Although the number of test subjects used in these studies was limited by the procedures necessary for individual engraftment, results for eight viruses were consistent with genotype groupings. Therefore, we are confident in our conclusions regarding in vivo differences between dengue virus genotypes, although we used only two viruses to represent each of these groups. Any variation in virus replication due to CB donor, transplantation level, or virus passage differences was accounted for by analysis of variance, and our data graphs include the standard error of the mean to demonstrate these ranges. However, we have limited our interpretations only to the statistically significant trends in differences between genotypes, to eliminate the effect of any measurable differences in reagents.
In terms of the validity of using this mouse model to mirror dengue virus pathogenesis in humans, these mice are still the only system described to date that mimics dengue fever (28
). Other systems, using immunocompromised and immunodeficient mice, have produced some clinical signs of dengue (viremia, fever, vascular leakage, or hemorrhaging), but only by using specific strains of virus that have high passage levels (and therefore genetic differences introduced by selection), routes of inoculation that are not natural (i.e., intravenous or intraperitoneal), or virus doses that are extremely high (8 to 9 log10
PFU). Kuruvilla et al. (14
) recently described the RAG2−/−
strain of mice that was engrafted with human fetal stem cells, and these mice responded with fever and viremia after inoculation with pooled dengue viruses of different serotypes; there was no rash or thrombocytopenia. However, one-half of the inocula (of one or three to four pooled DEN-2 viruses) were given via peritoneum (the other half were subcutaneous), and these were the mice that produced detectable (two- to fourfold over background) antibodies to dengue (3 of 16 mice had IgM at 4 weeks and 6 of 16 had IgG at 8 weeks p.i.). Our system offers the advantage of avoiding the use of embryonic stem cells, and we have confirmed that viruses of low passage number (two to four cell culture passes from one patient sample) do produce consistent clinical signs. Although the dose of virus used in the humanized NOD-scid IL2rγ null
mice described here was higher than what was used in our previous model (6 log10
versus 4.7 log10
), these are still doses that could theoretically be delivered by a single mosquito bite. We are currently performing studies where single infected mosquitoes will be tested for the limits of dengue virus transmission to these humanized mice.
The results for two West African strains shown here are the first clear in vivo phenotypic differences between this genotype and others belonging to serotype 2. Other authors have suggested that these sylvatic viruses might emerge to become the agents of new outbreaks, and because they are so distinct genetically, they might overcome protective immunity induced by vaccination (22
). The evidence shown here suggests that this is not the case, because the viremias are much shorter and the skin pathology implicitly measured here as rash is much lower. That is, these viruses seem to be at an evolutionary disadvantage, even more so than the American genotype viruses that have been displaced by the SE Asian viruses in many countries. We have shown that differences in replicative ability in human skin target cells and whole mosquitoes result in much lower viremias and vectorial capacity, respectively, and thus in drastically reduced virus transmission (1
). Other investigators in West Africa have shown that A. aegypti
mosquitoes collected there are poor vectors for the sylvatic viruses; these viruses have very low rates of mosquito dissemination and therefore reduced potential to be transmitted (8
). Whether vaccine preparations would protect against this genotype remains inconclusive, but there should be less concern because these viruses have not caused outbreaks in West Africa, and countries in that area have actually imported their epidemic viruses, while their sylvatic dengue foci are being eliminated by human environmental disruption (17
Results of ELISA experiments showed that some mice have detectable antibodies against dengue virus, but only when infected with the most virulent SE Asian strain (K0049). Infection with other genotypes failed to induce detectable levels of immunoglobulins in a screening assay. Since each experimental group contains mice that had received transplants of progenitor cells from different donors, implicit host genetic differences could influence the production of antibodies after a challenge; however, further analyses are needed to address this question. For example, we could use CD34+
cells expanded in vitro (10
) in order to have enough cells to carry out transplantation with a larger group of mice and directly compare different genotype strains. In addition, even the reported success in producing antibodies in other strains of humanized mice (13
) has been qualified by no or low titers of detectable antibodies. This could be due to the fact that B-cell homeostasis is abnormal in transplant recipient mice, and human B cells could be lost within 2 weeks, suggesting that the murine environment does not provide the necessary cytokine environment for their survival (10
). Even when we obtain higher engraftment levels, the numbers of human immune cells are very low in the total blood of these mice (see Fig. S3 in the supplemental material). Recent studies have demonstrated that treatment of these mice with human recombinant tumor necrosis factor alpha and human B-lymphocyte stimulator (BLyS/BAFF) can enhance engraftment of B and T cells and promote B-cell survival with a concomitant increase in antibodies against T-dependent and T-independent antigens (10
). Because of the key role the antibody response plays in the immunopathology of dengue, we are now establishing protocols to use these cytokines in our humanized NOD-scid IL2rγ null
mouse model, to improve the anti-dengue virus antibody titers. This would allow us to measure the role of the humoral response in sequential infections and to develop a mouse that could be used to test vaccine efficacy in a human immune system milieu.
Prospective clinical studies in humans have shown that there is a correlation between the dengue disease course (and sometimes severity) and viremia (23
); the results of our study are in agreement with that observation. Infected mice showed a correlation between viremia and fever peaks; in general, infected mice showed a rapid increase of temperature by day 2 p.i., with a slight decrease by day 8 to 10 p.i. and a second increase by day 12 p.i. The mouse group infected with the SE Asian genotype viruses showed the maximum fever peak (about 2°C higher than controls) and, along with the mice infected with American genotype viruses, showed a two-peak viremia and fever curves which were statistically different from controls. In contrast, mice infected with viruses of the Indian and West African genotypes showed only one peak of fever and viremia. In addition, the latter two genotypes produced lower levels of viremia and/or thrombocytopenia in these mice. These observations could provide evidence for lower virus replication or virulence in humans, which could also reflect some epidemiologic differences. There is evidence from serotype 2 and 3 viruses that specific genotypes have increased transmission, can displace other genotypes, and can be associated with epidemics of more severe disease (SE Asian genotype for DEN-2, genotype IIIb for DEN-3) (15
). The Indian, American, and West African genotypes of DEN-2 are currently being displaced by the SE Asian genotype in many regions affected by dengue, as evidenced by numerous, continually updated phylogenies (7
). The mice analyzed here therefore show clinical signs that might reflect this epidemiology, where more virus replication (higher and longer, when infected with SE Asian viruses) implies a greater opportunity to be transmitted. Gubler et al. reached similar conclusions in the 1970s, when studying dengue outbreaks in the Pacific Islands (11
). We believe we now have the laboratory systems in which to test these hypotheses.