus (JE) antiserum on the outcome of infection with Murray Valley encephalitis virus (MVE). We show that prior treatment of mice with subneutralizing concentrations of heterologous JE antiserum resulted in an increase in viraemia titres and in mortality following challenge with wild-type MVE. Our findings support the hypothesis that subneutralizing concentrations of antibody may enhance flavivirus infection and virulence in vivo. These findings are of potential importance for the design of JE vaccination programs in geographic areas in which MVE co-circulates. Should subneutralizing concentrations of antibody remain in the population following JE vaccination, it is possible that enhanced disease may be observed during MVE epidemics.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Murray Valley encephalitis virus (MVE) and Japanese encephalitis virus (JE) are closely related viruses belonging to the genus Flavivirus within the family Flaviviridae (Heinz et al., 2000) and share a high degree of antigenic cross-reactivity (Calisher et al., 1989). Both viruses cause mosquito-borne encephalitis of high mortality and with high rates of residual neurological morbidity among survivors (Bourke & Monath, 2001). MVE is endemic to northern Australia and Papua New Guinea and JE is endemic throughout the Asia-Pacific region (Mackenzie et al., 1994; Vaughn & Hoke, 1992). JE was first identified in the Australian geographic region in 1995, when a small epidemic was reported in the islands of the Torres Strait (et al., 1996). Subsequently, a case of JE infection was identified on the Australian mainland adjacent to the Torres Strait (Cape York Peninsula) in 1998 (et al., 1999). As a result of the emergence of JE into the Australian geographic region, it is now possible for JE and MVE to co-circulate within Australia (Mackenzie et al., 2002). In response to the JE outbreaks in north Queensland, residents of the Torres Strait were immunized against JE (Pyke et al., 2001). As a result, it is now possible that JE-exposed or immunized individuals in the Torres Strait will be infected with MVE at a later date.

There is currently no vaccine available to protect against MVE infection. Broom et al. (2000) examined several strategies to protect against MVE using a mouse model. These studies included (1) passive immunization of mice with homologous (MVE) immune serum and (2) induction of cross-protective immunity by immunization with the JE (Biken) vaccine. This study showed that JE immunization not only failed to protect mice from challenge with MVE, but also showed that JE-immunized mice died much sooner after MVE challenge than mock-immunized mice, suggesting that low concentrations of heterologous JE antibody may have enhanced MVE disease.

Although antibody-dependent enhancement (ADE) of flavivirus infection has been consistently demonstrated in cell culture-based studies, ADE has not been demonstrated conclusively in vivo. Despite this, a considerable amount of clinical and epidemiological evidence suggests that ADE is responsible for dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) in individuals with pre-existing immunity to heterologous dengue virus (DEN) serotypes (Halstead, 1988, 1989).

In this study, we present the findings of an investigation into the effect of passive immunization with heterologous (JE) antiserum on MVE infection in mice. We show that passive immunization of mice with subneutralizing concentrations of JE antiserum result in increased viraemia titres and enhanced mortality in mice challenged with wild-type MVE. Our findings support the hypothesis that subneutralizing concentrations of antibody may enhance flavivirus infection and virulence in vivo.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Virus and cells.
Vero cells (ATCC CCL 81) were cultured in Eagle's minimal essential medium (EMEM) containing 10 % foetal calf serum (FCS) at 37 °C/5 % CO2. MVE strain F3/51 (French et al., 1952) was used as a challenge virus in the mouse experiments. The Nakayama strain of JE (Mitamura et al., 1936) was used in the preparation of JE antiserum. Working stocks of virus were prepared by inoculation of Vero cells at an m.o.i. of 1 p.f.u. of virus per cell, and harvesting the supernatant when the cells showed >=80 % cytopathic effect.

