Hantaviruses : shedding, stability and induction of apoptosis

Hantaviruses are spread and maintained in different rodent and insectivore species worldwide. Humans are believed to be infected mainly by inhalation of aerosolized contaminated rodent excreta. The natural hosts are generally unaffected by the virus, whereas infection of humans can result in either hemorrhagic fever with renal syndrome (HFRS) in Europe and Asia, or hantavirus cardiopulmonary syndrome (HCPS) in the Americas. Infection can also be asymptomatic and the severity of disease partly depends on the hantavirus causing the infection. An increased capillary permeability is the main manifestation for the disease in humans, but the exact mechanisms behind the pathogenesis are unclear. In Sweden and other parts of northern Europe, Puumala (PUUV) hantavirus causes a relatively mild form of HFRS called nephropathia epidemica (NE).

In this thesis, we have followed hantavirus shedding from the host, observed its stability in the environment, studied cytopathogenicity as well as pathogenesis in humans and finally investigated the possibility of transmission between humans.

In detail, we used real-time RT-PCR to study how and when PUUV hantavirus is secreted from the natural host. We observed a clear peak in PUUV-RNA shed in saliva, urine and feces at around 3 weeks after experimental infection of bank voles. Further we showed that all types of excretions were infectious when inoculated intranasally in naïve bank voles, indicating that saliva can transfer the virus via other routes than biting. We also observed a remarkable ex vivo stability for Hantaan virus, with infectious virus observed after nearly hundred days incubation at 4ºC. Interestingly, this stability was not exceptional for hantaviruses when compared to vector-borne members of the Bunyaviridae family.

Further we wanted to study if the disease in humans could be due to induction of apoptosis and we measured apoptosis both in hantavirus-infected Vero-E6 cells and in hospitalized NE-patients. Increased apoptosis was not observed in vitro. However, we found increased levels of serum perforin, granzyme B, and the epithelial cell apoptosis marker caspase cleaved cytokeratin 18 in the patient samples, suggesting that tissue damage is immune-mediated and that apoptosis contributes significantly to the damage.

Andes hantavirus, spread in South America, is the only known hantavirus with evidence of person-to-person transmission. We detected PUUV-RNA in saliva from NE-patients. However, the patient saliva did not induce seroconversion in inoculated bank voles, indicating that it did not contain infectious virus. This might be due to the fact that human saliva, and especially the salivary component mucin, was able to partly inactivate hantavirus.

List of scientific papers

I. Hardestam J, Karlsson M, Falk K, Olsson G, Klingström J, Lundkvist Å (2008). Puumala hantavirus excretion kinetics in bank voles (Myodes gladiolus). [Accepted]
https://doi.org/10.3201/eid1408.080221

II. Hardestam J, Simon M, Hedlund KO, Vaheri A, Klingström J, Lundkvist A (2007). Ex vivo stability of the rodent-borne Hantaan virus in comparison to that of arthropod-borne members of the Bunyaviridae family. Appl Environ Microbiol. 73(8): 2547-51.
https://doi.org/10.1128/AEM.02869-06

III. Hardestam J, Klingström J, Mattsson K, Lundkvist A (2005). HFRS causing hantaviruses do not induce apoptosis in confluent Vero E6 and A-549 cells. J Med Virol. 76(2): 234-40.
https://doi.org/10.1002/jmv.20347

IV. Klingström J, Hardestam J, Stoltz M, Zuber B, Lundkvist A, Linder S, Ahlm C (2006). Loss of cell membrane integrity in puumala hantavirus-infected patients correlates with levels of epithelial cell apoptosis and perforin. J Virol. 80(16): 8279-82.
https://doi.org/10.1128/JVI.00742-06

V. Pettersson L, Klingström J, Hardestam J, Lundkvist A, Ahlm C, Evander M (2008). Hantavirus RNA in saliva from patients with hemorrhagic fever with renal syndrome. Emerg Infect Dis. 14(3): 406-11.
https://doi.org/10.3201/eid1403.071242

VI. Hardestam J, Pettersson L, Ahlm C, Evander M, Lundkvist Å, Klingström J (2008). Antiviral effect of human saliva against hantavirus. [Submitted]