Transmission and Pathogenesis of Hantavirus

TRANSMISSION AND PATHOGENESIS OF HANTAVIRUS

Lisa Pettersson

Department of Clinical Microbiology

Umeå 2015

Responsible publisher under Swedish law: the Dean of the Medical Faculty This work is protected by the Swedish Copyright Legislation (Act 1960:729) ISBN: 978-91-7601-225-3. ISSN: 0346-6612 New series nr: 1701 Cover photo by Lisa Pettersson Elektronic version available at http://umu.diva-portal.org/ Printed by: Print & Media Umeå, Sweden 2015

To Julia, Anton and Konstantin

Table of Contents

Table of Contents i Abstract iii Sammanfattning på svenska v Original Papers vii 1.Introduction 1 1.1 The hantavirus genus 1 1.1.1 The family Bunyaviridae 1 1.1.2 Hantaviruses and their hosts 1 1.1.3 The ongoing discovery of hantaviruses 4 1.2.The virion and the replication cycle 5 1.2.1 The virion 5 1.2.2 The replication cycle 6 1.3. Transmission of hantaviruses 8 1.3.1. Shedding of virus in the rodent host 8 1.3.2. Infectivity and stability of the virus 8 1.3.3. Rodent-to-rodent transmission 9 1.3.4. Rodent to human transmission 10 1.3.5 Human-to-human transmission 12 1.5. The diseases 15 1.6. Pathogenesis 16 1.6.1. Animal Models for Pathogenesis 16 1.6.2 The incubation period 17 1.6.3. Viral entry and dissemination in the human body 17 1.6.4. An overactive immune response 19 1.6.5 The humoral immune response 20 1.6.6. Viremia 21 1.7. Treatment 21 2. Aims 23 3. Results and discussion 24 3.1. Outbreak of Puumala virus infection, Sweden. 24 3.2. Influence of human saliva on PUUV infectivity 25 3.3. Viral load and humoral immune response in association with disease severity in Puumala hantavirus-infected patients―implications for treatment. 26 4. Conclusions 28 References 31

i ii Abstract

Hantaviruses are the causative agents of hemorrhagic fever with renal syndrome (HFRS) in Eurasia, and of hantavirus cardiopulmonary syndrome (HCPS) in the Americas. Transmission to humans usually occurs by inhalation of aerosolized virus-contaminated rodent excreta. To date, human-to-human transmission has only been described for the Andes hantavirus. The mode of transmission of Andes hantavirus is not yet known, but transmission through saliva has been suggested. In Sweden, we have one hantavirus that is pathogenic to humans, Puumala virus (PUUV), which is endemic in Central and Northern Europe. It induces a relatively mild form of HFRS, also called nephropathia epidemica (NE). The rodent reservoir is the bank vole (Myodes glareolus). The mechanism behind the pathogenesis of hantavirus is complex and probably involves both virus-mediated and host- mediated mechanisms. The aim of this project was to investigate the transmission mechanisms and pathogenesis of hantavirus disease in humans.

In our first study, we described the largest outbreak of PUUV so far in Sweden. We investigated factors that might be important for causing the outbreak, and suggested that a peak in the bank vole population together with concurrent extreme weather conditions most probably contributed to the outbreak.

Our next studies concentrated on human-to-human transmission of hantaviruses. We found PUUV RNA in saliva from PUUV- infected patients, suggesting that there is PUUV in the saliva of infected humans, although no person-to person transmission appears to occur with PUUV. In the studies that followed, we showed that human saliva and human salivary components could inhibit hantavirus replication. We also found PUUV-specific IgA in the saliva of PUUV-infected patients, which might prevent person-to-person transmission of the virus.

In the final study, we focused on the pathogenesis of NE. One hundred five patients were included in a prospective study. They were divided into a group with mild disease and a group with moderate or severe disease. We found that the immune response had a dual role in disease development. It was partly responsible for development of severe disease, with significantly higher amounts of neutrophils in severely ill patients, but it was also protective against severe disease, because patients with mild disease had higher levels of PUUV-specific IgG.

In conclusion, a peak in the bank vole population in combination with extreme weather will increase the risk of human infection, PUUV RNA is present in saliva, PUUV-specific IgA and salivary components inhibit person-to-person transmission of PUUV, and the immune response is important for the pathogenesis of PUUV and the severity of the disease.

iii iv Sammanfattning på svenska

HANTAVIRUS ÖVERFÖRING OCH PATOGENES

Hantavirus är en grupp av virus som finns hos gnagare som bär på viruset utan att själva bli märkbart sjuka. Varje hantavirus har anpassat sig till sin egen art av gnagare som de infekterar (kallas virusets reservoar). Hantaviruset kan överföras till människor från gnagare och kallas då för en zoonos eftersom detsprids från djur till människa. I människa orsakar hantavirus blödarfeber med njurpåverkan i Eurasien och blödarfeber med med hjärt och lungpåverkan i Nord- och Sydamerika.

I Sverige har vi bara ett hantavirus som är sjukdomsframkallande hos människor, Puumala-viruset som även finns i delar av övriga Europa. Det framkallar en relativt mild form av blödarfeber, som kallas sorkfeber eller Nephropathia epidemica. Puumala-virusets reservoar är skogssorken (Myodes glareolus).

Människor smittas oftast av hantavirus när de andas in infekterat damm som innehåller utsöndringar (avföring, urin eller saliv) från gnagare som har torkat in och sedan blivit luftburet. Vad man vet hittills så finns det bara ett hantavirus som smittar från person till person, för övriga hantavirus är människan en ”dead end”. Det virus som kan smitta från person till person heter Andes hantavirus och finns i Sydamerika. Andes hantavirus har en mus som reservoar från vilken människor kan smittas, sedan har smittan i vissa fall förts vidare från människa till människa, som tur är har dessa utbrott gått att stoppa. Fastän utbrotten har varit små har många personer dött, eftersom dödligheten är så hög, ungefär 30-40% av de diagnostiserade fallen dör. Hur Andes hantavirus överförs från människa till människa är inte känt men överföring genom saliv har föreslagits.

Hur viruset ger upphov till sjukdom hos människa är inte klarlagt. Studier talar för att mekanismen bakom sjukdomsutvecklingen (den så kallade patogenesen) hos hantavirusorsakade blödarfebrar är komplex. Sannolikt beror patogenesen både på egenskaper hos viruset och värden d.v.s. människan som är smittad av viruset. Vårt mål med detta projekt var att undersöka vad som hindrar överföring av Puumala hantavirus från människa till människa och att undersöka hur virusinfektionen påverkar sjukdomsutvecklingen hos människan.

v I vår första studie beskrev vi det största utbrottet av sorkfeber hittills i Sverige och vi undersökte faktorer som kan ha orsakat utbrottet. Vi föreslog att en topp i skogssorkpopulationen samtidigt med extremt varmt väder troligen bidrog till utbrottet. Utbrottet skedde i december och det extremt varma vädret medförde att snön smälte bort. Sorkarna bor vanligtvis under snön på vintern, vi tror att frånvaro av snötäcke fick sorkarna att söka sig till byggnader för att söka skydd och där kom i kontakt med människor.

Våra efterföljande studier fokuserade på överföring av hantavirus från människa till människa. Vi hittade Puumala-virusets arvsmassa (RNA) i saliv från sorkfeberpatienter, vilket tyder på att det finns Puumala-virus i saliven hos infekterade människor, även om ingen överföring från person till person verkar inträffa. I efterföljande studier visade vi att mänsklig saliv och mänskliga salivkomponenter minskar hantavirus smittsamhet. Vi fann också Puumala-virusspecifika IgA-antikroppar i saliven från sorkfeberpatienter, vilket kan förhindra överföring från person till person.

I den sista studien fokuserade vi på patogenesen hos människor efter hantavirusinfektion. 105 patienter ingick i en prospektiv studie och delades in i en grupp med mild sjukdom och en grupp med måttlig/svår sjukdom. Vi hittade en dubbel roll hos immunsvaret för sjukdomsutvecklingen. Immunsvaret var delvis ansvarig för utveckling av svår sjukdom med betydligt högre mängd neutrofiler hos svårt sjuka patienter, men det var också skyddande mot allvarlig sjukdom, eftersom patienter med en mild sjukdom hade högre nivåer av Puumalavirusspecifika IgG-antikroppar. Detta talar för att behandling med IgG-antikroppar specifikt riktade mot hantavirus skulle kunna vara effektiv hos hantavirusinfekterade patienter.

Sammanfattningsvis; en topp i skogssorkspopulationen i kombination med extremt väder ökar risken för infektion hos människor; Puumala-virus arvsmassa (RNA) finns i saliv; Puumala-virusspecifika IgA-antikroppar och salivkomponenter hämmar överföring av Puumalavirus från person till person; immunsvaret är viktigt för Puumala-virus patogenes och sjukdomens svårighetsgrad.

vi Original Papers

The thesis is based on the following papers, which will be referred to in the text by their Roman numbers.

I. PETTERSSON, L., BOMAN, J., JUTO, P., EVANDER, M. & AHLM, C. 2008a. Outbreak of Puumala virus infection, Sweden. Emerg Infect Dis, 14, 808-10.

II.PETTERSSON, L., KLINGSTRÖM, J., HARDESTAM, J., LUNDKVIST, A., AHLM, C. & EVANDER, M. 2008b. Hantavirus RNA in saliva from patients with hemorrhagic fever with renal syndrome. Emerg Infect Dis, 14, 406-11.

III. HARDESTAM, J., PETTERSSON, L., AHLM, C., EVANDER, M., LUNDKVIST, A. & KLINGSTROM, J. 2008. Antiviral effect of human saliva against hantavirus. J Med Virol, 80, 2122-6.

IV.PETTERSSON, L., RASMUSON, J., ANDERSSON, C., AHLM, C. & EVANDER, M. 2011. Hantavirus-specific IgA in saliva and viral antigen in the parotid gland in patients with hemorrhagic fever with renal syndrome. J Med Virol, 83, 864-70.

V.PETTERSSON, L., THUNBERG, T., ROCKLÖV, J., KLINGSTRÖM, J., EVANDER, M. & AHLM, C. 2014. Viral load and humoral immune response in association with disease severity in Puumala hantavirus-infected patients- -implications for treatment. Clin Microbiol Infect, 20, 235-41.

Paper III and IV were kindly provided by the publisher

vii

viii

1.Introduction

1.1 The hantavirus genus

1.1.1 The family Bunyaviridae

Hantaviruses belong to the family Bunyaviridae, which includes more than 350 viruses (Elliott, 2014) and is the largest family of animal viruses (Mertz, 2009). Members of the Bunyaviridae have common features, with similar virion structure, genome composition, and major proteins, and they also have similar replication strategies (Plyusnin et al., 2012). This will be discussed further in Chapter 2. The family is divided into five genera: Orthobunyavirus, Phlebovirus, Nairovirus, Hantavirus, and Tospovirus (Plyusnin et al., 2012). All genera except the Tospoviruses (which are plant viruses) are able to infect vertebrate hosts and to cause disease in humans (Mertz, 2009, Plyusnin et al., 2012). The genera Orthobunyavirus, Phlebovirus, and Nairovirus replicate alternately in vertebrates and arthropods. Humans are not thought to be a natural reservoir for any of the Bunyaviridae (Mertz, 2009, Plyusnin et al., 2012). Important human pathogens are represented in each genus of the Bunyaviridae, except for Tospovirus. For example, Puumala virus is a Hantavirus, Crimean-Congo hemorrhagic fever virus is a Nairovirus, Rift Valley fever virus is a Phlebovirus, and California encephalitis virus is an Orthobunyavirus.

1.1.2 Hantaviruses and their hosts

The natural hosts for hantaviruses are rodents and insectivores. Each hantavirus has its own specific host species (Vapalahti et al., 2003). In the host, the infection is persistent (Hardestam et al., 2008, Yanagihara et al., 1985, Hutchinson et al., 2000, Padula et al., 2004, Bernshtein et al., 1999) and goes on without any apparent symptoms (Bernshtein et al., 1999), although some studies have indicated that hantavirus infection has some adverse effects on the host (Kallio et al., 2007, Netski et al., 1999). The persistent infection in the reservoir host is attributed to the upregulation of regulatory responses and downregulation of proinflammatory responses (Li

and Klein, 2012). The evolution of the hantaviruses has followed that of their hosts, and their phylogenetic trees reflect each other (so-called virus- host co-divergence) (Sironen and Plyusnin, 2011). The fact that hantaviruses exist in both rodents and insectivores supports the hypothesis that hantaviruses are ancient viruses and already existed when the rodents and insectivores became separated on the evolutionary tree about 90‒100 million years ago (Plyusnin and Sironen, 2014). The main mechanism of hantavirus diversification is through genetic drift. However, genetic shift through segment reassortment and recombination can also occur. Both inter-species and intra-species reassortment has been shown (Sironen and Plyusnin, 2011). As with other RNA viruses, the error rate per replication cycle is high―but the evolutionary rate of hantaviruses is low. This can be explained by the fact that they have adapted to their rodent host for millions of years, and there is little selection pressure to change (Sironen and Plyusnin, 2011). However, when propagated in cell culture the hantavirus adapts to its new environment and has even been shown to lose its ability to infect the natural host (Lundkvist et al., 1997). Consistent with the co-divergence of their natural hosts, hantaviruses have been divided into groups according to their hosts. See Table 1.