Plaque assays.
Virus plaque assays were performed on Vero cell monolayers in 12-well tissue culture plates. Serial dilutions of virus were inoculated onto the cells for 1 h at 37 °C, the inoculum removed and the cells overlaid with methylcellulose containing EMEM/10 % FCS for 4–6 days until plaques were visible. Monolayers were then stained with 1 % (w/v) methylene blue in 10 % (v/v) formaldehyde and titres estimated by counting the number of plaques observed in each well.

Plaque reduction neutralization assays.
Neutralizing antibody titres in mouse sera were estimated by 50 % plaque reduction neutralization tests (PRNT50) Briefly, dilutions of antiserum were incubated for 1 h (37 °C) with 102 p.f.u. of virus prior to inoculation of Vero cells in 12-well tissue culture plates. The cells were then overlaid with methylcellulose/EMEM/10 % FCS. After 4–6 days incubation, the cells were stained with 1 % methylene blue/10 % formaldehyde and the plaques counted in each well. The PRNT50 titre was calculated as the reciprocal of the highest dilution of antiserum that reduced the number of plaques in each well by 50 %; each assay was performed in duplicate.

Generation of mouse antisera.
JE (Nakayama) antiserum was prepared by intraperitoneal (i.p.) inoculation of 8-week-old Swiss outbred mice (Animal Resources Centre, Perth, Australia) with 103 p.f.u. of heat-inactivated virus on day 0, followed by a boost with 103 p.f.u. of live virus on day 14. Serum was collected on day 28, pooled, heat-inactivated (56 °C, 30 min) and stored at -20 °C until use. An MVE hyperimmune ascitic fluid (HIAF), prepared in mice by McMinn et al. (1995), was used as a source of homologous antibody for the control groups. Non-immune serum obtained from 8-week-old Swiss mice was purchased from the Animal Resources Centre (Perth, Australia). The PRNT50 titres of the JE antiserum against MVE and JE were 1 : 40 and 1 : 320, respectively; the PRNT50 titres of the MVE HIAF against MVE and JE were both 1 : 800; the PRNT50 titres of the non-immune serum against MVE and JE were both <1 : 20.

ELISA.
MVE-reactive IgG titres in mouse serum were determined by indirect ELISA using MVE antigen prepared from infected Vero cell supernatant, following Hall et al. (1995). Antibody titres are expressed as the reciprocal of the highest dilution of sera producing substrate colour changes that resulted in an absorbance greater than 0·1 when measured in a spectrophotometer (Bio-Rad) at 415 nm and 490 nm.

Virus titration in mouse tissues.
Blood was obtained by cardiac puncture and the serum aliquoted and stored at -80 °C. Brains were dissected, prepared as 10 % suspensions in Hanks' balanced salt solution, pH 8, in ground glass tissue homogenizers (Corning) and stored at -80 °C. Virus titres in individual serum and brain samples were determined by plaque assay on Vero cells. The sensitivity of the plaque assay in brain and serum samples was determined to be 50 p.f.u. g-1 and 50 p.f.u. ml-1, respectively.

Passive immunization and challenge experiments.
Groups of thirty-four 18- to 20-day-old BALB/c mice were inoculated i.p. (250 µl) with various concentrations of JE antiserum 4 h before and 16 h after i.p. challenge with 103 p.f.u. of MVE-F3/51. Mice were treated with the following dilutions of JE antiserum: 10-fold above, equal to, 10-fold below or 100-fold below the PRNT50 titre of the JE antiserum against MVE-F3/51. Anti-MVE HIAF diluted to 80-fold above the PRNT50 titre for MVE was used as a positive control for mouse protection and non-immune serum was used as a negative control for mouse protection.

Three mice from each treatment group were sacrificed daily between 2 and 7 days after virus challenge in order to measure virus titres in the blood (viraemia) and brain, and to estimate humoral immune responses induced by virus challenge. The remaining mice in each treatment group were observed twice daily for 28 days after challenge and the onset of illness and deaths were recorded. Humane end-points were used throughout these experiments, with mice being euthanased at the first clinical signs of encephalitis. All mouse experiments were undertaken using protocols approved by the University of Western Australia Animal Experimentation Ethics Committee. Mice were kept on clean litter of sawdust and given access to food and water ad libitum.

