Hantaviruses, similar to several emerging zoonotic viruses, persistently infect their natural reservoir hosts, without causing overt signs of disease. Spillover to incidental human hosts results in morbidity and mortality mediated by excessive proinflammatory and cellular immune responses. The mechanisms mediating the persistence of hantaviruses and the absence of clinical symptoms in rodent reservoirs are only starting to be uncovered. Recent studies indicate that during hantavirus infection, proinflammatory and antiviral responses are reduced and regulatory responses are elevated at sites of increased virus replication in rodents. The recent discovery of structural and non-structural proteins that suppress type I interferon responses in humans suggests that immune responses in rodent hosts could be mediated directly by the virus. Alternatively, several host factors, including sex steroids, glucocorticoids, and genetic factors, are reported to alter host susceptibility and may contribute to persistence of hantaviruses in rodents. Humans and reservoir hosts differ in infection outcomes and in immune responses to hantavirus infection; thus, understanding the mechanisms mediating viral persistence and the absence of disease in rodents may provide insight into the prevention and treatment of disease in humans. Consideration of the coevolutionary mechanisms mediating hantaviral persistence and rodent host survival is providing insight into the mechanisms by which zoonotic viruses have remained in the environment for millions of years and continue to be transmitted to humans.
Citation: Easterbrook JD, Klein SL (2008) Immunological Mechanisms Mediating Hantavirus Persistence in Rodent Reservoirs. PLoS Pathog 4(11): e1000172. doi:10.1371/journal.ppat.1000172
Editor: Michael J. Buchmeier, University of California Irvine, United States of America
Published: November 28, 2008
Copyright: © 2008 Easterbrook, Klein. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: NIH R01 AI 054995.
Competing interests: The authors have declared that no competing interests exist.
Hantaviruses are negative sense, enveloped RNA viruses (family: Bunyaviridae) that are comprised of three RNA segments, designated small (S), medium (M), and large (L), which encode the viral nucleocapsid (N), envelope glycoproteins (GN and GC), and an RNA polymerase (Pol), respectively. More than 50 hantaviruses have been found worldwide . Each hantavirus appears to have coevolved with a specific rodent or insectivore host as similar phylogenetic trees are produced from virus and host mitochondrial gene sequences . Spillover to humans causes hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS), depending on the virus –. Although symptoms vary, a common feature of both HFRS and HCPS is increased permeability of the vasculature and mononuclear infiltration . Pathogenesis of HRFS and HCPS in humans is hypothesized to be mediated by excessive proinflammatory and CD8+ T cell responses (Table 1).
Table 1. Summary of Immune Responses in Humans during Hantavirus Infection.doi:10.1371/journal.ppat.1000172.t001
Table 2. Summary of Immune Responses in Rodents during Hantavirus Infection.doi:10.1371/journal.ppat.1000172.t002
In contrast to humans, hantaviruses persistently infect their reservoir hosts, presumably causing lifelong infections . Hantaviruses are shed in saliva, urine, and feces, and transmission among rodents or from rodents to humans occurs by inhalation of aerosolized virus in excrement or by transmission of virus in saliva during wounding ,. Although widely disseminated throughout the rodent host, high amounts of hantaviral RNA and antigen are consistently identified in the lungs of their rodent hosts, suggesting that the lungs may be an important site for maintenance of hantaviruses during persistent infection –. Hantavirus infection in rodents is characterized by an acute phase of peak viremia, viral shedding, and virus replication in target tissues, followed by a persistent phase of reduced, cyclical virus replication despite the presence of high antibody titers (Figure 1) –, –. The onset of persistent infection varies across hantavirus–rodent systems, but generally the acute phase occurs during the first 2–3 weeks of infection and virus persistence is established thereafter (Figure 1).
Figure 1. Kinetics of Hantavirus Infection in Rodents.