TABLE 1. Species in the hantavirus genus, recognized by the International Committee on Taxonomy of Viruses (ICTV) in the ninth report of the ICTV.

Hantaviruses carried by the family Cricetidae, subfamily Arvicolinae (voles and lemmings from all over the world) Puumala (PUUV) Myodes glareolus, bank vole Tula (TULV) Microtus arvalis, European common vole Topografov (TOPV) Lemmus sibiricus, lemming Khabarovsk (KHAV) Microtus maximowiczii Maximowicz vole Microtus Fortis, reed vole Prospect Hill (PHV) Microtus pennsylvanicus, meadow vole Isla Vista (ISLAV) Microtus californicus, Californian vole Hantaviruses carried by the family Cricetidae, subfamily Sigmodontinae (New world rats and mice) Andes (ANDV) Oligoryzomys longicaudatus, long-tailed pygmy rice rat

Bayou (BAYV) Oryzomys palustris marsh rice rat

Black Creek Canal Sigmodon hispidus, hispid cotton rat (BCCV)

Cano Delgadito (CADV) Sigmodon alstoni, Alston’s cotton rat

Laguna Negra (LANV) Calomys laucha, vesper mouse

Muleshoe (MULV) Sigmodon hispidus, hispid cotton rat

Rio Mamore (RIOMV) Oligoryzomys microtis, small-eared pygmy rice rat

Hantaviruses carried by the family Cricetidae, subfamily Neotominae (New World mice)

Sin Nombre (SNV) Peromyscus maniculatus, deer mouse New York (NYV) Peromyscus leucopus, white-footed mouse El Moro Canyon Reithrodontomys megalotis, western harvest (ELMCV) mouse Rio Segundo (RIOSV) Reithrodontomys mexicanus, Mexican harvest mouse Hantaviruses carried by family Muridae, subfamily Murinae (Old world rats and mice) Hantaan (HTNV) Apodemus agrarius coreae, dark-striped field mouse Dobrava (DOBV) Apodemus flavicollis, yellow-necked mouse Saaremaa (SAAV) Apodemus agrarius agrarius, striped field mouse Thailand (THAIV) Bandicota indica, great bandicoot rat Seoul (SEOV) Rattus norvegicus, Norway rat Rattus rattus, black rat Hantaviruses carried by insectivores Thottapalayam (TPMV) Suncus murinus, Asian house shrew Table references: (Plyusnin et al., 2012, Sironen and Plyusnin, 2011)

1.1.3 The ongoing discovery of hantaviruses

Hantaviruses do not grow easily in cell culture, and all initial attempts to isolate the causative agent from acutely ill patients were unsuccessful (Lee et al., 2014, French et al., 1981). Attempts to isolate the agent responsible for Korean hemorrhagic fever (KHF) started in 1952, when over 3,000 UN soldiers contracted the disease in the Korean War (French et al., 1981, Lee et al., 2014). It was suspected for a long time that KHF and similar diseases were caused by the same etiological agent, and that the diseases were acquired by contact with rodents and rodent excreta (Lee et al., 1978). Finally in 1976, the causative agent was detected with indirect immunofluorescence when sera from patients with KHF were applied to acetone-fixed lung sections of Apodemus agrarius mice (Lee et al., 1978). The virus was subsequently isolated in cell culture (Lee et al., 2014, French et al., 1981) and later named Hantaan virus (HTNV) after Hantaan river. In Sweden in 1935, Myhrman and Zetterholm described (independently of each other) a disease that later came to be called nephropathia epidemica (NE) (Myhrman, 1951). Researchers in Finland used the same methodology as Lee, and found that sera from NE patients reacted with lungs of bank voles (Myodes glareolus) which led to the discovery of Puumala virus (PUUV) (Brummer-Korvenkontio et al., 1980). Subsequently, other hantaviruses in Europe and Asia were discovered; for example, Dobrava virus (DOBV) was found in the yellow-necked mouse (Apodemus flavicollis) in Slovenia in 1992 (Avsic-Zupanc et al., 1992). In 1993, the first pathogenic hantavirus in the American continent was discovered when there was an outbreak of acute respiratory distress syndrome with high mortality in the Four Corners region of the southwestern United States. Antibodies in serum from the patients were reactive to hantaviruses using ELISA and IFA assays, suggesting that the illness was caused by a cross-reactive hantavirus. This was confirmed when patient tissues were examined; hantavirus genetic sequences were detected with RT-PCR and hantavirus nucleocapsid protein with immunohistochemistry (IHC) (Lee et al., 2014). Since 1993, several hantaviruses have been found in North and South America. The latest (ninth) report of the International Committee on Taxonomy of Viruses (ICTV) listed 23 species in the hantavirus genus and 30 provisional species (Table 1) (Plyusnin et al., 2012). A large proportion of these tentative species have insectivores (shrews and moles) as hosts. The Thottapalayam virus (TPMV) has an insectivore host (the shrew Suncus murinus) and for a long time it was thought to be an exception to the rest of the hantaviruses, since they all had rodent hosts. However, since 2006 several hantaviruses

have been discovered in shrews and moles in Africa, America, Europe, and Asia (Klempa et al., 2007, Song et al., 2007, Avsic-Zupanc et al., 2013). So far, it is not known whether these insectivore-borne viruses are pathogenic to humans (Sironen and Plyusnin, 2011, Avsic-Zupanc et al., 2013). Recently, “new” hantaviruses have been found in bats. It is not known whether bats are the natural host or just spill-over from another, unknown host (Avsic- Zupanc et al., 2013, Weiss et al., 2012).

1.2.The virion and the replication cycle

1.2.1 The virion

At first glance, hantaviruses have a simple structure. They are enveloped negative-stranded RNA viruses with only four structural proteins. Each protein is multifunctional, however, and examples will be given in this section of how these proteins enable attachment to the host cell and then entry, replication, translation, and finally budding of new virus particles from the cell surface. Hantavirus virions vary in shape and size; they are mainly round and 120‒160 nm in diameter, but elongated tubular particles can also be seen (Huiskonen et al., 2010, Battisti et al., 2011). Hantaviruses have a lipid envelope that is covered with spike structures, which extend through the envelope―protruding both on the inside and the outside (Huiskonen et al., 2010, Battisti et al., 2011). The spikes consist of the Gn and Gc glycoproteins bound to each other. The envelope encloses three segments of negative-sense, single-stranded RNA (vRNA) named after their different sizes. The S (small) segment encodes the nucleocapsid protein (the N protein), the M (medium) segment encodes the glycoprotein precursor that is cleaved further into the Gn and Gc glycoproteins, and the L (large) segment encodes the RNA polymerase (the RdRp). In the family Bunyaviridae, each genomic RNA forms a circular molecule that comes about by base pairing between inverted complementary sequences at the 3′ and 5′ ends of linear viral RNA (Jonsson et al., 2010). The N protein encapsidates and thereby protects the genomic RNA, forming three different-sized ribonucleoproteins (RNPs). It has been suggested that the N protein forms trimers around the viral RNA molecule, which then results in long multimers (Kaukinen et al., 2001). It is often assumed that the RdRp is associated with the RNP complex, but it is still not known whether or not the RdRp is part of the RNP complex (Jonsson et al., 2010)

Nucleocapsid protein (NP)

glycoprotein (Gn)

glycoprotein (Gc)

RNA dependent RNA polymerase (RdRp)

Nucleocapsid trimer encapsidating the negative stranded RNA

Figure 1. Hantavirus, kindly provided by Marie Lindkvist

1.2.2 The replication cycle

For hantaviruses as for all bunyaviruses, all stages of replication occur in the cytoplasm (Plyusnin et al., 2012). The first step of replication is attachment of the virus to the host cell. In vitro studies have implicated several cellular proteins as hantavirus receptors. Pathogenic hantaviruses have been shown to use β3-integrins as receptors while non-pathogenic hantaviruses use β1- integrins (Gavrilovskaya et al., 1999, Gavrilovskaya et al., 2002, Gavrilovskaya et al., 1998). The gC1qR/p32 glycoprotein and decay- accelerating factor (DAF)/CD55 have also been shown to be receptors for hantaviruses (Choi et al., 2008, Krautkramer and Zeier, 2008). After binding to its receptor and possibly co-receptors, the hantavirus enters the cell by clathrin-dependent receptor-mediated endocytosis and is delivered by the endocytic pathway to the endosomes and later lysosomes (Jin et al., 2002). Like all negative-sense RNA viruses, hantaviruses carry their own RNA- dependent RNA polymerase (RdRp), which transcribes the negative-sense

vRNA into positive-sense messenger RNA (mRNA) and replicates the vRNA (Jonsson and Schmaljohn, 2001). The RdRp needs primers to initiate the transcription of vRNA into mRNA; these primers are obtained by a cap-snatching mechanism that has also been described for other negative-sense segmented RNA viruses. For hantaviruses, this process starts when the N protein binds to host mRNA caps. The N protein protects the host cell mRNA caps from the host cell’s own cellular RNA degradation machinery, which actively removes caps and degrades cellular transcripts after the completion of translation (Mir et al., 2008, Cheng and Mir, 2012). Hantaviral RdRp and N proteins have been found to be membrane-associated and localized to the perinuclear region (Kukkonen et al., 2004, Ravkov and Compans, 2001). The membrane association of the RNA polymerase would suggest that RNA synthesis is also membrane-associated. The M segment is translated into a glycoprotein precursor, which is cleaved, glycosylated, and folded in the endoplasmic reticulum (ER) to form the Gn and Gc glycoproteins (Lober et al., 2001, Shi and Elliott, 2004). After translation and replication, the assembly of newly produced virus protein and genome takes place to form new viruses. The Bunyaviridae family undergo virus assembly at the Golgi complex (Pettersson and Melin, 1996). It is common for enveloped viruses to have matrix proteins that mediate the contact between the components of the virus, to make assembly possible. The Bunyaviridae lack matrix proteins; instead, the cytoplasmatic tails of the glycoproteins act as matrix proteins and interact with the N protein for packaging of the RNP into virus particles (Hepojoki et al., 2010, Overby et al., 2007, Strandin et al., 2013). The bunyaviruses obtain their membranes from the host cell when they are budding in the Golgi complex, and they are then transported in vacuoles to the plasma membrane where they are released by exocytosis (Pettersson and Melin, 1996). However, there is evidence that hantaviruses of the New World use an alternative route whereby they are assembled and released from the cell surface. Using electron microscopy, the Sin Nombre virus has been shown to bud from the plasma membrane (Goldsmith et al., 1995).

1.3. Transmission of hantaviruses

1.3.1. Shedding of virus in the rodent host

When a rodent is infected with hantavirus, there is an acute phase with a peak in virus shedding in excretions a few weeks after infection, and the viruses are efficiently transmitted from infected hosts to naïve cage mates (so-called horizontal intracage transmission) (Hardestam et al., 2008, Yanagihara et al., 1985, Bernshtein et al., 1999, Hutchinson et al., 2000, Padula et al., 2004). Then there is a prolonged chronic stage where the virus is usually present at lower levels or absent from secretions, and the transmission is lower (Bernshtein et al., 1999, Yanagihara et al., 1985). Periods of no shedding/transmission followed by recurrence of shedding/transmission have been observed (Padula et al., 2004, Hardestam et al., 2008).

1.3.2. Infectivity and stability of the virus

Three types of secretion/excretion (i.e. saliva, faeces, and urine) have been shown to be capable of transmitting virus from an infected rodent to an uninfected rodent. For example, Hardestam et al. (2008) found that intranasal inoculation with saliva, faeces, or urine from PUUV-infected bank voles could result in infection of naïve bank voles, indicating that all three transmission routes may occur in nature (Hardestam et al., 2008). It has been shown that hantavirus has prolonged survival outside the rodent host, i.e. prolonged ex vivo stability. In laboratory experiments, the ex vivo stability of hantavirus has been shown to persist much longer in wet environments than in dry environments, and also to persist longer in cold environments than in warm environments (Hardestam et al., 2007, Kallio et al., 2006). PUUV excreted by experimentally infected donor bank voles has been shown to be transmitted indirectly between rodents through contaminated bedding, and the virus maintained its infectivity for recipient voles for 12–15 days at room temperature (Kallio et al., 2006).