Statistical analysis.
Statistical analyses were performed using SigmaStat 2.0 (SPSS, Chicago, USA; www.spssscience.com), comparing each antiserum-treated group with its cognate mock-treated group. The Mann–Whitney and Student's t-tests were used to compare mean survival time, virus and antibody titres after virus challenge. The {chi}2 and Fisher's Exact tests were used to compare the percentage survival of mice after virus challenge. A statistically significant difference between groups was considered to have occurred when P values were <0·05.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Survival of 18- to 20-day-old BALB/c mice after MVE challenge
Previously, Broom et al. (2000) showed that mice passively immunized with serum obtained from JE (Biken)-immunized mice died much sooner after MVE challenge than mock-immunized mice. In order to confirm and extend this observation, we undertook a series of experiments in which mice were passively immunized with JE antiserum diluted to various concentrations above and below the PRNT50 titre against MVE prior to challenge with wild-type MVE. Survival data are shown in Table 1.


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Table 1. Mortality in 18- to 20-day-old BALB/c mice after i.p. challenge with MVE-F3/51

The challenge dose was 103 p.f.u. of MVE-F3/51. Mice were immunized by i.p. inoculation of antiserum 4 h before and 16 h after virus challenge.

 
Intraperitoneal challenge of mock-immunized mice with 103 p.f.u. of MVE-F3/51 caused a ‘background’ mortality of 29 %. Mice immunized with JE antiserum at a concentration that equalled the PRNT50 titre against MVE had a reduced mortality compared to control mice after i.p. challenge with MVE (7 % versus 29 %); however, this difference was not statistically significant. Of particular interest, immunization of mice with subneutralizing concentrations of JE antiserum (1 : 10 and 1 : 100 of the PRNT50 titre against MVE) resulted in increased mortality after challenge. The 71 % mortality observed in mice immunized with a 1 : 10 dilution of JE antiserum did not reach statistical significance; however, mice immunized with a 1 : 100 dilution of JE antiserum had a significantly increased mortality of 93 % (P<0·001) compared to mock-immunized mice (29 % mortality).

Humoral immune responses to MVE challenge
Humoral immune responses were measured in the serum of mock- and antiserum-treated mice between 2 and 7 days after challenge with MVE, and the results are shown in Table 2. All of the mock-immunized mice (Table 2, group 1) produced MVE-reactive IgG (1 : 27) on day 6 after i.p. challenge with MVE-F3/51, which increased to a titre of 1 : 160 at 7 days post-challenge. As expected, high-titre MVE-reactive IgG was also detected at all time-points in control mice passively immunized with MVE HIAF at a concentration 80-fold above the PRNT50 titre for MVE (Table 2, group 5). In addition, MVE-reactive IgG was detected at all time-points in mice immunized with high-titre JE antiserum (10-fold above the PRNT50 titre for MVE) (Table 2, group 2). It is likely that the IgG detected in these mice was residual passively transferred antibody.


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Table 2. MVE-reactive IgG titres in 18- to 20-day-old BALB/c mice after i.p. challenge with MVE-F3/51

MVE-reactive IgG titres were determined by ELISA. Each value in the table represents the average MVE antibody titre in three mice; individual titres varied less than 4-fold. The challenge dose was 103 p.f.u. of MVE-F3/51. Mice were immunized by i.p. inoculation of antiserum 4 h before and 16 h after virus challenge.

 
MVE-reactive IgG was also detected at low titre (1 : 20) on days 2, 3 and 4 post-challenge in mice immunized with JE antiserum at a concentration equal to the PRNT50 titre for MVE (Table 2, group 3); it is likely that the antibody detected on days 2–4 post-challenge was residual passively transferred antibody. However, on day 5 post-challenge, MVE-reactive IgG was not detectable in this group of mice (<1 : 20) and again became detectable on days 6 (1 : 20) and 7(1 : 40) post-challenge, suggesting that these mice had mounted a humoral immune response to the challenge virus, but at an average titre 4-fold below that of mock-immunized mice (P<0·005).