Adapted from Lee et al.  and others –,,,, the kinetics of relative hantaviral load in blood (red), saliva (green), and lung tissue (blue) and antibody responses (black) during the acute and persistent phases of infection are represented. The amount of genomic viral RNA, infectious virus titer, and/or relative amount of viral antigen have been incorporated as relative hantaviral load. The antibody response is integrated as the relative amount of anti-hantavirus IgG and/or neutralizing antibody titers.doi:10.1371/journal.ppat.1000172.g001
Hantavirus infection alone does not cause disease, as reservoir hosts and non-natural hosts (e.g., hamsters infected with Sin Nombre virus [SNV] or Choclo virus) may support replicating virus in the absence of overt disease ,,,,,. Our primary hypothesis is that certain immune responses that are mounted in humans during hantavirus infection are suppressed in rodent reservoirs to establish and maintain viral persistence, while preventing disease (Table 2). During the coevolution of hantaviruses with their reservoir hosts, the viruses may have evolved mechanisms to enhance persistence, including immune evasion, direct suppression or alteration of host antiviral, proinflammatory, and cellular immune responses, and induction of host regulatory responses. Alternatively, hosts may have evolved adaptations, including immunological responsiveness to steroid hormones and pre-existing host genetic factors, to regulate the detrimental effects of infection, which also may affect persistence of hantaviruses.
Virus-Mediated Responses to Hantaviruses
Mechanisms of immune evasion, including viral mutation and segment reassortment, may contribute to viral persistence. Cyclical rises of Seoul virus (SEOV) L segment deletions and S segment insertions in regions necessary for initiation of transcription are observed in vitro ; whether mutated RNAs can self-repair and whether this occurs in vivo remains to be determined. Quasispecies have been identified in vivo in deer mice (Peromyscus maniculatus) and European common voles (Microtus arvalis) infected with SNV and Puumala virus (PUUV), respectively, with nucleotide and amino acid mutations identified in immunodominant regions of the GN protein ,. Segment reassortment of hantaviruses has been identified in Vero E6 cells that are co-infected with related strains of SNV and in vivo in deer mice infected with SNV ,. Recent data also suggest that a newly discovered hantavirus in Paraguay may be a reassortment between Laguna Negra or Rio Mamoré viruses with Pergamino or Maciel viruses . Quasispecies and reassortants likely contribute to the evolution of new species of hantaviruses, but also may be involved in immune evasion.
Direct alteration of host cellular responses.
Hantaviruses can infect macrophages and endothelial cells in rodents and humans ,,,. These cell types have been identified primarily by morphological analyses or by non-specific cell separation. Recently, SEOV N protein was definitively identified in macrophages and endothelial cells in lung tissue of infected rats using cell-specific antibodies . Hantaan virus (HTNV) infects human dendritic cells (DCs) in vitro and induces DC activation and maturation . Whether rodent DCs are infected by hantaviruses and whether rodent macrophage and DC activity is suppressed by hantavirus infection to cause persistence remains unknown. Several viruses in the Bunyaviridae family encode a non-structural protein (NSs) that suppresses antiviral immune responses in infected cells . The S segment of PUUV and Tula virus (TULV) has an open reading frame (ORF) for NSs, a protein that reduces the expression of IFN-β in human lung fibroblasts and contributes to virus survival in vitro ,. Immunoreceptor tyrosine-based activation motifs (ITAMs) that bind kinases to regulate immune and endothelial cell function have been identified in the GN protein of hantaviruses ,. Additionally, the cytoplasmic tails of HCPS-causing hantavirus G proteins inhibit the expression of type I IFN responses in human umbilical vein endothelial cells (HUVECs) and human lung microvascular endothelial cells (HMVEC-Ls) in vitro ,. Whether hantavirus proteins and RNA have immunomodulatory activity in rodent reservoirs remains unknown.
Host-Mediated Responses to Hantaviruses
Innate antiviral and proinflammatory responses.
Infection of HMVEC-Ls and HUVECs indicate that at least some of the hantaviruses that cause disease in humans (e.g., HTNV, Andes virus [ANDV], SNV, New York-1 virus [NY-1V], PUUV, and SEOV) delay induction of factors in the type I IFN pathway (e.g., production of IFN-α, IFN-β, and MxA) as compared with hantaviruses that cause no known disease in humans (e.g., TULV and Prospect Hill virus [PHV]) , –. Delayed production of antiviral responses in humans may contribute to more efficient replication of pathogenic hantaviruses than non-pathogenic hantaviruses. Despite delayed induction of type I IFNs, human cells mount innate antiviral responses that also may contribute to viral clearance.