1.3.3. Rodent-to-rodent transmission

It is yet not known whether hantavirus in rodents is mainly transmitted through indirect modes of transmission (aerosols) or direct modes of transmission (physical contact), and it is still unclear which route of shedding is most important for rodent-to-rodent transmission. There has been extensive research on the subject, and the results have been conflicting. Different kinds of optimal transmission strategies for different hantaviruses may have evolved during the long co-evolution between hantaviruses and their rodent hosts. Different species of rodent have different social behavior: some rodents live in groups and have friendly grooming behavior while other rodent species are more aggressive and tend to injure each other. The different rodent species also live in different climates and environments, which could also influence the mode of transmission. Yanagihara et al. (1985) proposed that saliva is the most important route of transmission of PUUV by bank voles. In their studies, horizontal intracage transmission coincided with virus shedding in saliva; the shedding of virus in urine and faeces occurred later. These authors speculated that the horizontal transmission of PUUV during the period of virus shedding in saliva is compatible with the grooming behavior of bank voles (Yanagihara et al., 1985). This result conflicts with the work of Hardestam et al. (2008), who also investigated PUUV shedding in the bank vole, and found simultaneous peaks of shedding in saliva, urine, and faeces (on days 11‒28, 14‒28, and 11‒28, respectively) (Hardestam et al., 2008). It is not only saliva and close contact that have been proposed to be important routes of transmission of PUUV. As mentioned Kallio et al. (2006) showed that PUUV was transmitted indirectly between rodents through contaminated bedding, The amount of saliva secreted into the environment is likely to be very low, so the indirect transmission through contaminated bedding was probably due to shedding of the virus in urine and faeces (Kallio et al., 2006).

Padula et al. (2004) suggested that Andesvirus (ANDV) may be mainly transmitted by saliva or saliva aerosols―rather than by faeces and urine of Sigmodontine rodents. They demonstrated horizontal intracage transmission between the rodents, but transmission was not found between animals that were separated by wire mesh, and the rodents were not infected from excrement-tainted bedding. They observed that the rodents mostly had non-aggressive contact, and they found no association between wounding and transmission (Padula et al., 2004).

For rodents that have aggressive behavior, wounding has been proposed as a way of transmission. For example Hinson et al. (2004) performed a study on wild Norway rats and found that wounded male rats were more likely to have detectable Seoul virus (SEOV) in tissues―and antibodies to SEOV―than uninjured males. Wounded males also shed more virus in excrement than uninjured males. Consequently, they suggested that wounding is the primary mode of SEOV transmission in male Norway rats (Hinson et al., 2004). In a study on Sin Nombre virus (SNV) in wild deer mice, Safronetz et.al. (2008) found high levels of SNV RNA in blood from seropositive rodents and they speculated that direct blood-borne transmission through aggressive behavior might contribute significantly to the transmission of SNV between rodents (Safronetz et al., 2008).

Studies have indicated that hantaviruses are not transmitted vertically, i.e. from mother to fetus during pregnancy or to pups during birth. Newborn rodents from persistently infected seropositive mothers have been shown to be protected from infection (Bernshtein et al., 1999, Dohmae and Nishimune, 1995). This is due to transfer of antibodies from the mother to the offspring via the placenta and through lactation (Dohmae and Nishimune, 1995). As the juveniles become older, the maternal antibodies to hantavirus disappear and later there is a reappearance of antibodies due to horizontal infection (Bernshtein et al., 1999, Borucki et al., 2000).

1.3.4. Rodent to human transmission

Humans are infected by hantaviruses mainly through inhalation of aerosolized virus-contaminated rodent excreta and the higher the number of infected rodents, the higher the risk of human infection (Jonsson et al., 2010, Olsson et al., 2003, Olsson et al., 2009). Exposure to the virus- contaminated excreta most commonly occurs indoors when rodents have entered buildings, exposure outdoors also occurs when for example doing activities such as gardening and handling hay (Olsson et al., 2003, Hjelle and Glass, 2000).

The ecology of bank voles and PUUV ‒ effects on NE occurrence in Europe

Bank voles in both northern and southern Europe transmit PUUV to humans. However, in the boreal zone of northern Europe, bank voles live under different conditions than the bank voles of southern Europe. In northern Europe, the bank vole population undergoes cycles with an increase over 3‒4 years and then there is a sudden decrease in bank vole numbers, a so-called “crash” (Kallio et al., 2006, Olsson et al., 2010, Jonsson et al.,

2010). In western and central Europe, there are generally no cyclic fluctuations in bank vole density, and seasonal variation in vole density partly depends on high production of beech and oak seeds (mast years) (Tersago et al., 2009). Consequently the incidence of NE in northern Sweden shows a periodic pattern that follows the population dynamics of the bank vole, with approximately 3-year intervals between peaks ― in contrast to Belgium where heavy mast production precedes NE peaks (Olsson et al., 2003, Clement et al., 2009).

During the winter in northern Europe, the so-called subnivean space (the space between the ground and the snow cover) is very important for bank voles. It provides them with insulation, access to food, and shelter from predators (Khalil et al., 2014). Kallio et al. (2006) showed that low temperature and high moisture prolonged virus longevity. They argued that the subnivean space protects the virus from UV light and drying during the winter. They speculated that the survival of PUUV may partly explain the regional differences in the human epidemiology of NE in Europe. In northern Europe there are thousands of cases diagnosed annually, whereas in the temperate, more populated part of Europe, the annual number of human cases amounts to hundreds (Kallio et al., 2006)

Climate effects on rodent to human transmission

Climate can have an impact on the incidence of hantavirus infections in humans in several different ways. Climate influences the survival of rodents, for example by influencing the amount of food and survival during the winter. When there are many rodents, humans are more likely to become infected. Climate can also influence the survival of the virus outside the host; with a lot of virus surviving outside the host both the rodent-to-rodent transmission and the rodent-to-human transmission increases. In a Belgian study, lower winter temperatures appeared to be strongly linked to higher prevalence of PUUV in bank voles. This can be explained by longer PUUV survival outside the host,. There was also an association between human NE cases and moist soils, which can also be explained by longer PUUV survival outside the host (Linard et al., 2007). This is in line with the results of in vitro studies of hantavirus survival outside the host under different conditions (Kallio et al., 2006).

El Niño events have been implicated as an important driver for SNV in both deer mice and humans in the southwestern United States (Hjelle and Glass, 2000, Engelthaler et al., 1999). A study in Sweden by Palo found no relationship between the North Atlantic Oscillation (NAO) and the number

of cases of HFRS in humans (Palo, 2009). The conflicting results of the studies in Sweden and North America underscore the need for more studies in this area (Palo, 2009, Dearing and Dizney, 2010). Palo suggested that the NAO index is perhaps too coarse a tool, as it translates into several weather variables such as precipitation and temperature. Studies on the incidence of NE in Belgium implicated warmer temperatures as the central factor responsible for increase in NE cases in that area. Tersago et al. (2009) found that warmer summer temperatures promote mast seed production of broadleaf trees thereby driving up food availability, which leads to an increase in vole populations, also warmer autumn temperatures were found to be associated with increased survival of voles. The fact that the combined effect of hotter summer and autumn seasons is matched by a growing trend of epidemics of NE in Belgium in recent years suggests that the increase in NE may be an effect of global warming (Clement et al., 2009). To date, there have been no attempts to investigate whether NE is associated with climate change in northern Europe (Hedlund et al., 2014). The Intergovernmental Panel on Climate Change (IPCC) predicts that ecosystems in the Arctic and subarctic area are likely to be highly affected by climate change (IPCC, Hedlund et al., 2014). The IPCC has reported that the temperature increase is greater at higher northern latitudes and that the average Arctic temperatures have increased at almost twice the average global rate in the past 100 years. It predicts that the subarctic region will also be affected, especially with warmer temperatures during winter and with more precipitation all year round (Hedlund et al., 2014, IPCC).

1.3.5 Human-to-human transmission

Humans were long considered to be a dead end for hantavirus infections. However, in 1996 in Argentina there was an outbreak of HCPS in which 16 persons were involved. Starting with one index patient, there was a cluster of cases including both healthcare workers and patients at the local hospital of a small town in the southwest part of the country . One of the patients was transported to Buenos Aires 1,400 km away, for intensive care. There, a doctor who was treating the patient fell ill with HCPS, indicating human-to- human transmission. Later, epidemiological and molecular evidence for person-to-person transmission was obtained (Wells et al., 1997, Padula et al., 1998). The causative hantavirus was found to be ANDV. Two parts of the virus genome were sequenced and all the cases in the outbreak showed the same sequence, including the doctor in Buenos Aires. The more geographically distant the cases, the more unlikely it is that there will be the same variant in the local rodent population (Padula et al., 1998). After this

outbreak, human-to-human transmission has been implicated during other outbreaks of ANDV―for example, in Chile (Toro et al., 1998, Ferres et al., 2007). A prospective study of household contacts of index case patients with HCPS showed that sex partners were at the greatest risk of person-to-person transmission. However, person-to-person transmission also occurred in people with close, non-sexual contact. Exposure to saliva was identified as a significant risk factor for person-to-person transmission (Ferres et al., 2007).

Antiviral activity of saliva

Why does ANDV spread from person to person but other hantaviruses do not? As mentioned, exposure to saliva has been identified as a risk factor for person-to-person transmission of ANDV. This indicates that other hantaviruses may be inhibited in some way in saliva and do not therefore spread from person to person. Fultz (1986) demonstrated that pretreatment of HIV-1 with saliva reduced the virus infectivity (Fultz, 1986). After this observation, there have been numerous studies on the antiviral activity of saliva―mostly on HIV-1, but also on other viruses (Shugars, 1999). There have only been a few studies of the antiviral activity of saliva on hantaviruses, which will be dealt with below. It has been found in different studies that saliva has several antiviral properties including virus-specific antibody responses, aggregation of virus particles by high-molecular-mass glycoproteins, and blocking of viral or cellular targets by inhibitory endogenous proteins (Shugars, 1999). Virus-specific antibodies have been found for several viruses, for example Dengue virus (Vazquez et al., 2007). Specific IgA antibodies have also been detected in saliva from patients with acute HCPS caused by ANDV (Padula et al., 2000). Saliva contains mucins, which are glycoproteins that have a “bottle-brush” appearance with a backbone of filamentous protein usually carrying hundreds of complex oligosaccharide structures (Linden et al., 2008). The mucins are rich in terminal sialic acids (Cohen et al., 2013). There are at least 16 human mucins, six of which are secreted and ten of which are membrane bound. Glands in the mouth secrete three types of mucins, MUC5B, MUC19, and MUC7 (Linden et al., 2008). MUC7 is the only monomeric mucin. The mucins express many of the oligosaccharide structures found on the cell surface, so microbes can attach to the mucin instead of the cell (Linden et al., 2008, Cohen et al., 2013). Mucin in human saliva has been shown to protect cells from infection with influenza A virus in vitro (Cohen et al., 2013). The mechanism behind this is that the salivary mucin presents sialylated decoys to influenza A virus. However, influensa A virus can circumvent this

mechanism; it carries with it an enzyme―a neuroaminidase―that has a sialidase activity to free virions from sialylated host mucin decoys (Cohen et al., 2013).

1.4. Epidemiology of NE in Sweden

The bank vole can be found throughout Sweden, but NE is only present in the northern and central parts of the country. It is still debated why this discrepancy exists (Olsson et al., 2010). South of latitude 59°N, the presence of antibodies to PUUV in rodents and humans is very rare while north of this latitude, the prevalence is high (12.9% in farmers and 6.8% in reference individuals (with both farmers and reference individuals living in rural areas) (Ahlm et al., 1998). One study investigated the seroprevalence of PUUV in inhabitants of the two northernmost counties of Sweden; this was found to be 5.4%. There was no statistically significant difference in seropositivity between men and women. The age distribution showed significantly increasing seropositivity with increasing age, and a higher seroprevalence in those living in rural areas than in those living in urban areas (Ahlm et al., 1994). Physicians in Sweden are obliged to report confirmed cases of NE. Based on these reported cases and the seroepidemiological studies, it has been estimated that approximately one in eight PUUV-infected individuals are diagnosed with a laboratory test (Ahlm et al., 1994). This discrepancy could be due to mild symptoms not requiring medical care, or to atypical symptoms not being recognized as NE by physicians. In northern Sweden, exposure to PUUV commonly happens through cleaning or redecorating the residence, handling firewood, gardening, or handling hay (Olsson et al., 2003). Most cases of NE are in the fall and winter and as mentioned the incidence follows the population dynamics of the bank vole, with approximately 3-year intervals between peaks (Olsson et al., 2003).

1.5. The diseases

Hantaviruses cause two diseases, hemorrhagic fever with renal syndrome (HFRS)―caused by Old World hantaviruses on the European and Asian continents―and hemorrhagic cardiopulmonary syndrome (HCPS)―caused by the New World hantaviruses in North and South America. In both diseases, the virus can be detected in endothelial cells throughout the body without causing any obvious signs of CPE (Zaki et al., 1995, Rasmuson et al., 2011). Capillary permeability and vascular leakage are central to disease development, and they explain many signs and symptoms, for example hypotension and chock, abdominal pains due to retroperitoneal edema, and pleural effusion (Mustonen et al., 2013, Zaki et al., 1995). In HCPS, the capillary leakage commonly causes pulmonary edema, which can also occur but is rare in HFRS (Rasmuson et al., 2011, Mustonen et al., 2013, Zaki et al., 1995).