MVE-reactive IgG was not detected in mice immunized with JE antiserum at a concentration 10-fold below the PRNT50 titre for MVE (Table 2, group 4) from 2–5 days post-challenge, inclusive. MVE-reactive IgG was first detected in this group (at a titre of 1 : 20) on day 6 post-challenge, similar to the mock-immunized mice, and increased to a titre of 1 : 67 at 7 days post-challenge, which was significantly lower than for mock-immunized mice (P<0·005). Thus, passive immunization of mice with JE antiserum appeared to inhibit the development of humoral immune responses after MVE challenge.

Virus titres in mouse serum following MVE challenge
Virus titres were measured in the blood (viraemia) of mock- or JE antiserum-immunized mice between 2 and 7 days after MVE challenge. Viraemia was detected between 2 and 4 days post-challenge, peaking on day 3 in all treatment groups. Individual mouse and mean viraemia titres for each treatment group are shown as a dot plot in Fig. 1. A large variation in viraemia titres was observed between individual mice. However, very low or undetectable viraemia was observed in mice immunized with MVE HIAF or JE antiserum at a concentration 10-fold above the PRNT50 titre for MVE. The viraemia titre increased in inverse proportion to the concentration of neutralizing antibody received by the immunized mice. Prior immunization of mice with JE antiserum at a concentration 10-fold below the PRNT50 titre for MVE resulted in approximately 10-fold increases in the mean viraemia titre compared to mock-immunized mice on day 2 (average titres 1·4x103 p.f.u. ml-1 versus 1·3x102 p.f.u. ml-1) and day 3 (average titres 3·2x104 p.f.u. ml-1 versus 2·9x103 p.f.u. ml-1) after virus challenge.



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Fig. 1. Viraemia titres in 18- to 20-day-old BALB/c mice following i.p. challenge with 103 p.f.u. of MVE-F3/51. Groups of three mice were immunized with the indicated concentrations of antiserum 4 h before and 16 h after MVE challenge. At the indicated times post-challenge, mice were anaesthetized and blood was collected by cardiac puncture. Each point on the graph represents the viraemia titre of an individual mouse. Viraemia titres were determined by plaque assay on Vero cells. Titres are expressed as p.f.u. ml-1. The limit of sensitivity for detection of virus by the plaque assay is 50 p.f.u. ml-1 (denoted by the dashed line); mouse sera in which virus was undetectable have values plotted below the dotted line. Mouse groups in which viraemia was detected have the mean titre denoted on the graph by a cross (+). {square}, Non-immune serum; {blacksquare}, JE antiserum diluted to 10-fold above the PRNT50 titre for MVE; {circ}, JE antiserum diluted to equal the PRNT50 titre for MVE; {bullet}, JE antiserum diluted to 10-fold below the PRNT50 titre for MVE; {triangleup}, MVE HIAF diluted to 80-fold above the PRNT50 titre for MVE.

 
Virus replication in mouse brain after MVE challenge
Virus replication in mouse brain was determined between 2 and 7 days after virus challenge, and the results are shown in Fig. 2. The graph shows the average virus titre in three mouse brain samples at the specified times post-challenge and the bars indicate the maximum and minimum virus titres for each treatment group at each time-point.