In the lungs of rats with high amounts of virus (i.e., male rats), the expression of pattern recognition receptors (i.e., Rig-I and Tlr7) is reduced or remains unchanged throughout SEOV infection, suggesting that inhibition of viral recognition may contribute to the establishment of persistent infection . Furthermore, antiviral (e.g., IFN-β, Mx2, and IFN-γ) and proinflammatory (e.g., IL-1β, TNF-α, and NOS2) responses are reduced or unaltered during infection in the lungs of male rats, which also may contribute to hantavirus persistence ,,. In contrast, in the spleen, a peripheral immune organ that supports low amounts of virus, the expression and production of proinflammatory and antiviral factors are elevated during acute SEOV infection and subsequently return to baseline ,. Thus, rats infected with SEOV do not appear to be globally immunosuppressed, but rather have a site-specific reduction of proinflammatory responses. There is no evidence that male rats that are naturally infected with SEOV are more likely to acquire additional pathogens, further illustrating that infected rats are not immunocompromised . Conversely, natural populations of deer mice that have antibody against SNV elicit a lower response to phytohemagglutinin (i.e., a measure of immunocompetence) than their uninfected counterparts, suggesting that SNV causes some degree of immunosuppression in deer mice . Administration of exogenous IL-1β, which elevates circulating IL-1β and Il6 and Tnfα expression in the lungs within physiological ranges, does not affect SEOV persistence in male rats nor does it cause observable disease . Thus, extremely high proinflammatory responses observed during acute infection in humans may be necessary for viral clearance at the expense of causing potentially fatal proinflammatory-mediated disease.
CD4+ and CD8+ T cell responses.
Cellular immune responses, in particular CD8+ T cells, contribute to clearance of hantaviruses in humans at the expense of causing disease ,. Following inoculation with SEOV, nude (i.e., T cell deficient) rats have more virus in target tissues and shed more infectious virus than do their immunocompetent counterparts and die 10 weeks after inoculation, indicating that T cells contribute to the control of virus replication and survival in a reservoir host . During the acute phase of SNV infection, deer mice have observable Th1 and Th2 responses (i.e., elevated expression of Ifnγ, Gata3 [i.e., the hallmark Th2 transcription factor], Il4, and Il5) in cultured CD4+ T cells, which are not evident in CD4+ T cells isolated from persistently infected deer mice . In severe-combined immunodeficient (SCID) mice (Mus musculus), the transfer of functional CD8+ T cells is necessary for clearance of HTNV . Persistence of HTNV in newborn BALB/c mice is correlated with a decrease in HTNV-specific CD8+ T cell numbers and activity, as measured by IFN-γ production, further suggesting a role of functional CD8+ T cell responses in viral clearance ,. Not only are laboratory mice non-natural hosts, but SCID and newborn mice do not have fully functional immune systems, so these models do not accurately represent viral persistence in immunocompetent rodent reservoir populations. The effect of infection on hantavirus-specific CD8+ T cells in adult rodent reservoir hosts requires examination.
Regulatory T cell responses.
Regulatory T cell responses suppress proinflammatory and effector T cell responses locally at the site of infection to allow pathogen persistence, as well as to mitigate proinflammatory-mediated pathogenesis . Recent studies have demonstrated that regulatory T cells contribute to SEOV and SNV persistence in rats and deer mice, respectively ,. Expression of Foxp3 mRNA and proportions of CD4+CD25+FoxP3+ regulatory T cells are elevated locally at a site of elevated SEOV replication (i.e., in the lungs) in male rats during persistent SEOV infection ,. Functional inactivation of regulatory T cells reduces the amount of SEOV RNA present in the lungs and the proportion of animals shedding viral RNA in saliva . In the lungs, the expression and production of TGF-β is elevated and TNF-α is suppressed during persistent infection; both cytokine expression patterns are dependent on the presence of functional regulatory T cells . Similarly, CD4+ T cells isolated from deer mice during the persistent phase of SNV infection have higher expression of Tgfβ than do CD4+ T cells isolated from deer mice during the acute phase of infection . The production of IL-10 is consistently reduced throughout SEOV and during the persistent phase of SNV infection in rats and deer mice, respectively, revealing that IL-10 does not contribute to regulatory T cell–mediated hantaviral persistence ,,. Because responses to hantavirus infection in humans involve overproduction of proinflammatory cytokines, it is consistent that “regulatory T cell activity” (i.e., T cells which reduce ConA-induced proliferation of PBMCs) is suppressed during symptomatic HTNV infection in humans ; whether suppressed regulatory T cell responses contribute to disease in humans requires consideration.