HFRS

The incubation period is approximately 3 weeks, ranging from 10 days to 6 weeks (Jonsson et al., 2010). The severity of HFRS varies depending on the causative hantavirus. In general, HNTV and DOBV cause severe disease, SEOV causes more moderate disease, and PUUV causes a relatively mild disease (Jonsson et al., 2010, Lee, 1996, Avsic-Zupanc et al., 1999). In the largest study to date on PUUV infection based on > 22,000 NE cases, the case fatality proportion was 0.08% (Makary et al., 2010). This can be compared with a case fatality rate of 15% for HFRS caused by HTNV, which is significantly reduced to approximately 5‒7% if the patient is treated with critical care and hemodialysis (Mertz, 2009). In the individual, the different types of hantaviruses can cause everything from subclinical to lethal disease (Jonsson et al., 2010). Typically, HFRS occurs in five distinct phases: febrile, hypotensive, oliguric, polyuric, and convalescent (Jonsson et al., 2010). Common clinical findings are fever, chills, malaise, myalgia, pain in the lumbar and abdominal regions, headache, dizziness, vomiting, diarrhea, and blurred vision (Avsic-Zupanc et al., 1999, Settergren et al., 1989, Huttunen et al., 2011, Saksida et al., 2011). There are also hemorrhagic manifestations such as epistaxis, petechiae and conjunctival injections, melaena, hematemesis, and macroscopic hematuria (Saksida et al., 2011, Settergren et al., 1989). Death from HFRS is in many cases caused by renal insufficiency, shock, and/or hemorrhagic manifestations (Avsic-Zupanc et al., 1999, Jonsson et al., 2010, Huttunen et al., 2011). Death can also be due to multiple organ failure (Huttunen et al., 2011, Rasmuson et al., 2011). As for

HCPS, the cause of death can in some cases also be due to cardiopulmonary distress (Rasmuson et al., 2011). In a literature review, Huttunen et al. (2011) showed that NE is milder in children than in adults, and that severe complications are reported only in adult patients.

HCPS

The incubation period is generally 9‒33 days (mean 14‒17 days) (Jonsson et al., 2010). The severity of disease and symptoms vary with the strain of infecting hantavirus (Jonsson et al., 2010, Toro et al., 1998). The disease starts in a similar way to HFRS, with a febrile prodomal phase involving fever and symptoms such as myalgia, headache, backache, abdominal pain, diarrhea, nausea, and vomiting (Mertz, 2009, Jonsson et al., 2010). This febrile prodromal phase is followed by the cardiopulmonary or shock phase, which is initiated by an abrupt onset of cough and shortness of breath followed by the sudden onset of non-cardiogenic pulmonary edema and in some cases by shock (Mertz, 2009). In hospitalized patients, death almost always results from shock with very low cardiac output and from cardiac arrhythmias. Death due to respiratory failure is rare due to treatment with mechanical ventilation (Mertz, 2009). The case fatality rate is approximately 40‒50% in North America and death takes place within a median of 5 days after onset of disease (Khan et al., 1996). Surviving patients enter the diuretic phase with rapid clinical improvement, which is then followed by the more prolonged convalescent phase (Mertz, 2009).

1.6. Pathogenesis

1.6.1. Animal Models for Pathogenesis

The pathogenesis of hantavirus disease in humans remains unclear, largely because of the lack animal models. Since hantavirus in rodents does not reflect the human disease, there have been no rodent models of disease available to study the pathogenesis in vivo. PUUV was used to infect cynomolgus macaques―which resulted in typical signs of HFRS (Klingstrom et al., 2002). In that study, wild-type virus was used and the authors speculated that because of this, the disease model reflected the human disease, in contrast to earlier attempts. These authors had previously shown that the infectivity of PUUV is dramatically reduced for the natural reservoir, the bank vole when the virus has been adapted to cell culture (Lundkvist et al., 1997).

Until recently, the only model of HCPS was the Syrian hamster, which, after infection with ANDV, develops severe disease that recapitulates the cardiopulmonary phase of HCPS in humans (Safronetz et al., 2011b). Last year (2014), Safronetz et al. developed an NHP model for HCPS based on infection of rhesus macaques with wild-type SNV (propagated in the natural host of SNV, the deer mouse). (Safronetz et al., 2014).

1.6.2 The incubation period

The incubation period of hantaviruses is relatively long: 2‒4 weeks (see section 4). The onset of disease is sudden, with flu-like symptoms that rapidly progress to―in some cases―severe or even deadly disease. What happens in the infected individual during the incubation period and why the symptoms start so suddenly is largely unknown. Hantavirus RNA has been found in the serum of patients up to three weeks before onset of disease, and virus has been isolated from serum before the onset (Rasmuson et al., 2011, Galeno et al., 2002, Ferres et al., 2007). In a prospective study in humans, none had IgG or IgM antibody detected before the onset of prodromal symptoms (Ferres et al., 2007). However, at onset of disease most patients have specific antibodies to the virus (Ferres et al., 2007), so the immune defense has obviously already been in contact with the virus for some time and has elicited a specific response, but without producing any symptoms. In the NHP model for HCPS, viremia was the earliest indicator of disease and was first detected 4–10 days before the onset of respiratory distress and immediately before the observed hematological abnormalities and activated host immune responses.

1.6.3. Viral entry and dissemination in the human body

The very first barrier that the virus encounters when entering the body is the mucus gel that covers respiratory epithelial cells. In the lower respiratory tract, trapped microbes are removed by the ciliated cells that continually move the mucus with their cilia―away from the lungs to the throat (Flint et al., 2009, Linden et al., 2008). The lowest parts of the respiratory tract, the alveoli, lack both cilia and mucus and macrophages lining the alveoli are important for digestion of microbes (Flint et al., 2009). Mucin glycoproteins produced by mucus-producing cells in the epithelium or submucosal glands are the major macromolecular constituents of mucus (Linden et al., 2008).

When the virus has passed the mucus gel, it encounters yet another barrier―the respiratory epithelium. The respiratory epithelium has tight junction integrity and forms a particle-impermeable barrier. It is not yet clear how hantavirus can traverse this barrier. Hantavirus has been observed in untyped pneumocytes of virus-infected patients (Nuovo et al., 1996). Rowe and Pekosz (2006) showed in an in vitro study that ANDV can infect club cells (Clara) and goblet cells, but not ciliated cells of the respiratory endothelium (in this case, hamster tracheal epithelial cells) (Rowe and Pekosz, 2006). Despite being infected, these cells showed no cytopathic effect (CPE) and the epithelial tight junction integrity was intact. The virus was able to infect via both the apical membrane and the basolateral membrane, and subsequently virus particles were secreted bidirectionally. The authors speculated that the bidirectional spread allows the virus to disseminate beyond the epithelium.

Once the respiratory epithelium has been traversed, the virus spreads to the lung endothelium and more distant locations in the body. Dendritic cells and monocytes/macrophages have been proposed to play a part in this step of infection. Dendritic cells and monocytes/macrophages can be infected in vitro (Raftery et al., 2002, Temonen et al., 1995). In vivo, abundant amounts of dendritic cells with hantavirus inside were found (by staining) in lymph nodes of SNV-infected patients (Zaki et al., 1995) and monocytes and macrophages with hantavirus antigen inside have been found in the lung interstitium of hantavirus-infected patients (Zaki et al., 1995, Rasmuson et al., 2011). It is not clear whether this is phagocytosed virus or whether it results from virus replication in the cells.

Once spread, the virus can be found in endothelial cells of capillaries and small vessels in numerous organs and tissues of the body (Nuovo et al., 1996, Zaki et al., 1995, Rasmuson et al., 2011).. Despite the extensive presence of hantavirus antigens in endothelial cells, there is little evidence of cytopathic effect in endothelium (Zaki et al., 1995). Far more SNV can be found in lung endothelial cells than in other tissues (Zaki et al., 1995, Nuovo et al., 1996), with hardly any of the cells of the lung microcirculation remaining uninfected (Zaki et al., 1995). Hantaviruses causing HFRS have tropism for cells in the kidneys, and they are found in podocytes in the glomeruli (Krautkramer et al., 2011) and in tubular epithelial cells (Poljak and Avsic Zupanc, 1994, Krautkramer et al., 2011) of infected patients. In HCPS patients, virus can also be found in tubular epithelial cells but usually to a low degree (Zaki et al., 1995, Nuovo et al., 1996).

1.6.4. An overactive immune response

An in vitro study of hantavirus infection of endothelial cells showed that there was no increased permeability of the confluent cell layer. However, the infection induced the production of chemokines RANTES and IP-10, and the production of these chemokines was enhanced by IFN-γ (Sundstrom et al., 2001). The authors proposed the theory that the chemokines attract antigen- specific activated T-cells. The T-cells produce IFN-γ, which enhances the production of chemokines even more―and then even more T-cells are recruited.

Studies on biopsy specimens showed inflammatory cellular infiltration close to infected lung endothelial cells in HCPS patients and close to infected kidney cells in HFRS patients. The infiltrations of inflammatory cells in the kidneys of HFRS patients included lymphocytes (mainly CD8+ T-cells), monocytes, macrophages, and polymorphonuclear leukocytes (including eosinophilic granulocytes and neutrophils) (Temonen et al., 1996). In the lungs of HCPS patients, mononuclear cell infiltrate was mainly composed of a mixture of T-lymphocytes (an increased proportion of CD8+ T-cells was seen) and macrophage/monocytic cells (Zaki et al., 1995). High levels of SNV-specific CD8+ T-cells have been found in the circulation of infected patients (Kilpatrick et al., 2004). When compared to other viral diseases, the levels are exceptionally high; only infection with EBV has shown equally high levels (Kilpatrick et al., 2004). The authors speculated that TNF-α and IFN-γ produced by activated SNV-specific T-cells when recognizing virus on SNV-infected pulmonary endothelial cells contribute to the observed capillary leakage during HCPS.

Kidney biopsy samples from HFRS patients have shown increased expression of cytokines (TNF-α , TGF-β, and PDGF) in the peritubular areas (Temonen et al., 1996). Elevated serum levels of TNF-α have been observed in both HCPS patients and HFRS patients (Borges et al., 2008, Saksida et al., 2011). TNF-α is a pro-inflammatory cytokine that causes increased vascular permeability (among other things). 物Very high serum levels of IL-6 have been found in fatal cases of HCPS Very high serum levels of IL-6 have been found in fatal cases of HCPS (Borges et al., 2008). In that study, the authors found a positive correlation between IL-6 and the serum concentration of NO, and they speculated that IL-6 may be important for inhibition of cardiac function and induction of hypotension in HCPS. High IL-6 levels in serum have also been shown to be associated with a more severe disease in NE patients (Outinen et al., 2010).

Elevated levels of IFN-γ, IL-12, and TNF-β in HCPS patients would indicate an elevated Th1 response (Borges et al., 2008). The Th1 response plays a key role in antiviral immunity, but it can also produce an immunopathogenic effect. Both IL-5 and IL-6 were elevated in HCPS patients, suggesting that the Th2 response is also activated (Borges et al., 2008).

High levels of the anti-inflammatory cytokine IL-10 were found in serum from both HCPS patients and HFRS patients (Borges et al., 2008, Saksida et al., 2011). Production of IL-10 might be induced due to the high level of inflammatory cytokines, and could lead to an unfavorable immunosuppression (Saksida et al., 2011).

In summary, some studies have indicated that infection of endothelial cells induces production of chemokines, which attract immune cells. These immune cells―especially CD8+ T-cells―are thought to release an excess of cytokines (a so-called cytokine storm), which increases endothelial permeability.

1.6.5 The humoral immune response

In studies on patients with HCPS, low titers of specific IgG have been linked to more severe disease progression in SNV-infected patients (Bharadwaj et al., 2000, MacNeil et al., 2010). A study on HFRS showed a tendency of lower titers of specific IgG in DOBV-infected patients with severe disease than in patients with milder disease (Saksida et al., 2008). Regarding specific IgM, there have been conflicting reports. Bharadway et al. found no association between SNV IgM titer and disease severity, while MacNeil et al. found an association between SNV IgM and survival of HCPS patients (Bharadway 2000, MacNeil 2010). Bharadway et al. (2000) also investigated SNV-specific IgA levels in relation to disease severity, but no correlation was found.

1.6.6. Viremia

Studies in HFRS and HCPS patients have indicated that a high virus load in serum is associated with severe disease. Saksida et.al. (2008) found that the mean number of DOBV RNA copies was higher in patients with severe disease than in those with milder disease, and they suggested that the virus load might be associated with the severity of disease (Saksida et al., 2008). For HFRS caused by HNTV, patients with severe disease have been found to have higher virus loads than those with milder disease (Yi et al., 2013). There have also been studies on HCPS patients. Terajima et al. (1999) found a significant association between high levels of viremia on the one hand and low platelet counts and high hematocrit values on the other, and they found that fatal cases tended to have higher viral RNA levels than survivors (Terajima et al., 1999). A later study found a statistically significant association between plasma SNV RNA levels at admission to hospital and disease severity (Xiao et al., 2006).

