Development of a transmission model for dengue virus

Abstract

Background: Dengue virus (DENV) research has historically been hampered by the lack of a susceptible vertebrate transmission model. Recently, there has been progress towards such models using several varieties of knockout mice, particularly those deficient in type I and II interferon receptors. Based on the critical nature of the type I interferon response in limiting DENV infection establishment, we assessed the permissiveness of a mouse strain with a blunted type I interferon response via gene deficiencies in interferon regulatory factors 3 and 7 (IRF3/7 -/- -/-) with regards to DENV transmission success. We investigated the possibility of transmission to the mouse by needle and infectious mosquito, and subsequent transmission back to mosquito from an infected animal during its viremic period.

Methods: Mice were inoculated subcutaneously with non-mouse adapted DENV-2 strain 1232 and serum was tested for viral load and cytokine production each day. Additionally, mosquitoes were orally challenged with the same DENV-2 strain via artificial membrane feeder, and then allowed to forage or naïve mice. Subsequently, we determined acquisition potential by allowing naïve mosquitoes on forage on exposed mice during their viremic period.

Results: Both needle inoculation and infectious mosquito bite(s) resulted in 100% infection. Significant differences between these groups in viremia on the two days leading to peak viremia were observed, though no significant difference in cytokine production was seen. Through our determination of transmission and acquisition potentials, the transmission cycle (mouse-to mosquito-to mouse) was completed. We confirmed that the IRF3/7 -/- -/- mouse supports DENV replication and is competent for transmission experiments, with the ability to use a non-mouse adapted DENV-2 strain. A significant finding of this study was that this IRF3/7 -/- -/- mouse strain was able to be infected by and transmit virus to mosquitoes, thus providing means to replicate the natural transmission cycle of DENV.

Conclusion: As there is currently no approved vaccine for DENV, public health monitoring and a greater understanding of transmission dynamics leading to outbreak events are critical. The further characterization of DENV using this model will expand knowledge of key entomological, virological and immunological components of infection establishment and transmission events.

Figure 1

Type I interferon production signals through IRF3 and IRF7. Double stranded RNA is recognized by RIG-I and MDA5 in the cytoplasm and double stranded RNA is recognized in the endosome by TLR3. The resulting signal cascades all utilize IRF 3 and 7 to first promote transcription of IFN-β, which positively feeds back via STAT1/STAT2/IRF9 to promote transcription of IRF7, which promotes transcription of IFN-α.

Figure 2

Gel showing replicative strand amplification at 48 hours post inoculation with DENV-2 1232. Left to Right: Ladder position (~900 bp), Positive control- Freeze thawed DENV-2 1232 infected Vero cell culture; Negative control- Uninfected, age matched Vero cell culture; Samples 1 and 2: Samples from inoculation site and draining lymph node of mice inoculated with DENV-2 1232, respectively.

Figure 3

Mean viremia and IFN-gamma levels of DENV-2 1232 needle inoculated mice. Mean viremia and IFN-γ levels (+/− SEM) are centered around viremia peak day (peak = P0). IFN-γ levels peaked the day following peak viremia.

Figure 4

DENV-2 1232 viremia levels of mosquito vs. needle inoculated mice. Mean viremia (+/− SEM) in mosquito-inoculated mice was detectable for one day more than needle inoculated mice (5 days vs. 4 days, respectively). Viremia levels between the two treatment groups were statistically different (as indicated by *) at all times points except peak viremia day, P0.

Figure 5

Cytokine responses in needle-inoculated vs. mosquito-inoculated infections. A) IFN-γ. The IFN-γ response (+/− SEM) was significantly higher in the needle inoculated group on days P0 and P1, as indicated by *. B) TNF-α. Means of serum TNF-α (+/− SEM) were not significantly different between mosquito and needle inoculated mice. C) IL-4. Means of serum IL-4 (+/−SEM) were not significantly different between mosquito and needle inoculated mice.

Figure 6

Recapitulation of the DENV transmission cycles. On day 3 post infection (dpi), a viremic mouse was fed on by naïve mosquitoes (I), resulting in three disseminated infections from naïve mosquito exposure. The resulting body and leg titers from these mosquitoes are shown in the table (II). A naïve mouse was then exposed to these mosquitoes 16 dpi and the resulting viremia from that successful transmission is shown (III).