Hantaviruses persist in their rodent hosts despite the presence of neutralizing antibody. Antibody against hantaviruses is usually detectable after the first 2 weeks of infection, rapidly increases for the next 4–6 weeks, and declines, but remains detectable presumably for the lifetime of the rodent (Figure 1) , –,,. Hantavirus-specific antibody responses, although not capable of eliminating virus, can serve a protective role against infection. Maternal antibody protects offspring of hantavirus-infected dams during the first 2 months of life (i.e., when the immune system is not fully developed) in various rodent reservoirs –. Not only are young rodents protected from hantavirus infection, but young male and female bank voles with maternal antibody against PUUV also mature earlier, suggesting that reproductive success may be increased in bank voles with, as compared to bank voles without, maternal antibody . How hantaviruses evade antibody responses in their rodent hosts remains to be answered.
Sex differences and sex steroids.
In natural populations of rodent reservoirs, males are more frequently infected with hantaviruses and are more likely to engage in aggressive encounters than are females, which may result in elevated exposure and transmission of hantaviruses among males , –. In laboratory settings, when given the same challenge, male rats have more SEOV RNA and antigen in target organs and saliva than do females ,. Removal of the testes in males (i.e., reduction of androgens to non-detectable levels) reduces, whereas removal of the ovaries in females (i.e., reduction of estrogens and progesterone to non-detectable levels) increases SEOV RNA loads as compared with their intact counterparts . Consistent with sex differences in SEOV load, the expression of innate antiviral (e.g., Tlr7, Myd88, Rig-I, Visa, Ifnβ, and Mx2) and proinflammatory (e.g., Tnfα and Ccl5) factors is higher in the lungs of female than male rats ,,,. Similarly, immunocompetence, as measured by swelling in response to PHA, is higher in female than in male deer mice during SNV infection . Conversely, the expression and production of regulatory factors, including Foxp3 and TGF-β, is elevated in the lungs of males as compared with those of females . These sexually dimorphic immune responses may be dependent on estradiol in females and testosterone in males, as gonadectomy reverses these differences ,. It is plausible that reduced innate and proinflammatory defenses and elevated regulatory responses combined with an increased propensity to engage in aggression may contribute to increased maintenance and transmission of hantaviruses among male as opposed to among female rodents. Whether there exists a sexual dimorphism in the risk of zoonotic transmission of hantaviruses should continue to be considered.
Sex differences in response to hantavirus infection in humans are beginning to receive attention. During acute PUUV infection in humans, circulating concentrations of CXCL8 and CXCL10 are higher, whereas concentrations of IL-9 and GM-CSF are lower in men than in women . Although a similar proportion of men and women have antibodies against PUUV , men are more likely to develop symptoms of disease (i.e., be hospitalized) during PUUV infection than are women ,. Whether sexually dimorphic immune responses during hantavirus infection cause differences in the severity of disease between men and women requires further investigation.
Glucocorticoids are potent immunosuppressive steroid hormones that can suppress proinflammatory and cellular responses and have been administered to patients with HFRS or HCPS to reduce immunopathology ,. Infection of humans with either HTNV or PUUV causes pituitary and adrenal necrosis, which may contribute to the reduced concentrations of cortisol and elevated proinflammatory responses in patients during the acute phase of infection ,. In rats, circulating concentrations of corticosterone are reduced during SEOV infection in males, but not in females . Males with reduced concentrations of glucocorticoids have more SEOV RNA in the lungs than do males with elevated concentrations . No such relationship between glucocorticoid concentration and SEOV load is observed in females . Low concentrations of corticosterone correlate with elevated regulatory responses (i.e., expression of Foxp3 and production of TGF-β) and expression of the glycogenase, matrix metalloprotease (Mmp)-9, in the lungs of male, but not female, rats during SEOV infection . Increased production of MMP-9 may disrupt the basement membrane and extracellular matrices in tissues to increase virus dissemination in male rats . Whether hantaviruses alter glucocorticoids in other reservoir hosts requires further investigation. Based on our data, administration of corticosteroids to patients with HFRS or HCPS would not be expected to cause chronic hantavirus infection and may even reduce viral dissemination in tissues.