1.7. Treatment

Ribavirin

Ribavirin is a broad-spectrum guanosine analog with activity against a number of DNA and RNA viruses. An in vitro study on HNTV showed that ribavirin causes a high frequency of mutation during viral RNA synthesis (so-called “error catastrophe”), and that the transcripts produced are not functional (Severson et al., 2003). Safronetz et al. (2011) demonstrated that ribavirin is highly active against ANDV, both in vitro and in vivo, using a lethal hamster model of HPS (Safronetz et al., 2011a). Ribavirin given as post-exposure prophylaxis within three days after infection was 100% effective in preventing lethal HPS disease in the hamster model. However, when therapy was initiated at five days post infection, its efficacy dropped dramatically. Safronetz et al. suggested that ribavirin should be considered for patients with early clinical symptoms of HCPS or for people who are exposed, for example to prevent human-to-human transmission of ANDV. For HFRS, ribavirin has been shown to be effective. A prospective, randomized, double-blind placebo-controlled clinical trial in China showed that intravenous ribavirin therapy had a positive effect, with reduced morbidity and mortality observed in the treatment group when compared to

the placebo control group (Huggins et al., 1991). A study in Korea (which was not placebo-controlled) suggested that treatment with ribavirin early in HFRS may reduce renal complications (Rusnak et al., 2009). For HCPS, the positive effect of ribavirin in the in vitro and in vivo studies on hamsters has not been confirmed in clinical trials, possibly because treatment was initiated too late in the course of the disease. The first study on ribavirin was an open-label study in the United States, which did not suggest an appreciable effect of ribavirin (Chapman et al., 1999). Later, a placebo-controlled double-blind trial in North America had the same result (Mertz et al., 2004). The authors wanted to enroll the study subjects in the prodrome phase, but this was not possible because HCPS was generally not considered by the healthcare system until the onset of the cardiopulmonary phase. The study indicated that ribavirin is probably not effective for the treatment of HCPS after the onset of the cardiopulmonary phase, and the author speculated that ribavirin could still have an effect if given in the prodrome phase (Mertz et al., 2004).

Methylprednisolone There is evidence of an immune-mediated pathogenesis of HCPS. Vial et al. (2013) performed a randomized, double-blind placebo-controlled clinical trial in Chile to assess the safety and efficacy of intravenous methylprednisolone in patients with HCPS in the cardiopulmonary phase (Vial et al., 2013). However, they did not find any significant difference in mortality between the treatment groups.

Neutralizing antibodies Since a low antibody titer and high viral load have been associated with more severe disease, hantavirus-specific antibodies could be a possible treatment; see section 1.6.5. Passive immunization in monkeys, using serum from monkeys previously infected with hantavirus, induced protection―suggesting that convalescent-phase antibodies may be a possible therapy that can induce immediate protection against HFRS and HPS (Klingstrom et al., 2008). Neutralizing antibodies will be discussed further in Chapter 3.

2. Aims

To characterize and describe possible causes of the largest outbreak of hantavirus infection in Sweden so far, including demographics (gender and age) and mortality rate, and to investigate how many of the NE cases diagnosed were hospitalized (paper I).

To enhance preparedness for hantavirus outbreaks (paper I).

To evaluate the possibility of person-to-person transmission of PUUV through saliva (paper II).

To study the influence of human saliva on hantavirus infectivity (papers III and IV).

To characterize factors that may be associated with a severe course of NE (paper V).

3. Results and discussion

3.1. Outbreak of Puumala virus infection, Sweden.

In December of 2007, the largest outbreak of NE in Sweden suddenly started. The large outbreak was totally unexpected, since there were no more bank voles than in previous peak years. The outbreak coincided with extremely warm weather. The snow cover had melted, so that there was only a crust of ice covering the ground. In paper I, we put forward the hypothesis that the size of the rodent population is not the only factor that determines the size of a hantavirus epidemic; climate factors may also have contributed to the large outbreak. We speculated that the bank voles that normally live under the protecting snow cover had sought refuge in barns, houses, and other buildings when the snow cover melted, thereby increasing the exposure of the human population at risk. However, this was only one observation during one outbreak, and the hypothesis needed to be tested during several seasons to be proven correct. If the hypothesis is correct, there would be a higher incidence of NE in winters that have higher temperatures and thinner snow cover. Recently, the hypothesis was proven to be correct. Khalil et al (2014) showed in a model that the risk of NE is higher in winters with many rainy days (i.e. warm winters with a thin or absent snow cover, since there are normally no rainy days―only snowy days―during a cold winter in northern Sweden). The model was based on weather conditions, bank vole densities, and the incidence of NE in northern Sweden over several years (1990‒2012).

The IPCC predicts that as the temperatures rise due to climate change, the likelihood of precipitation falling as rain rather than snow will increase (Trenberth et al., 2007). This prediction together with the study by Khalil et al. indicates that NE may be more common in the future. There is an increasing trend of PUUV infections in Finland and Sweden (Makary et al., 2010, Olsson et al., 2009). Climate change may be one factor associated with the increased numbers of NE cases in northern Europe. However, no-one has assessed long-term trends to investigate whether the higher incidence of NE and climate change may be associated (Roda Gracia et al., 2014).

The demographics during the outbreak were similar to earlier observations. For example, in the largest study of NE so far, based on all NE cases in

Finland 1995‒2008 (> 22,000 cases), the incidence was higher in males than in females: 62% of the cases were males (Makary et al., 2010). Similarly, in our study more men (58%) than women (42%) had NE. In the same study from Finland, the highest incidences were observed in the 35- to 49-year and the 50- to 64-year age groups, which is similar what we found in our study where individuals aged 35‒74 years had the highest incidence.

During the outbreak, as many as 30% of the diagnosed cases were hospitalized. This shows that a hantavirus outbreak puts a significant burden on the healthcare system. In Finland, 52% of NE cases are hospitalized (Makary et al., 2010). This is probably not because patients in Finland have more severe disease, but rather because of different healthcare politics in the countries. Finland has approximately twice as many hospital beds per 1,000 inhabitants than Sweden (OECD, 2013). The outbreak coincided with the annual influenza and calicivirus peaks in January―adding to the already difficult situation at the infectious diseases clinic and the clinical microbiology laboratory. Hopefully, our article has increased the awareness of the healthcare system and its preparedness for the possibility of a hantavirus outbreak..

3.2. Influence of human saliva on PUUV infectivity

We found viral RNA in the saliva of PUUV-infected patients (paper II), indicating that there is PUUV in the saliva of PUUV-infected patients. Several factors may influence why PUUV and most other hantaviruses cannot spread from person to person while ANDV has this ability. Factors such as a) the mode of secretion of hantavirus into the saliva, b) the sensitivity of hantaviruses to unspecific proteins in human saliva, and c) neutralization of virus by virus-specific antibodies may be important. a) In our study on PUUV-infected patients, the virus antigen was only visible in the endothelial cells of the salivary glands (paper IV). However, hantavirus antigen has been found in the secretory cells of salivary glands in the natural host (Yanagihara et al., 1985). In patients infected with ANDV, the antigen is also visible in the secretory cells of salivary glands (Navarrete et al., 2007). It is possible that a larger number of virions are shed into the saliva from the secretory cells than from the endothelial cells, and this could partly explain the ability of ANDV to transmit from person to person.

b) The infectivity of ANDV has been shown to be less affected by the saliva of uninfected individuals than that of HNTV and PUUV in in vitro experiments (Hardestam et al., 2009). Since the saliva did not contain any hantavirus- specific antibodies, there must be an unspecific property of human saliva that ANDV manages to overcome to a greater extent than HNTV and PUUV, and probably also other hantaviruses. In paper III, we found that HNTV was inhibited by mucin. No-one has yet investigated whether ANDV is resistant to mucin, or whether there is a major difference between ANDV and other hantaviruses regarding their resistance to mucin. Linden et al. (2008) speculated that differences in mucin glycosylation between mammalian species may underlie some of the differences in infectivity/pathogenicity of individual microbial pathogens (Linden et al., 2008). c) As discussed in paper IV, there is also a hantavirus-specific defense, in the form of PUUV-specific IgA in the saliva of PUUV-infected patients. We found an inverse relationship between the presence of IgA and viral RNA in the saliva. We speculated that there may be a window of opportunity for infection via saliva when virus is present, before antibodies appear. Recently, a report from Argentina described an outbreak in five people where the index patient was negative for IgG and IgM when he was admitted to hospital (Martinez-Valdebenito et al., 2014). However they did not analyze salivary IgA. That patient infected two of the medical staff, one of whom died. This illustrates that patients may be more contagious before seroconversion, but this needs to be studied further.

3.3. Viral load and humoral immune response in association with disease severity in Puumala hantavirus-infected patients―implications for treatment.

Using multivariate regression analysis, we found that a low titer of IgG and a high white blood cell (WBC) count were associated with more severe disease in NE patients. We found high viral loads in the patients with more severe disease, early on in the disease course. However, few samples were drawn on the first days of disease (day 0‒3), and we could not show statistically that a high virus load is associated with a more severe disease outcome. At the same time as we carried out our study, another study was done by Korva et al. (2013) with similar results to ours. They also found the mean RNA level in PUUV-infected patients to be approximately 1 × 105 copies/ml (Korva et al., 2013). As we did, they also found the plasma of PUUV-infected patients to be RNA-positive for a longer time than just a few days. This had been assumed but had never been thoroughly investigated for PUUV until these two studies were done.

A high WBC count was associated with a more severe course of the disease. When we looked closer at the types of WBCs, the neutrophils were the only cells that were significantly associated with moderate-to-severe disease. Our study was the first to show an association between neutrophils and severity of hantavirus disease in patients. Although neutrophilia has been used as a diagnostic tool especially for HCPS (Zaki et al., 1995), there have been surprisingly few studies on neutrophils and hantavirus pathogenesis. Shortly after our study was published, Koma et al. (2014) showed that the occurrence of pulmonary edema and the pulmonary vascular permeability were significantly suppressed by neutrophil depletion in HTNV-infected SCID mice, indicating that neutrophils are involved in the vascular hyperpermeability and development of pulmonary edema in hantavirus infection. Another study published shortly after our study demonstrated that HTNV strongly stimulated neutrophils to release neutrophil extracellular traps (NETs) in vitro (Raftery et al., 2014). These authors also showed that infection with PUUV, DOBV, and Tula virus (TULV) induced high systemic levels of circulating NETs in patients. Our study and these studies indicate that neutrophils are important for the pathogenesis of hantaviruses, and further studies are needed.

Our results can be useful in the clinical setting: 90% of the patients were PCR-positive for PUUV RNA at an early time point of disease. Hopefully, this information will urge physicians and laboratories to analyze more samples by PCR for diagnostic purposes, especially in critically ill patients. We have shown that critically ill patients are more likely to have a low or undetectable antibody titer, and for those patients PCR analysis could be very important in making the right diagnosis. Our study also indicated that a low IgG level and a high WBC count can be used as predictors for a more severe disease course.

Currently, there is no established treatment for hantavirus-infected patients. Our study showed high virus loads at an early time point in patients with severe disease might indicate that there is a window of opportunity for treatment with anivirals such as Ribavirin.Recently, there was a study from Chile where ANDV-infected patients had been treated with immune plasma (Vial et al., 2014). The case fatality rate was 14% in the treated study group (29 patients), as compared to a case fatality rate of 32% for the non-treated cases during the same period. There were no serious adverse events associated with the immune plasma infusion. The results in the Chilean study are very promising, and in our study we found that PUUV-specific IgG is protective against severe disease, which suggests that treatment with passive immunization could be efficient against PUUV infection as well.

4. Conclusions

Puumala virus can suddenly cause unexpected and large human outbreaks with considerable effects on human health and public health services.

The size of the rodent population and climate factors may have contributed to the largest Puumala virus outbreak in Sweden.

Puumala virus RNA can be detected in the saliva of infected patients indicating that saliva can be a possible route of human-to-human infection.

Human saliva reduced hantavirus infectivity and mucin was identified as a salivary component that inhibited hantavirus infection.

Puumala virus specific IgA was found in the saliva of Puumala virus infected patients. The presence of secretory IgA antibodies was significantly inversely associated with the presence of Puumala RNA in saliva, suggesting that the virus could be contagious before appearance of salivary IgA.

A majority of Puumala virus infected patients have detectable Puumala virus RNA in their serum until day 9 after symptom debut.

The immune response has a dual role for disease development. It is in part responsible for development of severe disease with significantly higher amounts of neutrophils in severely ill patients, but it is also protective against severe disease, because patients with a mild disease had higher levels of Puumala virus specific IgG.