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Fig. 2. Growth kinetics of MVE-F3/51 in the brain after i.p. inoculation of 18- to 20-day-old BALB/c mice. Groups of three mice were immunized with the indicated concentrations of antiserum 4 h before and 16 h after inoculation of MVE-F3/51. At the indicated times post-challenge, mice were euthanased and the brains removed. The infectivity of 10 % brain homogenates was titrated by plaque formation on Vero cells. The points on the graph are the average of three individual mouse brain titres and the bars indicate the maximum and minimum titre at each time-point. Titres are expressed as p.f.u. g-1. The limit of sensitivity for detection by the plaque assay is 50 p.f.u. g-1 (denoted by the dashed line); brain homogenates in which virus was undetectable have the values plotted below the dotted line. {square}, Non-immune serum; {blacksquare}, JE antiserum diluted to 10-fold above the PRNT50 titre for MVE; {circ}. JE antiserum diluted to equal the PRNT50 titre for MVE; {bullet}, JE antiserum diluted to 10-fold below the PRNT50 titre for MVE; {triangleup}, MVE HIAF diluted to 80-fold above the PRNT50 titre for MVE.

 
Consistent with previous studies (McMinn et al., 1995), MVE first appeared in the brains of mock-immunized mice 5 days after i.p. challenge with MVE-F3/51 and brain virus titres peaked (average titre approximately 109 p.f.u. g-1) on day 6 post-challenge. As expected, immunization of mice with MVE HIAF or JE antiserum at a concentration 10-fold above the PRNT50 titre against MVE prevented brain infection (average titres <50 p.f.u. g-1 for both groups at all time-points) compared to mock-immunized mice. By contrast, mice immunized with JE antiserum at concentrations equal to or 10-fold below the PRNT50 titre for MVE were not protected from brain infection after MVE challenge. No statistically significant differences were observed in either the timing of virus invasion or in peak virus titres within the brain between the mock-immunized and immunized mouse groups.


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
We have shown that passive immunization of mice with JE antiserum at concentrations 10-fold and 100-fold below the PRNT50 titre for MVE resulted in increased mortality after MVE challenge compared to mock-immunized mice. For mice immunized with JE antiserum at a concentration 10-fold below the PRNT50 titre for MVE, the increased mortality was associated with higher viraemia titres and attenuated humoral immune responses after challenge compared to mock-immunized mice. By contrast, passive immunization of mice with JE antiserum at a concentration equal to the PRNT50 titre for MVE appeared to protect mice from MVE challenge.

Although the phenomenon of ADE of flavivirus infection has been well described in cell culture-based studies (Hawkes, 1964; Halstead & O'Rourke, 1977; Gollins & Porterfield, 1984), to date, a rigorous demonstration of ADE of flavivirus infection in vivo has not been reported. Halstead et al. (1967) observed that the emergence of DHF and DSS in Asia was associated with the simultaneous circulation of multiple DEN serotypes, resulting in sequential DEN infections in large numbers of individuals. Furthermore, Russell et al. (1967) argued that severe forms of DHF and DSS were more prevalent in people with serological evidence of prior DEN infection, a phenomenon that they termed the ‘second-infection’ hypothesis. The model for development of DHF and DSS was further refined by Halstead et al. (1973), who proposed that low-titre (subneutralizing or ‘enhancing’) antibody remaining from a prior DEN infection was responsible for the development of severe disease upon re-infection with a heterologous DEN serotype (the ‘immune-enhancement’ hypothesis). This hypothesis provided a link between the laboratory observations of ADE by Hawkes (1964) and the epidemiological observations of severe DEN infections in Asia (Halstead et al., 1978).

Whilst an extrapolation of in vitro evidence for ADE of flavivirus infection has been used to explain the epidemiological link between DHF and DSS in people with serological evidence of prior DEN infection, there is a lack of experimental evidence for ADE of flavivirus infection in vivo. However, an ‘early death’ phenomenon has been observed in several animal models, in which animals with pre-existing humoral immunity succumb to virus challenge earlier than animals without pre-existing immunity (reviewed in Morens, 1994). The recent observation of an ‘early death’ phenomenon in JE-immunized mice challenged with MVE (Broom et al., 2000) is of particular relevance to this study.