Host genetic factors may contribute to susceptibility to and outcome of hantavirus infection in both humans and rodent hosts. Patients with TNF polymorphisms associated with elevated or reduced TNF-α transcription are predisposed to exhibit more severe disease during PUUV infection ,. Possession of certain HLA haplotypes (i.e., HLA B8-DR3, C4A*Q0, and DRB1*0301) is a risk factor for severe disease during PUUV infection in humans, illustrating that antigen presentation and T cell responses likely contribute to the severity of disease ,. Several MHC alleles may alter susceptibility in rodent reservoirs, specifically Cgl-DQA-09, which is positively, and Cgl-DQA-05 and Cgl-DQA-12, which are negatively, associated with the likelihood of bank voles being infected with PUUV . Because uninfected bank voles may never have been exposed to PUUV, whether these specific MHC alleles alter susceptibility following a known exposure to PUUV remains to be determined.
Hantaviruses and their rodent reservoirs represent highly coevolved systems in which virulence and host responses have been adapted to ensure survival of both the virus and the host. The current literature suggests that hantavirus persistence is mediated by both the virus and the host. Although several potential mechanisms mediating the persistence of hantaviruses in their rodent hosts have been discovered in recent years, there are a number of intriguing questions that remain to be addressed:
- Do hantaviral NSs inhibit type I IFN responses in rodents and therefore contribute to viral persistence?
- Is DC and macrophage activity (e.g., antigen presentation, cytokine production, and T cell activation) suppressed by hantavirus infection in rodent reservoirs?
- Does suppression of excessive proinflammatory cytokine responses (e.g., TNF-α) prevent disease in rodents at the cost of causing viral persistence?
- Is the activity of hantavirus-specific CD8+ T cells suppressed during infection to mediate viral persistence in rodents?
- How is regulatory T cell activity induced by hantaviruses in reservoir hosts?
- What is the mechanism of regulatory T cell–mediated hantaviral persistence (e.g., suppression of proinflammatory and/or CD8+ T cell activity)?
- Do host genetic factors, in addition to MHC alleles, contribute to the susceptibility of rodents to hantaviruses?
- What is the role of non-immune mediators, including MMP-9  and receptor use for cellular entry ,, in hantaviral dissemination and persistence in rodents?
We propose that comparing immune responses in rodents to those in humans may provide insight into ways to prevent pathology in humans. Although advances are being made in the development of a hantavirus vaccine, there currently is no FDA-approved vaccine or drug for prevention or treatment of hantaviral disease ,. Elevated regulatory T cell responses in rodents contribute to hantavirus persistence, possibly by suppressing proinflammatory responses (i.e., production of TNF-α) ,. Regulatory T cell responses during hantavirus infection have not been well characterized in humans, but may be downregulated  and contribute to symptoms of HFRS and HCPS. Targeted manipulation of regulatory T cell responses by adoptive transfer of regulatory T cells, administration of anti-TNFα therapy, or treatment with glucocorticoids may control the “cytokine storm” that is initiated when hantaviruses infect humans and cause severe immunopathology. Understanding the mechanisms mediating viral persistence in the absence of disease in reservoir hosts may contribute to advances in the treatment of HFRS and HCPS in humans.
- 1. Klein SL, Calisher CH (2007) Emergence and persistence of hantaviruses. Curr Top Microbiol Immunol 317: 217–252.
- 2. Plyusnin A, Morzunov SP (2001) Virus evolution and genetic diversity of hantaviruses and their rodent hosts. Curr Top Microbiol Immunol 256: 47–75.
- 3. Zaki SR, Greer PW, Coffield LM, Goldsmith CS, Nolte KB, et al. (1995) Hantavirus pulmonary syndrome. Pathogenesis of an emerging infectious disease. Am J Pathol 146: 552–579.
- 4. Khaiboullina SF, St Jeor SC (2002) Hantavirus immunology. Viral Immunol 15: 609–625.
- 5. Nichol ST, Spiropoulou CF, Morzunov S, Rollin PE, Ksiazek TG, et al. (1993) Genetic identification of a hantavirus associated with an outbreak of acute respiratory illness. Science 262: 914–917.
- 6. Meyer BJ, Schmaljohn CS (2000) Persistent hantavirus infections: characteristics and mechanisms. Trends Microbiol 8: 61–67.
- 7. Glass GE, Childs JE, Korch GW, Leduc JW (1988) Association of intraspecific wounding with hantaviral infection in wild rats (Rattus norvegicus). Epidemiol Infect 101: 459–472.