Our results support the use of passive immunization as a potential treatment for HFRS/HCPS.

Acknowledgements

I would like to thank all of you who have supported me during these years and contributed to this thesis! I would like to acknowledge;

First of all the patients participating in the study.

My supervisor Magnus Evander, thank you for being such a great supervisor! Positive, enthusiastic and supportive! Always taking the time to discuss research and answer questions.

My co-supervisor Clas Ahlm who also is so enthusiastic and encouraging! It has been great having you as a co-supervisor!

My co-supervisor Annika Allard, for your great sense of humor and for always taking the time for questions. Thank you for your support!

All co-authors including my supervisors and Jens Boman, Per Juto, Jonas Klingström, Jonas Hardestam, Åke Lundkvist, Johan Rasmuson, Charlotta Andersson, Therese Thunberg and Joacim Rocklöv.

Lena Rova thank you for teaching me how to make antigens, ELISA and IF diagnostics.

Irene Eriksson and Maj Bylund thank you for great technical assistance!

Per Juto thank you for teaching me so much about NE and Puumala virus diagnostics!

Urban Kumlin and Göran Wadell thank you for your contagious enthusiasm for viruses!

Members of the “bunyavirus-group”. It seems like research on bunyaviruses attracts really nice people! Anne-Marie Connolly-Andersen, Göran Bucht and Maria Baudin and those who have moved on Mari Lindkvist, Nahla Mohamed and Jonas Näslund. Therese Thunberg it has been really great working with you! It has been really great working with you too Johan Rasmuson!

I would like to acknowledge everyone at the clinic of infectious diseases who helped to gather information and samples.

I would like to thank all my colleagues for their support!

I am really fortunate to work at a department where we have a really good atmosphere and everyone helps each other, that is both in “staten” and “landstinget”. Thank you all for your support!

My family and friends who have encouraged and supported me!

References

AHLM, C., LINDERHOLM, M., JUTO, P., STEGMAYR, B. & SETTERGREN, B. 1994. Prevalence of serum IgG antibodies to Puumala virus (haemorrhagic fever with renal syndrome) in northern Sweden. Epidemiol Infect, 113, 129-36.

AHLM, C., THELIN, A., ELGH, F., JUTO, P., STIERNSTROM, E. L., HOLMBERG, S. & TARNVIK, A. 1998. Prevalence of antibodies specific to Puumala virus among farmers in Sweden. Scand J Work Environ Health, 24, 104-8.

AVSIC-ZUPANC, T., PETROVEC, M., FURLAN, P., KAPS, R., ELGH, F. & LUNDKVIST, A. 1999. Hemorrhagic fever with renal syndrome in the Dolenjska region of Slovenia--a 10-year survey. Clin Infect Dis, 28, 860-5.

AVSIC-ZUPANC, T., SAKSIDA, A. & KORVA, M. 2013. Hantavirus Infections. Clin Microbiol Infect.

AVSIC-ZUPANC, T., XIAO, S. Y., STOJANOVIC, R., GLIGIC, A., VAN DER GROEN, G. & LEDUC, J. W. 1992. Characterization of Dobrava virus: a Hantavirus from Slovenia, Yugoslavia. J Med Virol, 38, 132-7.

BATTISTI, A. J., CHU, Y. K., CHIPMAN, P. R., KAUFMANN, B., JONSSON, C. B. & ROSSMANN, M. G. 2011. Structural studies of Hantaan virus. J Virol, 85, 835-41.

BERNSHTEIN, A. D., APEKINA, N. S., MIKHAILOVA, T. V., MYASNIKOV, Y. A., KHLYAP, L. A., KOROTKOV, Y. S. & GAVRILOVSKAYA, I. N. 1999. Dynamics of Puumala hantavirus infection in naturally infected bank voles (Clethrinomys glareolus). Arch Virol, 144, 2415-28.

BHARADWAJ, M., NOFCHISSEY, R., GOADE, D., KOSTER, F. & HJELLE, B. 2000. Humoral immune responses in the hantavirus cardiopulmonary syndrome. J Infect Dis, 182, 43-8.

BORGES, A. A., CAMPOS, G. M., MORELI, M. L., MORO SOUZA, R. L., SAGGIORO, F. P., FIGUEIREDO, G. G., LIVONESI, M. C. & MORAES FIGUEIREDO, L. T. 2008. Role of mixed Th1 and Th2 serum cytokines on pathogenesis and prognosis of hantavirus pulmonary syndrome. Microbes Infect, 10, 1150-7.

BORUCKI, M. K., BOONE, J. D., ROWE, J. E., BOHLMAN, M. C., KUHN, E. A., DEBACA, R. & ST JEOR, S. C. 2000. Role of maternal antibody in natural infection of Peromyscus maniculatus with Sin Nombre virus. J Virol, 74, 2426-9.

BRUMMER-KORVENKONTIO, M., VAHERI, A., HOVI, T., VON BONSDORFF, C. H., VUORIMIES, J., MANNI, T., PENTTINEN, K., OKER-BLOM, N. & LAHDEVIRTA, J. 1980. Nephropathia epidemica: detection of antigen in bank voles and serologic diagnosis of human infection. J Infect Dis, 141, 131-4.

CHAPMAN, L. E., MERTZ, G. J., PETERS, C. J., JOLSON, H. M., KHAN, A. S., KSIAZEK, T. G., KOSTER, F. T., BAUM, K. F., ROLLIN, P. E., PAVIA, A. T., HOLMAN, R. C., CHRISTENSON, J. C., RUBIN, P. J., BEHRMAN, R. E., BELL, L. J., SIMPSON, G. L. & SADEK, R. F. 1999. Intravenous ribavirin for hantavirus pulmonary syndrome: safety and tolerance during 1 year of open-label experience. Ribavirin Study Group. Antivir Ther, 4, 211-9.

CHENG, E. & MIR, M. A. 2012. Signatures of host mRNA 5' terminus for efficient hantavirus cap snatching. J Virol, 86, 10173-85.

CHOI, Y., KWON, Y. C., KIM, S. I., PARK, J. M., LEE, K. H. & AHN, B. Y. 2008. A hantavirus causing hemorrhagic fever with renal syndrome requires gC1qR/p32 for efficient cell binding and infection. Virology, 381, 178-83.

CLEMENT, J., VERCAUTEREN, J., VERSTRAETEN, W. W., DUCOFFRE, G., BARRIOS, J. M., VANDAMME, A. M., MAES, P. & VAN RANST, M. 2009. Relating increasing hantavirus incidences to the changing climate: the mast connection. Int J Health Geogr, 8, 1.

COHEN, M., ZHANG, X. Q., SENAATI, H. P., CHEN, H. W., VARKI, N. M., SCHOOLEY, R. T. & GAGNEUX, P. 2013. Influenza A penetrates host mucus by cleaving sialic acids with neuraminidase. Virol J, 10, 321.

DEARING, M. D. & DIZNEY, L. 2010. Ecology of hantavirus in a changing world. Ann N Y Acad Sci, 1195, 99-112.

DOHMAE, K. & NISHIMUNE, Y. 1995. Protection against hantavirus infection by dam's immunity transferred vertically to neonates. Arch Virol, 140, 165-72.

ELLIOTT, R. M. 2014. Orthobunyaviruses: recent genetic and structural insights. Nat Rev Microbiol, 12, 673-85.

ENGELTHALER, D. M., MOSLEY, D. G., CHEEK, J. E., LEVY, C. E., KOMATSU, K. K., ETTESTAD, P., DAVIS, T., TANDA, D. T., MILLER, L., FRAMPTON, J. W., PORTER, R. & BRYAN, R. T. 1999. Climatic and environmental patterns associated with hantavirus pulmonary syndrome, Four Corners region, United States. Emerg Infect Dis, 5, 87-94.

FERRES, M., VIAL, P., MARCO, C., YANEZ, L., GODOY, P., CASTILLO, C., HJELLE, B., DELGADO, I., LEE, S. J. & MERTZ, G. J. 2007. Prospective evaluation of household contacts of persons with hantavirus cardiopulmonary syndrome in chile. J Infect Dis, 195, 1563-71.

FLINT, S. J., ENQUIST, L. W., RACANIELLO, V. R. & A.M., S. 2009. Principles of Virology, Washington, DC, ASM Press.

FRENCH, G. R., FOULKE, R. S., BRAND, O. A., EDDY, G. A., LEE, H. W. & LEE, P. W. 1981. Korean hemorrhagic fever: propagation of the etiologic agent in a cell line of human origin. Science, 211, 1046-8.

FULTZ, P. N. 1986. Components of saliva inactivate human immunodeficiency virus. Lancet. England.

GALENO, H., MORA, J., VILLAGRA, E., FERNANDEZ, J., HERNANDEZ, J., MERTZ, G. J. & RAMIREZ, E. 2002. First human isolate of Hantavirus (Andes virus) in the Americas. Emerg Infect Dis, 8, 657-61.

GAVRILOVSKAYA, I. N., BROWN, E. J., GINSBERG, M. H. & MACKOW, E. R. 1999. Cellular entry of hantaviruses which cause hemorrhagic fever with renal syndrome is mediated by beta3 integrins. J Virol, 73, 3951-9.

GAVRILOVSKAYA, I. N., PERESLENI, T., GEIMONEN, E. & MACKOW, E. R. 2002. Pathogenic hantaviruses selectively inhibit beta3 integrin directed endothelial cell migration. Arch Virol, 147, 1913-31.

GAVRILOVSKAYA, I. N., SHEPLEY, M., SHAW, R., GINSBERG, M. H. & MACKOW, E. R. 1998. beta3 Integrins mediate the cellular entry of hantaviruses that cause respiratory failure. Proc Natl Acad Sci U S A, 95, 7074-9.

GOLDSMITH, C. S., ELLIOTT, L. H., PETERS, C. J. & ZAKI, S. R. 1995. Ultrastructural characteristics of Sin Nombre virus, causative agent of hantavirus pulmonary syndrome. Arch Virol, 140, 2107-22.

HARDESTAM, J., KARLSSON, M., FALK, K. I., OLSSON, G., KLINGSTROM, J. & LUNDKVIST, A. 2008. Puumala hantavirus excretion kinetics in bank voles (Myodes glareolus). Emerg Infect Dis, 14, 1209-15.

HARDESTAM, J., LUNDKVIST, A. & KLINGSTROM, J. 2009. Sensitivity of Andes hantavirus to antiviral effect of human saliva. Emerg Infect Dis, 15, 1140-2.

HARDESTAM, J., SIMON, M., HEDLUND, K. O., VAHERI, A., KLINGSTROM, 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, 2547-51.

HEDLUND, C., BLOMSTEDT, Y. & SCHUMANN, B. 2014. Association of climatic factors with infectious diseases in the Arctic and subarctic region--a systematic review. Glob Health Action, 7, 24161.

HEPOJOKI, J., STRANDIN, T., WANG, H., VAPALAHTI, O., VAHERI, A. & LANKINEN, H. 2010. Cytoplasmic tails of hantavirus glycoproteins interact with the nucleocapsid protein. J Gen Virol, 91, 2341-50.

HINSON, E. R., SHONE, S. M., ZINK, M. C., GLASS, G. E. & KLEIN, S. L. 2004. Wounding: the primary mode of Seoul virus transmission among male Norway rats. Am J Trop Med Hyg, 70, 310-7.

HJELLE, B. & GLASS, G. E. 2000. Outbreak of hantavirus infection in the Four Corners region of the United States in the wake of the 1997-1998 El Nino-southern oscillation. J Infect Dis, 181, 1569-73.

HUGGINS, J. W., HSIANG, C. M., COSGRIFF, T. M., GUANG, M. Y., SMITH, J. I., WU, Z. O., LEDUC, J. W., ZHENG, Z. M., MEEGAN, J. M., WANG, Q. N. & ET AL. 1991. Prospective, double-blind, concurrent, placebo-controlled clinical trial of intravenous ribavirin therapy of hemorrhagic fever with renal syndrome. J Infect Dis, 164, 1119-27.

HUISKONEN, J. T., HEPOJOKI, J., LAURINMAKI, P., VAHERI, A., LANKINEN, H., BUTCHER, S. J. & GRUNEWALD, K. 2010. Electron cryotomography of Tula hantavirus suggests a unique assembly paradigm for enveloped viruses. J Virol, 84, 4889-97.

HUTCHINSON, K. L., ROLLIN, P. E., SHIEH, W. J., ZAKI, S., GREER, P. W. & PETERS, C. J. 2000. Transmission of Black Creek Canal virus between cotton rats. J Med Virol, 60, 70-6.

HUTTUNEN, N. P., MAKELA, S., POKKA, T., MUSTONEN, J. & UHARI, M. 2011. Systematic literature review of symptoms, signs and severity of serologically confirmed nephropathia epidemica in paediatric and adult patients. Scand J Infect Dis, 43, 405-10.

