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 PUBLICATIONS I. Jonas Hardestam, Malin Karlsson, Kerstin Falk, Gert Olsson, Jonas Klingström and Åke Lundkvist. Puumala hantavirus excretion kinetics in bank voles (Myodes glareolus) Emerging Infectious Diseases, Accepted II. Jonas Hardestam, Melinda Simon, Kjell-Olof Hedlund, Antti Vaheri, Jonas Klingström and Åke Lundkvist Ex vivo stability of the rodent-borne Hantaan virus in comparison to that of arthropod-borne members of the Bunyaviridae family. Applied and Environmental Microbiology. 2007, 73: 2547-51. PMID: 17337567 III. Jonas Hardestam, Jonas Klingström, Karin Mattsson and Åke Lundkvist HFRS causing hantaviruses do not induce apoptosis in confluent Vero E6 and A-549 cells. Journal of Medical Virology. 2005, 76: 234-240. PMID: 15834879 IV. Jonas Klingström, Jonas Hardestam, Malin Stoltz, Bartek Zuber, Åke Lundkvist, Stig Linder, and Clas Ahlm Loss of cell membrane integrity in Puumala hantavirus-infected patients correlates with levels of epithelial cell apoptosis and perforin. Journal of Virology. 2006, 80: 8279-8282. PMID: 16873286 V. Lisa Pettersson, Jonas Klingström, Jonas Hardestam, Åke Lundkvist, Clas Ahlm och Magnus Evander. Hantavirus RNA in saliva from patients with hemorrhagic fever with renal syndrome. Emerging Infectious Diseases. 2008, 14: 406-411. PMID: 18325254 VI. Jonas Hardestam, Lisa Pettersson, Clas Ahlm, Magnus Evander, Åke Lundkvist and Jonas Klingström. Antiviral effect of human saliva against hantavirus. Submitted CONTENTS 1 Hantavirus background ...............................................................................1 1.1 The discovery of a new zoonotic agent .............................................1 1.2 The hantaviruses................................................................................1 1.2.1 Hantavirus in Sweden............................................................8 1.3 Structure and replication....................................................................9 1.4 Clinical syndromes ..........................................................................11 1.4.1 HFRS...................................................................................11 1.4.2 HCPS...................................................................................11 1.5 Treatment, prevention and control ..................................................12 1.5.1 Antiviral drugs.....................................................................12 1.5.2 Vaccines ..............................................................................12 1.5.3 Passive immunization..........................................................12 1.5.4 Control.................................................................................13 2 Transmission .............................................................................................14 2.1 Between hosts and from host to human...........................................14 2.1.1 Host factors..........................................................................14 2.1.2 Host population density.......................................................16 2.1.3 Environmental factors .........................................................17 2.1.4 Shedding of virus.................................................................17 2.1.5 Virus ex vivo stability ..........................................................18 2.2 Host to human transmission ............................................................18 3 Immune response and pathogenesis in humans ........................................20 3.1 Why do we get sick (and not the rodents)? .....................................20 3.2 The role of endothelial cells in pathogenesis ..................................20 3.3 Human immune response against hantavirus infection...................21 3.3.1 Innate immunity ..................................................................21 3.3.2 Cytokines.............................................................................22 3.3.3 Humoral response................................................................22 3.3.4 Adaptive cellular response ..................................................23 3.3.5 Immune complexes and complement activation .................23 4 Apoptosis ..................................................................................................24 4.1 History .............................................................................................24 4.2 Apoptosis pathways.........................................................................24 4.3 Viruses and apoptosis ......................................................................26 4.3.1 Hantaviruses and apoptosis .................................................27 5 Person-to-person transmission ..................................................................29 5.1 Andes hantavirus .............................................................................29 5.1.1 How is the virus transmitted between humans? ..................30 5.2 Other hantaviruses? .........................................................................30 5.3 Anti-viral effect of human saliva.....................................................31 6 Aims ..........................................................................................................33 7 Results and discussion.............................................................................. 34 7.1 The shedding of Puumala virus from the natural host (paper I) ..... 34 7.2 Ex vivo stability of Hantaan Virus (paper II) .................................. 34 7.3 Absence of apoptosis in vitro (paper III) ........................................ 35 7.4 Induction of apoptosis in vivo (paper IV) ....................................... 36 7.5 Puumala virus RNA in human saliva (papers V and VI)................ 37 7.6 Anti hantavirus effect of human saliva (paper VI) ......................... 38 8 Populärvetenskaplig sammanfattning på svenska (summary in Swedish)39 9 Acknowledgements .................................................................................. 42 10 References ................................................................................................ 45 LIST OF ABBREVIATIONS ANDV Andes virus CCHFV Crimean Congo hemorrhagic fever virus CD cluster of differentiation CK 18 cytokeratin 18 CPE cytopathic effect cRNA complementary RNA CTL cytotoxic T-lymphocyte DAF decay-accelerating factor DISC death inducing signalling complex DNA deoxyribonucleic acid DOBV Dobrava virus EBV Epstain-Barr virus ELISA enzyme-linked immunosorbent assay EM electron microscopy ER endoplasmic reticulum FADD Fas-associated death domain Gc carboxyl-terminal part of glycoprotein precursor Gn amino-terminal part of glycoprotein precursor gp glycoprotein GPC glycoprotein precursor GrB granzyme B HCPS hantavirus cardiopulmonary syndrome HEK human embryonic kidney HFRS hemorrhagic fever with renal syndrome HIV human immunodeficiency virus HLA human leucocyte antigen HUVEC human umbilical vein endothelial cells ICAD inhibitor of caspase activated deoxyribonuclease IFN interferon Ig immunoglobulin IL interleukin im intramuscular in intranasal ip intraperitoneal LDH lactate dehydrogenase mRNA messenger RNA MxA myxovirus resistance A NE nephropathia epidemica NK natural killer NO nitric oxide NYV New York virus PAMP pathogen-associated molecular pattern PARP poly (ADP-ribose) polymerase PHV Prospect Hill virus PRR pattern recognition receptor PUUV Puumala virus RdRp RNA-dependent RNA-polymerase RNA ribonucleic acid RT-PCR reverse transcriptase polymerase chain reaction S, M, L-segment small, medium, large segment SAAV Saaremaa virus sc subcutaneous SEOV Seoul virus SFSV Sandfly fever Sicilian virus SLPI secretory leucocyte protease inhibitor SNV Sin Nombre virus TNF tumor necrosis factor TNF-R tumor necrosis factor receptor TRADD TNF receptor-associated death domain TRAIL TNF-related apoptosis inducing ligand TRAIL-R TNF-related apoptosis inducing ligand receptor TULV Tula virus TUNEL terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling UV ultraviolet VEGF vascular endothelial growth factor vRNA viral RNA For a complete list of hantavirus abbreviations see table 1.