Can non-human primates serve as models for investigating dengue disease pathogenesis?

Abstract

Dengue Virus (DV) infects between 50 and 100 million people globally, with public health costs totaling in the billions. It is the causative agent of dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), vector-borne diseases that initially predominated in the tropics. Due to the expansion of its mosquito vector, Aedes spp., DV is increasingly becoming a global problem. Infected individuals may present with a wide spectrum of symptoms, spanning from a mild febrile to a life-threatening illness, which may include thrombocytopenia, leucopenia, hepatomegaly, hemorrhaging, plasma leakage and shock. Deciphering the underlining mechanisms responsible for these symptoms has been hindered by the limited availability of animal models that can induce classic human pathology. Currently, several permissive non-human primate (NHP) species and mouse breeds susceptible to adapted DV strains are available. Though virus replication occurs in these animals, none of them recapitulate the cardinal features of human symptomatology, with disease only occasionally observed in NHPs. Recently our group established a DV serotype 2 intravenous infection model with the Indian rhesus macaque, which reliably produced cutaneous hemorrhages after primary virus exposure. Further manipulation of experimental parameters (virus strain, immune cell expansion, depletion, etc.) can refine this model and expand its relevance to human DF. Future goals include applying this model to elucidate the role of pre-existing immunity upon secondary infection and immunopathogenesis. Of note, virus titers in primates in vivo and in vitro, even with our model, have been consistently 1000-fold lower than those found in humans. We submit that an improved model, capable of demonstrating severe pathogenesis may only be achieved with higher virus loads. Nonetheless, our DV coagulopathy disease model is valuable for the study of select pathomechanisms and testing DV drug and vaccine candidates.

Keywords: bone marrow; dengue virus; disease pathogenesis; hemorrhage; non-human primate; platelet; platelet-lymphocyte aggregate; rhesus macaque.

Figure 1

Hematomas are seen in intravenously inoculated rhesus macaques. (A) Indian rhesus macaques were injected intravenously with 1 × 10⁷ PFUs of DV2 16681 as previously reported (Onlamoon et al., 2010). Hematomas of various degrees of severity were present on Days 3 till 14 PI. Prominent ecchymoses were visible in two young male animals, RNell and RYc11, on Day 7. (B) Four Chinese rhesus macaques were injected intravenously (n = 2) or subcutaneously (n = 2) with 1 × 10⁷ PFUs of DV2 16681 strain. Hemorrhaging was only observed in 1 of 2 IV-injected monkeys (GT49), depicted in the picture above on Day 6 PI. No hematomas were observed in subcutaneously inoculated macaques.

Figure 2

Peak DV titers in rhesus macaque BMs is markedly lower than that of humans. BMs were acquired and infected as previously described (Clark et al., 2012). Samples from Days 1 through 14 were quantified by realtime PCR. Human (red) and monkey (green) titers are depicted in RNA copy numbers per ml. The in vitro experimentation of whole BM indicates that human BM is able to produce far more virus than monkey BM. Titers appear to max out on average closer to Day 1 in monkey BM but reach their peak (~1000-fold higher) on Day 3 PI in humans.

Figure 3

Dynamics of lymphocyte-platelet aggregates (LymPA) during DV infection. Indian and Chinese rhesus macaques were infected as detailed in Figure 1. In addition, the Chinese macaques were challenged 2 months later with DV3 strain Hawaii. Peripheral blood samples obtained on Days 1 through 14 were subjected to flow cytometric analysis with CD45, CD41, CD61, and CD62P fluorescent antibodies. The frequencies of CD45⁺CD41⁺CD61⁺CD62P⁻ cells over time is graphed. (A) Panels to illustrate the gating strategy employed to analyze lymphocyte-platelet aggregates (LymPA). (B) The kinetics of LymPA in Indian rhesus macaques. The top graph displays LymPA frequencies from 3 individual macaques and the bottom graph, the average population frequency from 5 primates. The LymPA population is down-regulated during DV infection in Indian rhesus macaques. (C) LymPA kinetics in subcutaneously and intravenously infected Chinese rhesus macaques during primary DV2 (green line) and secondary DV3 infection (red line). The frequency of LymPA increases late after primary but not after secondary infection.