IPCC Core Writing Team Contribution of Working Groups I, II and III to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change

In: PACHAURI, R. K. & REISINGER, A. (eds.) Climate Change 2007: Synthesis Report. Geneva, Switzerland.

JIN, M., PARK, J., LEE, S., PARK, B., SHIN, J., SONG, K. J., AHN, T. I., HWANG, S. Y., AHN, B. Y. & AHN, K. 2002. Hantaan virus enters cells by clathrin-dependent receptor-mediated endocytosis. Virology, 294, 60-9.

JONSSON, C. B., FIGUEIREDO, L. T. & VAPALAHTI, O. 2010. A global perspective on hantavirus ecology, epidemiology, and disease. Clin Microbiol Rev, 23, 412-41.

JONSSON, C. B. & SCHMALJOHN, C. S. 2001. Replication of hantaviruses. Curr Top Microbiol Immunol, 256, 15-32.

KALLIO, E. R., KLINGSTROM, J., GUSTAFSSON, E., MANNI, T., VAHERI, A., HENTTONEN, H., VAPALAHTI, O. & LUNDKVIST, A. 2006. Prolonged survival of Puumala hantavirus outside the host: evidence for indirect transmission via the environment. J Gen Virol, 87, 2127-34.

KALLIO, E. R., VOUTILAINEN, L., VAPALAHTI, O., VAHERI, A., HENTTONEN, H., KOSKELA, E. & MAPPES, T. 2007. Endemic hantavirus infection impairs the winter survival of its rodent host. Ecology, 88, 1911-6.

KAUKINEN, P., KOISTINEN, V., VAPALAHTI, O., VAHERI, A. & PLYUSNIN, A. 2001. Interaction between molecules of hantavirus nucleocapsid protein. J Gen Virol, 82, 1845-53.

KHALIL, H., OLSSON, G., ECKE, F., EVANDER, M., HJERTQVIST, M., MAGNUSSON, M., LOFVENIUS, M. O. & HORNFELDT, B. 2014. The importance of bank vole density and rainy winters in predicting nephropathia epidemica incidence in northern sweden. PLoS One, 9, e111663.

KHAN, A. S., KHABBAZ, R. F., ARMSTRONG, L. R., HOLMAN, R. C., BAUER, S. P., GRABER, J., STRINE, T., MILLER, G., REEF, S., TAPPERO, J., ROLLIN, P. E., NICHOL, S. T., ZAKI, S. R., BRYAN, R. T., CHAPMAN, L. E., PETERS, C. J. & KSIAZEK, T. G. 1996. Hantavirus pulmonary syndrome: the first 100 US cases. J Infect Dis, 173, 1297-303.

KILPATRICK, E. D., TERAJIMA, M., KOSTER, F. T., CATALINA, M. D., CRUZ, J. & ENNIS, F. A. 2004. Role of specific CD8+ T cells in the severity of a fulminant zoonotic viral hemorrhagic fever, hantavirus pulmonary syndrome. J Immunol, 172, 3297-304.

KLEMPA, B., FICHET-CALVET, E., LECOMPTE, E., AUSTE, B., ANISKIN, V., MEISEL, H., BARRIERE, P., KOIVOGUI, L., TER MEULEN, J. & KRUGER, D. H. 2007. Novel hantavirus sequences in Shrew, Guinea. Emerg Infect Dis, 13, 520-2.

KLINGSTROM, J., PLYUSNIN, A., VAHERI, A. & LUNDKVIST, A. 2002. Wild-type Puumala hantavirus infection induces cytokines, C-reactive protein, creatinine, and nitric oxide in cynomolgus macaques. J Virol, 76, 444-9.

KLINGSTROM, J., STOLTZ, M., HARDESTAM, J., AHLM, C. & LUNDKVIST, A. 2008. Passive immunization protects cynomolgus macaques against Puumala hantavirus challenge. Antivir Ther, 13, 125-33.

KORVA, M., SAKSIDA, A., KEJZAR, N., SCHMALJOHN, C. & AVSIC-ZUPANC, T. 2013. Viral load and immune response dynamics in patients with haemorrhagic fever with renal syndrome. Clin Microbiol Infect, 19, E358-66.

KRAUTKRAMER, E., GROULS, S., STEIN, N., REISER, J. & ZEIER, M. 2011. Pathogenic old world hantaviruses infect renal glomerular and tubular cells and induce disassembling of cell- to-cell contacts. J Virol, 85, 9811-23.

KRAUTKRAMER, E. & ZEIER, M. 2008. Hantavirus causing hemorrhagic fever with renal syndrome enters from the apical surface and requires decay-accelerating factor (DAF/CD55). J Virol, 82, 4257-64.

KUKKONEN, S. K., VAHERI, A. & PLYUSNIN, A. 2004. Tula hantavirus L protein is a 250 kDa perinuclear membrane-associated protein. J Gen Virol, 85, 1181-9.

LEE, H. W. 1996. Epidemiology and Pathogenesis of Hemorrhagic Fever with Renal Syndrome. In: ELLIOTT, R. M. (ed.) The Bunyaviridae. New York: Plenum Press.

LEE, H. W., LEE, P. W. & JOHNSON, K. M. 1978. Isolation of the etiologic agent of Korean Hemorrhagic fever. J Infect Dis, 137, 298-308.

LEE, H. W., VAHERI, A. & SCHMALJOHN, C. S. 2014. Discovery of hantaviruses and of the Hantavirus genus: personal and historical perspectives of the Presidents of the International Society of Hantaviruses. Virus Res, 187, 2-5.

LI, W. & KLEIN, S. L. 2012. Seoul virus-infected rat lung endothelial cells and alveolar macrophages differ in their ability to support virus replication and induce regulatory T cell phenotypes. J Virol, 86, 11845-55.

LINARD, C., TERSAGO, K., LEIRS, H. & LAMBIN, E. F. 2007. Environmental conditions and Puumala virus transmission in Belgium. Int J Health Geogr, 6, 55.

LINDEN, S. K., SUTTON, P., KARLSSON, N. G., KOROLIK, V. & MCGUCKIN, M. A. 2008. Mucins in the mucosal barrier to infection. Mucosal Immunol, 1, 183-97.

LOBER, C., ANHEIER, B., LINDOW, S., KLENK, H. D. & FELDMANN, H. 2001. The Hantaan virus glycoprotein precursor is cleaved at the conserved pentapeptide WAASA. Virology, 289, 224-9.

LUNDKVIST, A., CHENG, Y., SJOLANDER, K. B., NIKLASSON, B., VAHERI, A. & PLYUSNIN, A. 1997. Cell culture adaptation of Puumala hantavirus changes the infectivity for its natural reservoir, Clethrionomys glareolus, and leads to accumulation of mutants with altered genomic RNA S segment. J Virol, 71, 9515-23.

MACNEIL, A., COMER, J. A., KSIAZEK, T. G. & ROLLIN, P. E. 2010. Sin Nombre virus-specific immunoglobulin M and G kinetics in hantavirus pulmonary syndrome and the role played by serologic responses in predicting disease outcome. J Infect Dis, 202, 242-6.

MAKARY, P., KANERVA, M., OLLGREN, J., VIRTANEN, M. J., VAPALAHTI, O. & LYYTIKAINEN, O. 2010. Disease burden of Puumala virus infections, 1995-2008. Epidemiol Infect, 138, 1484-92.

MARTINEZ-VALDEBENITO, C., CALVO, M., VIAL, C., MANSILLA, R., MARCO, C., PALMA, R. E., VIAL, P. A., VALDIVIESO, F., MERTZ, G. & FERRES, M. 2014. Person-to-person household and nosocomial transmission of andes hantavirus, Southern Chile, 2011. Emerg Infect Dis, 20, 1629-36.

MERTZ, G. J. 2009. Bunyaviridae, Bunyaviruses, Phleboviruses, Nairoviruses, and Hantaviruses. In: RICHMAN, D. D., WHITLEY, R. J. & HAYDEN, F. G. (eds.) Clinical Virology. Third Edition ed.

MERTZ, G. J., MIEDZINSKI, L., GOADE, D., PAVIA, A. T., HJELLE, B., HANSBARGER, C. O., LEVY, H., KOSTER, F. T., BAUM, K., LINDEMULDER, A., WANG, W., RISER, L., FERNANDEZ, H. & WHITLEY, R. J. 2004. Placebo-controlled, double-blind trial of intravenous ribavirin for the treatment of hantavirus cardiopulmonary syndrome in North America. Clin Infect Dis, 39, 1307-13.

MIR, M. A., DURAN, W. A., HJELLE, B. L., YE, C. & PANGANIBAN, A. T. 2008. Storage of cellular 5' mRNA caps in P bodies for viral cap-snatching. Proc Natl Acad Sci U S A, 105, 19294- 9.

MUSTONEN, J., MAKELA, S., OUTINEN, T., LAINE, O., JYLHAVA, J., ARSTILA, P. T., HURME, M. & VAHERI, A. 2013. The pathogenesis of nephropathia epidemica: new knowledge and unanswered questions. Antiviral Res, 100, 589-604.

MYHRMAN, G. 1951. Nephropathia epidemica a new infectious disease in northern Scandinavia. Acta Med Scand, 140, 52-6.

NAVARRETE, M., PIZARRO, E., MÉNDEZ, C., SALAZAR, P., PADULA, P. & ZAROR, L. 2007. Hantavirus ANDV distribution in human lungs and salivary glands. The VII International Conference on HFRS, HPS and Hantaviruses, June 13–15, 2007. Buenos Aires, Argentina.

NETSKI, D., THRAN, B. H. & ST JEOR, S. C. 1999. Sin Nombre virus pathogenesis in Peromyscus maniculatus. J Virol, 73, 585-91.

NUOVO, G. J., SIMSIR, A., STEIGBIGEL, R. T. & KUSCHNER, M. 1996. Analysis of fatal pulmonary hantaviral infection in New York by reverse transcriptase in situ polymerase chain reaction. Am J Pathol, 148, 685-92.

OECD. 2013. Health at a Glance 2013:OECD Indicators [Online]. http://dx.doi.org/10.1787/health_glance-2013-en: OECD Publishing.

OLSSON, G. E., DALERUM, F., HORNFELDT, B., ELGH, F., PALO, T. R., JUTO, P. & AHLM, C. 2003. Human hantavirus infections, Sweden. Emerg Infect Dis, 9, 1395-401.

OLSSON, G. E., HJERTQVIST, M., LUNDKVIST, A. & HORNFELDT, B. 2009. Predicting high risk for human hantavirus infections, Sweden. Emerg Infect Dis, 15, 104-6.

OLSSON, G. E., LEIRS, H. & HENTTONEN, H. 2010. Hantaviruses and their hosts in Europe: reservoirs here and there, but not everywhere? Vector Borne Zoonotic Dis, 10, 549-61.

OUTINEN, T. K., MAKELA, S. M., ALA-HOUHALA, I. O., HUHTALA, H. S., HURME, M., PAAKKALA, A. S., PORSTI, I. H., SYRJANEN, J. T. & MUSTONEN, J. T. 2010. The severity of Puumala hantavirus induced nephropathia epidemica can be better evaluated using plasma interleukin-6 than C-reactive protein determinations. BMC Infect Dis, 10, 132.

OVERBY, A. K., PETTERSSON, R. F. & NEVE, E. P. 2007. The glycoprotein cytoplasmic tail of Uukuniemi virus (Bunyaviridae) interacts with ribonucleoproteins and is critical for genome packaging. J Virol, 81, 3198-205.

PADULA, P., FIGUEROA, R., NAVARRETE, M., PIZARRO, E., CADIZ, R., BELLOMO, C., JOFRE, C., ZAROR, L., RODRIGUEZ, E. & MURUA, R. 2004. Transmission study of Andes hantavirus infection in wild sigmodontine rodents. J Virol, 78, 11972-9.

PADULA, P. J., EDELSTEIN, A., MIGUEL, S. D., LOPEZ, N. M., ROSSI, C. M. & RABINOVICH, R. D. 1998. Hantavirus pulmonary syndrome outbreak in Argentina: molecular evidence for person-to-person transmission of Andes virus. Virology, 241, 323-30.

PADULA, P. J., ROSSI, C. M., DELLA VALLE, M. O., MARTINEZ, P. V., COLAVECCHIA, S. B., EDELSTEIN, A., MIGUEL, S. D., RABINOVICH, R. D. & SEGURA, E. L. 2000. Development and evaluation of a solid-phase enzyme immunoassay based on Andes hantavirus recombinant nucleoprotein. J Med Microbiol, 49, 149-55.

PALO, R. T. 2009. Time series analysis performed on nephropathia epidemica in humans of northern Sweden in relation to bank vole population dynamic and the NAO index. Zoonoses Public Health, 56, 150-6.

PETTERSSON, R. F. & MELIN, L. 1996. Synthesis, Assembly, and Intracellular Transport of Bunyaviridae Membrane Proteins. In: ELLIOTT, R. M. (ed.) The Bunyaviridae. New York: Plenum Press.

