eNews July 10, 2012

Humanized NSG mice: an improved model of dengue virus disease

It may surprise you that dengue disease is the most important insect-transmitted viral illness in the world. That's because the ranges of the dengue virus and its mosquito vectors have expanded considerably. Yet, the pathogenesis of dengue disease is not well understood. There are three major reasons for this: 1) the lack of a good animal model; 2) co-circulation of four DENV serotypes; and 3) increased disease severity following a secondary infection with a different serotype. A research team led by Drs. Rebeca Rico-Hesse and Javier Mota-Sanchez from the Department of Virology & Immunology, Texas Biomedical Research Institute, San Antonio, has been working to improve a mouse model of dengue disease for several years.  In 2012, the team used humanized NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (005557) (hu-NSG) mice to produce the first animal model for evaluating human immunity to a dengue virus infection transmitted by mosquito bites (Cox et al. 2012). The model will help researchers better understand key features, including the effects of the mosquito vectors of dengue disease.

Dengue disease

Dengue disease is transmitted to humans by the bites of Aedes aegypti or Aedes albopictus mosquitoes. Each year, millions of people in over 100 countries – mostly tropical and subtropical – are infected. As mentioned above, there are four different dengue virus serotypes. Infection with one serotype usually confers lifelong immunity to that particular serotype but only short-term immunity to the other serotypes. Subsequent infection with a different serotype increases the possibility of severe disease complications.

Dengue disease occurs in two phases – dengue fever (DF) and dengue hemorrhagic fever (DHF). Both occur exclusively in humans. Three to 14 days after being bitten by an infected mosquito, a person may develop muscle, joint and abdominal pain, a fever, erythema (reddish skin caused by dilated blood vessels and increased blood flow beneath the skin), a measles-like rash, a low platelet count and viremia. DHF mostly affects children. It manifests suddenly as plasma leakage at the end of the DF stage. It can lead to low blood fluids, fluid build-up in the lung and chest cavities, shock, liver failure and brain damage. Although the two disease phases can resolve in as little as two days, complete recovery can take weeks to months. Approximately 5% of DHF patients die, usually from hypotensive (low blood pressure) shock, due to a delay in recognizing and treating the plasma leakage.

As mentioned earlier, the occurrence of dengue disease has increased markedly due to the range expansion of the virus and its mosquito vectors. Currently, there is no vaccine for dengue disease. Treatments depend on disease severity and include hydration, administration of intravenous fluids, and blood transfusions (The World Health Organization, Wikipedia, and Cox et al. 2012).

The new model

Rico-Hesse and Mota-Sanchez had previously developed a hu-NSG model of dengue disease (Mota and Rico-Hesse 2009, 2011). Although this model develops clinical signs of dengue disease, infection is induced not by mosquito bites but by intradermal injections of low-passage, wild-type dengue virus isolates. In contrast, the new model is infected by Aedes aegypti mosquitoes. To generate the model, sublethally irradiated newborn NSG mice are intra-hepatically injected with human cord blood-derived hematopoietic stem cells and, as a result, develop a near-human immune system (McDermott et al. 2010). Female Aedes aegypti mosquitoes infected with a DENV-2 strain K0049 virus – a strain isolated from a DHF patient in Kamphaeng Phet, Thailand, in 1995 – are starved for 24 hours and then allowed to feed on the footpads and/or ears of anesthetized hu-NSG mice. As controls, some mice are exposed to uninfected mosquitoes and later inoculated with the K0049 DENV strain. Others are inoculated with a mixture of virus and saliva from uninfected mosquitos.

Hu-NSG mice model key features of dengue disease pathogenesis

The results by Rico-Hesse and her colleagues accentuate that a good model of dengue disease must include DENV transmission by natural vectors (i.e., mosquitoes). Their key findings are summarized below:

  • Humanized NSG mice develop a near-human immune system. They are being widely used to research multiple human infectious diseases.
    hu-NSG mice bitten by 4-5 mosquitoes develop the most representative and consistent clinical signs (viremia, erythema, and fever) of human dengue disease.
  • Mosquito bite-infected hu-NSG mice develop greater viremia (similar to that developed by adult Vietnamese dengue patients), erythema and thrombocytopenia than those infected by direct viral injections.
  • Viremia of mosquito bite-infected hu-NSG mice may be enhanced by uninfected mosquito bites distal to the site of infection (demonstrating the aggravating effects of mosquito saliva).
  • Like humans, hu-NSG mice bitten by uninfected mosquitoes produce a variety of human cytokines, but, if bitten by infected mosquitoes, they produce significantly higher levels of IFNG and IL2RA (direct viral injections elicit a weaker cytokine response).
  • Whereas few virally injected hu-NSG mice develop anti-DENV antibodies, nearly half of infected mosquito-bitten hu-NSG mice develop them.
  • hu-NSG mice do not develop viremia as a result of dengue re-infection and are protected by cytokines and dengue-specific antibodies produced up to six months after the first infection, suggesting that they have functional memory against DENV for up to eight months after transplantation with human stem cells.

In summary, like previous researchers, Rico-Hesse and her colleagues found that mosquito saliva exacerbates the immune response to and enhances DENV infection. Once infected, most hu-NSG mice secrete pro-inflammatory cytokines similar to those secreted by DENV-infected humans. They also produce DENV-specific antibodies but only after being infected by mosquito bites. In effect, though hu-NSG mice lack some components of the human immune system and natural human DENV cellular targets that could modify dengue infection or disease, they can help scientists better understand key aspects of dengue disease, including the effect of the mosquito vector.