Hantaviruses : animal models, immunology and pathogenesis

Abstract

Hantaviruses cause two serious and often fatal human zoonotic diseases: hemorrhagic fever with renal syndrome (HFRS) in Eurasia, and hantavirus cardiopulmonary syndrome (HCPS) in the Americas. Hantaviruses are rodent-borne and are transmitted to humans via inhalation of contaminated rodent excreta. It is well known that the fatality after hantavirus infection varies depending on the causative virus. However, the mechanisms of the pathogenesis are at present mainly unknown, but as hantavirus infection of cells per se is nonpathogenic, it is believed that the diseases are mainly immune-mediated. Accumulating data support this view: a strong and specific immune response is induced at the onset of disease, high levels of circulating cytokines, especially interleukin 6 (IL-6), IL-10 and tumor necrosis factor alpha (TNF-alpha), have been detected in Puumala virus (PUUV) infected patients, elevated NO-production has been found in PUUV and Sin Nombre virus (SNV) infected patients, and very high levels of anti-SNV specific cytotoxic T cells have been detected in SNV-infected patients.

As of today there is no specific treatment available for HFRS/HCPS, and although vaccines against Hantaan virus (HTNV) exist, there is a clear need for safe effective vaccines and therapeutics. A major obstacle in studying the medical aspects of HFRS/HCPS has been the lack of adequate animal models mirroring human disease that can provide tools for studies on immune responses, pathogenesis, antivirals and vaccine candidates. The aims of this thesis were to develop hantavirus animal models, and to further use the models to study passive immunization, vaccinations and infection. We also investigated if NO was elevated in our animal models, and the possible antiviral effect of NO on hantaviruses.

When cynomolgus macaques were infected with wild-type PUUV they showed typical signs of mild HFRS including fever, lethargy, anorexia and proteinuria/hematuria. The levels of NO, IL6, IL-10 and TNF-alpha, C-reactive protein and creatinine were elevated, and the antibody responses also mimicked those observed in PUUV-infected patients. By using this model, we tested if passive immunizations, with a cocktail of two neutralizing monoclonal antibodies (mAbs), could protect monkeys from a subsequent infection. Of the two monkeys given mAbs, one was totally protected from all symptoms, as well as from elevated levels of IL-6, IL-10 and TNF-alpha, while the other monkey developed more severe symptoms than the two control monkeys, and showed clearly elevated levels of IL-6 and IL-10, as well as of TNF-alpha.

PUUV-spillover infections in wild-caught rodents of different species were proven, and we could also experimentally infect various rodent species with PUUV, Tula virus, Dobrava virus (DOBV) and Saaremaa virus (SAAV). Immunization of mice with recombinant DOBV nucleocapsid protein (rDOBV N) emulsified in Alum, Freund's or PBS, were followed by DOBVchallenge. Although rDOBV N together with Alum and Freund's induced strong immune responses, only rDOBV N given with Freund's adjuvant protected against virus challenge, suggesting that the strong anti-N protein T helper 2 type of immune response induced by Alum is not protective in mice.

Infection of suckling mice with DOBV, but not the closely related SAAV, was lethal An elevated level of NO-production was detected in the lethally DOBV-infected mice, but not in SAAVinfected or control mice. Adult mice infected for 49-60 days with HTNV, DOBV, SAAV and PUUV seroconverted, but showed no lethality or symptoms.

By using exogenous NO-donors, and endogenously produced NO, we showed that NO has antiviral effects against hantavirus replication and mature virions. In contrast to the findings in HFRS/HCPS patients, no systemically elevated levels of NO was observed in asymptomatic HTNV-infected adult mice.