PLYUSNIN, A., BEATY, B. J., ELLIOTT, R. M., GOLDBACH, R., KORMELINK, R., LUNDKVIST, Å., SCHMALJOHN, C. S. & TESH, R. B. 2012. Bunyaviridae. In: KING, A. M. Q., ADAMS, M. J. & CARSTENS, E. B. (eds.) Virus Taxonomy: Classification and Nomenclature of Viruses. Ninth Report of the International Committee on Taxonomy of Viruses,.

PLYUSNIN, A. & SIRONEN, T. 2014. Evolution of hantaviruses: co-speciation with reservoir hosts for more than 100 MYR. Virus Res, 187, 22-6.

POLJAK, M. & AVSIC ZUPANC, T. 1994. Immunohistochemical detection of Hantaan virus antigen in renal tissue from patient with hemorrhagic fever with renal syndrome. Nephron, 67, 252.

RAFTERY, M. J., KRAUS, A. A., ULRICH, R., KRUGER, D. H. & SCHONRICH, G. 2002. Hantavirus infection of dendritic cells. J Virol, 76, 10724-33.

RAFTERY, M. J., LALWANI, P., KRAUTKRMER, E., PETERS, T., SCHARFFETTER- KOCHANEK, K., KRUGER, R., HOFMANN, J., SEEGER, K., KRUGER, D. H. & SCHONRICH, G. 2014. beta2 integrin mediates hantavirus-induced release of neutrophil extracellular traps. J Exp Med, 211, 1485-97.

RASMUSON, J., ANDERSSON, C., NORRMAN, E., HANEY, M., EVANDER, M. & AHLM, C. 2011. Time to revise the paradigm of hantavirus syndromes? Hantavirus pulmonary syndrome caused by European hantavirus. Eur J Clin Microbiol Infect Dis, 30, 685-90.

RAVKOV, E. V. & COMPANS, R. W. 2001. Hantavirus nucleocapsid protein is expressed as a membrane-associated protein in the perinuclear region. J Virol, 75, 1808-15.

RODA GRACIA, J., SCHUMANN, B. & SEIDLER, A. 2014. Climate Variability and the Occurrence of Human Puumala Hantavirus Infections in Europe: A Systematic Review. Zoonoses Public Health.

ROWE, R. K. & PEKOSZ, A. 2006. Bidirectional virus secretion and nonciliated cell tropism following Andes virus infection of primary airway epithelial cell cultures. J Virol, 80, 1087-97.

RUSNAK, J. M., BYRNE, W. R., CHUNG, K. N., GIBBS, P. H., KIM, T. T., BOUDREAU, E. F., COSGRIFF, T., PITTMAN, P., KIM, K. Y., ERLICHMAN, M. S., REZVANI, D. F. & HUGGINS, J. W. 2009. Experience with intravenous ribavirin in the treatment of hemorrhagic fever with renal syndrome in Korea. Antiviral Res, 81, 68-76.

SAFRONETZ, D., DREBOT, M. A., ARTSOB, H., COTE, T., MAKOWSKI, K. & LINDSAY, L. R. 2008. Sin Nombre virus shedding patterns in naturally infected deer mice (Peromyscus maniculatus) in relation to duration of infection. Vector Borne Zoonotic Dis, 8, 97-100.

SAFRONETZ, D., HADDOCK, E., FELDMANN, F., EBIHARA, H. & FELDMANN, H. 2011a. In vitro and in vivo activity of ribavirin against Andes virus infection. PLoS One, 6, e23560.

SAFRONETZ, D., PRESCOTT, J., FELDMANN, F., HADDOCK, E., ROSENKE, R., OKUMURA, A., BRINING, D., DAHLSTROM, E., PORCELLA, S. F., EBIHARA, H., SCOTT, D. P., HJELLE, B. & FELDMANN, H. 2014. Pathophysiology of hantavirus pulmonary syndrome in rhesus macaques. Proc Natl Acad Sci U S A, 111, 7114-9.

SAFRONETZ, D., ZIVCEC, M., LACASSE, R., FELDMANN, F., ROSENKE, R., LONG, D., HADDOCK, E., BRINING, D., GARDNER, D., FELDMANN, H. & EBIHARA, H. 2011b. Pathogenesis and host response in Syrian hamsters following intranasal infection with Andes virus. PLoS Pathog, 7, e1002426.

SAKSIDA, A., DUH, D., KORVA, M. & AVSIC-ZUPANC, T. 2008. Dobrava virus RNA load in patients who have hemorrhagic fever with renal syndrome. J Infect Dis, 197, 681-5.

SAKSIDA, A., WRABER, B. & AVSIC-ZUPANC, T. 2011. Serum levels of inflammatory and regulatory cytokines in patients with hemorrhagic fever with renal syndrome. BMC Infect Dis, 11, 142.

SETTERGREN, B., JUTO, P., TROLLFORS, B., WADELL, G. & NORRBY, S. R. 1989. Clinical characteristics of nephropathia epidemica in Sweden: prospective study of 74 cases. Rev Infect Dis, 11, 921-7.

SEVERSON, W. E., SCHMALJOHN, C. S., JAVADIAN, A. & JONSSON, C. B. 2003. Ribavirin causes error catastrophe during Hantaan virus replication. J Virol, 77, 481-8.

SHI, X. & ELLIOTT, R. M. 2004. Analysis of N-linked glycosylation of hantaan virus glycoproteins and the role of oligosaccharide side chains in protein folding and intracellular trafficking. J Virol, 78, 5414-22.

SHUGARS, D. C. 1999. Endogenous mucosal antiviral factors of the oral cavity. J Infect Dis, 179 Suppl 3, S431-5.

SIRONEN, T. & PLYUSNIN, A. 2011. Genetics and Evolution of Hantaviruses. In: PLYUSNIN, A. & ELLIOTT, R. M. (eds.) Bunyaviridae Molecular and Cellular Biology. Norfolk, UK: Caister Academic Press.

SONG, J. W., GU, S. H., BENNETT, S. N., ARAI, S., PUORGER, M., HILBE, M. & YANAGIHARA, R. 2007. Seewis virus, a genetically distinct hantavirus in the Eurasian common shrew (Sorex araneus). Virol J, 4, 114.

STRANDIN, T., HEPOJOKI, J. & VAHERI, A. 2013. Cytoplasmic tails of bunyavirus Gn glycoproteins-Could they act as matrix protein surrogates? Virology, 437, 73-80.

SUNDSTROM, J. B., MCMULLAN, L. K., SPIROPOULOU, C. F., HOOPER, W. C., ANSARI, A. A., PETERS, C. J. & ROLLIN, P. E. 2001. Hantavirus infection induces the expression of RANTES and IP-10 without causing increased permeability in human lung microvascular endothelial cells. J Virol, 75, 6070-85.

TEMONEN, M., LANKINEN, H., VAPALAHTI, O., RONNI, T., JULKUNEN, I. & VAHERI, A. 1995. Effect of interferon-alpha and cell differentiation on Puumala virus infection in human monocyte/macrophages. Virology, 206, 8-15.

TEMONEN, M., MUSTONEN, J., HELIN, H., PASTERNACK, A., VAHERI, A. & HOLTHOFER, H. 1996. Cytokines, adhesion molecules, and cellular infiltration in nephropathia epidemica kidneys: an immunohistochemical study. Clin Immunol Immunopathol, 78, 47-55.

TERAJIMA, M., HENDERSHOT, J. D., 3RD, KARIWA, H., KOSTER, F. T., HJELLE, B., GOADE, D., DEFRONZO, M. C. & ENNIS, F. A. 1999. High levels of viremia in patients with the Hantavirus pulmonary syndrome. J Infect Dis, 180, 2030-4.

TERSAGO, K., VERHAGEN, R., SERVAIS, A., HEYMAN, P., DUCOFFRE, G. & LEIRS, H. 2009. Hantavirus disease (nephropathia epidemica) in Belgium: effects of tree seed production and climate. Epidemiol Infect, 137, 250-6.

TORO, J., VEGA, J. D., KHAN, A. S., MILLS, J. N., PADULA, P., TERRY, W., YADON, Z., VALDERRAMA, R., ELLIS, B. A., PAVLETIC, C., CERDA, R., ZAKI, S., SHIEH, W. J., MEYER, R., TAPIA, M., MANSILLA, C., BARO, M., VERGARA, J. A., CONCHA, M., CALDERON, G., ENRIA, D., PETERS, C. J. & KSIAZEK, T. G. 1998. An outbreak of hantavirus pulmonary syndrome, Chile, 1997. Emerg Infect Dis, 4, 687-94.

TRENBERTH, K. E., JONES, P. D., AMBENJE, P., BOJARIU, R., EASTERLING, D., TANK, A. K., PARKER, D., RAHIMZADEH, F., RENWICK, J. A., RUSTICUCCI, M. & ZHAI, B. S. A. P. 2007. Observations: Surface and Atmospheric Climate Change. Climate Change 2007: The Physical Science Basis

Contribution of Working Group I to the Fourth Assessment Report of the Intergovernmental Panel on Climate Change. Cambridge: Cambridge University Press.

VAPALAHTI, O., MUSTONEN, J., LUNDKVIST, A., HENTTONEN, H., PLYUSNIN, A. & VAHERI, A. 2003. Hantavirus infections in Europe. Lancet Infect Dis, 3, 653-61.

VAZQUEZ, S., CABEZAS, S., PEREZ, A. B., PUPO, M., RUIZ, D., CALZADA, N., BERNARDO, L., CASTRO, O., GONZALEZ, D., SERRANO, T., SANCHEZ, A. & GUZMAN, M. G. 2007. Kinetics of antibodies in sera, saliva, and urine samples from adult patients with primary or secondary dengue 3 virus infections. Int J Infect Dis, 11, 256-62.

VIAL, P. A., VALDIVIESO, F., CALVO, M., RIOSECO, M. L., RIQUELME, R., ARANEDA, A., TOMICIC, V., GRAF, J., PAREDES, L., FLORENZANO, M., BIDART, T., CUIZA, A., MARCO, C., HJELLE, B., YE, C., HANFELT-GOADE, D., VIAL, C., RIVERA, J. C., DELGADO, I. & MERTZ, G. J. 2014. A non-randomized multicentre trial of human immune plasma for treatment of hantavirus cardiopulmonary syndrome by ANDV. Antivir Ther.

VIAL, P. A., VALDIVIESO, F., FERRES, M., RIQUELME, R., RIOSECO, M. L., CALVO, M., CASTILLO, C., DIAZ, R., SCHOLZ, L., CUIZA, A., BELMAR, E., HERNANDEZ, C., MARTINEZ, J., LEE, S. J. & MERTZ, G. J. 2013. High-dose intravenous methylprednisolone for hantavirus cardiopulmonary syndrome in Chile: a double-blind, randomized controlled clinical trial. Clin Infect Dis, 57, 943-51.

WEISS, S., WITKOWSKI, P. T., AUSTE, B., NOWAK, K., WEBER, N., FAHR, J., MOMBOULI, J. V., WOLFE, N. D., DREXLER, J. F., DROSTEN, C., KLEMPA, B., LEENDERTZ, F. H. & KRUGER, D. H. 2012. Hantavirus in bat, Sierra Leone. Emerg Infect Dis, 18, 159-61.

WELLS, R. M., SOSA ESTANI, S., YADON, Z. E., ENRIA, D., PADULA, P., PINI, N., MILLS, J. N., PETERS, C. J. & SEGURA, E. L. 1997. An unusual hantavirus outbreak in southern Argentina: person-to-person transmission? Hantavirus Pulmonary Syndrome Study Group for Patagonia. Emerg Infect Dis, 3, 171-4.

XIAO, R., YANG, S., KOSTER, F., YE, C., STIDLEY, C. & HJELLE, B. 2006. Sin Nombre viral RNA load in patients with hantavirus cardiopulmonary syndrome. J Infect Dis, 194, 1403-9.

YANAGIHARA, R., AMYX, H. L. & GAJDUSEK, D. C. 1985. Experimental infection with Puumala virus, the etiologic agent of nephropathia epidemica, in bank voles (Clethrionomys glareolus). J Virol, 55, 34-8.

YI, J., XU, Z., ZHUANG, R., WANG, J., ZHANG, Y., MA, Y., LIU, B., ZHANG, C., YAN, G., ZHANG, F., YANG, A. & JIN, B. 2013. Hantaan virus RNA load in patients having hemorrhagic fever with renal syndrome: correlation with disease severity. J Infect Dis, 207, 1457-61.

ZAKI, S. R., GREER, P. W., COFFIELD, L. M., GOLDSMITH, C. S., NOLTE, K. B., FOUCAR, K., FEDDERSEN, R. M., ZUMWALT, R. E., MILLER, G. L., KHAN, A. S. & ET AL. 1995. Hantavirus pulmonary syndrome. Pathogenesis of an emerging infectious disease. Am J Pathol, 146, 552- 79.