To our knowledge, this study provides the first unequivocal demonstration of ADE of flavivirus infection and virulence in vivo. Subneutralizing concentrations of heterologous (JE) antiserum resulted in elevated viraemia titres and increased mortality in BALB/c mice after i.p. challenge with wild-type MVE. By contrast, Kreil & Eibl (1997) were unable to demonstrate ADE in a model of tick-borne encephalitis virus (TBE) infection in BALB/c mice, despite using a TBE antiserum concentration (5-fold lower than the PRNT50 titre for TBE) and virus inoculum (103 p.f.u.) identical to those found to cause a 100-fold enhancement of TBE infection in peritoneal macrophages derived from BALB/c mice (Kreil & Eibl, 1997). It is possible that the relatively high titre of homologous (TBE) antiserum (5-fold below the PRNT50 titre for TBE) used by Kreil & Eibl (1997) in comparison to our study (heterologous JE antiserum diluted 10-fold and 100-fold below the PRNT50 titre for MVE) may have been responsible for the different outcomes observed in these two studies. This is consistent with the observations of Morens et al. (1987), who showed that inhibition of virus infection occurs when the ratio of neutralizing antibody to virus is high, whereas enhancement occurs when this ratio is reversed.

Despite our observation that mice passively immunized with JE antiserum at a concentration 10-fold below the PRNT50 titre for MVE had increased mortality and viraemia titres compared to mock-immunized mice, no significant differences in either the timing of virus entry or in the magnitude of virus replication in the brain were detected in these mice compared to controls. Thus, the mechanism for the enhanced mortality of mice observed in this study has not been resolved and may be due to factors other than increased uptake of virus into monocytes and/or enhanced brain infection. Furthermore, our observation that mice passively immunized with JE antiserum at concentrations 10-fold above (Table 2, group 2), equal to (Table 2, group 3) and 10-fold below (Table 2, group 4) the PRNT50 titre for MVE had attenuated humoral immune responses compared to controls suggests that altered immune responses may have played a role in the apparent increase of MVE virulence after passive immunization with subneutralizing concentrations of JE antiserum. Although mice that received JE antiserum at a concentration equal to the PRNT50 titre for MVE had attenuated humoral immune responses, the presence of low-titre MVE-specific IgG up to 4 days after challenge appeared to protect these mice from challenge with wild-type MVE. By contrast, passive immunization of mice with a 10-fold lower concentration of JE antiserum, which did not result in the presence of measurable passively transferred antibody at any time after challenge, also appeared to lead to an attenuated humoral immune response and to enhanced mortality after challenge with wild-type MVE. Quantitative and qualitative studies of humoral and cell-mediated immune responses to MVE infection in passively immunized mice should help to identify the mechanism of virulence enhancement in this model.

In conclusion, our findings support the hypothesis that subneutralizing concentrations of specific antibody enhance flavivirus infection and virulence in vivo, although the mechanism for the virulence enhancement remains unclear. Nevertheless, our data have important implications for JE immunization campaigns in areas where MVE and JE viruses co-circulate, such as the Torres Strait and Cape York Peninsula regions of northern Australia (Mackenzie et al., 2002). Should subneutralizing concentrations of JE antibody remain in the population following immunization, it is possible that MVE infections of enhanced severity may occur during periods of MVE epidemic activity.


   ACKNOWLEDGEMENTS
 
This study was funded by a grant from the Health Department of Western Australia. The authors wish to thank Simone Ross and Owen Walker for providing excellent technical assistance with the care of the animals used in this study. We also thank Dr Cheryl Johansen and other members of the University of Western Australia Arbovirus Research Laboratory for their support and advice.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bourke, D. & Monath, T. (2001). Flaviviruses. In Fields Virology, 4th edn, vol. 1, pp 1043–1126. Edited by D. M. Knipe & P. M. Howley. Philadelphia: Lippincott Williams & Wilkins.

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Received 18 November 2002; accepted 3 March 2003.