- 8. Kariwa H, Fujiki M, Yoshimatsu K, Arikawa J, Takashima I, et al. (1998) Urine-associated horizontal transmission of Seoul virus among rats. Arch Virol 143: 365–374.
- 9. Kariwa H, Kimura M, Yoshizumi S, Arikawa J, Yoshimatsu K, et al. (1996) Modes of Seoul virus infections: persistency in newborn rats and transiency in adult rats. Arch Virol 141: 2327–2338.
- 10. Hinson ER, Shone SM, Zink MC, Glass GE, Klein SL (2004) Wounding: the primary mode of Seoul virus transmission among male Norway rats. Am J Trop Med Hyg 70: 310–317.
- 11. Klein SL, Marson AL, Scott AL, Ketner G, Glass GE (2002) Neonatal sex steroids affect responses to Seoul virus infection in male but not female Norway rats. Brain Behav Immunity 16: 736–746.
- 12. Botten J, Mirowsky K, Kusewitt D, Bharadwaj M, Yee J, et al. (2000) Experimental infection model for Sin Nombre hantavirus in the deer mouse (Peromyscus maniculatus). Proc Natl Acad Sci U S A 97: 10578–10583.
- 13. Botten J, Mirowsky K, Kusewitt D, Ye CY, Gottlieb K, et al. (2003) Persistent Sin Nombre virus infection in the deer mouse (Peromyscus maniculatus) model: Sites of replication and strand-specific expression. J Virol 77: 1540–1550.
- 14. Yanagihara R, Amyx HL, Gajdusek DC (1985) Experimental infection with Puumala virus, the etiologic agent of nephropathia epidemica, in bank voles (Clethrionomys glareolus). J Virol 55: 34–38.
- 15. Lee HW, Lee PW, Baek LJ, Song CK, Seong IW (1981) Intraspecific transmission of Hantaan virus, etiologic agent of Korean Hemorrhagic fever, in the rodent. Am J Trop Med Hyg 30: 1106–1112.
- 16. Lee PW, Yanagihara R, Gibbs CJ Jr, Gajdusek DC (1986) Pathogenesis of experimental Hantaan virus infection in laboratory rats. Arch Virol 88: 57–66.
- 17. Tanishita O, Takahashi Y, Okuno Y, Tamura M, Asada H, et al. (1986) Persistent infection of rats with hemorrhagic fever with renal syndrome virus and their antibody responses. J Gen Virol 67: 2819–2824.
- 18. Hutchinson KL, Rollin PE, Peters CJ (1998) Pathogenesis of a North American hantavirus, Black Creek Canal virus, in experimentally infected Sigmodon hispidus. Am J Trop Med Hyg 59: 58–65.
- 19. Hannah MF, Bajic VB, Klein SL (2008) Sex differences in the recognition of and innate antiviral responses to Seoul virus in Norway rats. Brain Behav Immun 22: 503–516.
- 20. Klein SL, Bird BH, Glass GE (2001) Sex differences in immune responses and viral shedding following Seoul virus infection in Norway rats. Am J Trop Med Hyg 65: 57–63.
- 21. Eyzaguirre EJ, Milazzo ML, Koster FT, Fulhorst CF (2008) Choclo virus infection in the Syrian golden hamster. Am J Trop Med Hyg 78: 669–674.
- 22. Wahl-Jensen V, Chapman J, Asher L, Fisher R, Zimmerman M, et al. (2007) Temporal analysis of Andes virus and Sin Nombre virus infections of Syrian hamsters. J Virol 81: 7449–7462.
- 23. Feuer R, Boone JD, Netski D, Morzunov SP, St Jeor SC (1999) Temporal and spatial analysis of Sin Nombre virus quasispecies in naturally infected rodents. J Virol 73: 9544–9554.
- 24. Plyusnin A, Cheng Y, Lehvaslaiho H, Vaheri A (1996) Quasispecies in wild-type Tula hantavirus populations. J Virol 70: 9060–9063.
- 25. Rodriguez LL, Owens JH, Peters CJ, Nichol ST (1998) Genetic reassortment among viruses causing hantavirus pulmonary syndrome. Virology 242: 99–106.
- 26. Li D, Schmaljohn AL, Anderson K, Schmaljohn CS (1995) Complete nucleotide sequences of the M and S segments of two hantavirus isolates from California: evidence for reassortment in nature among viruses related to hantavirus pulmonary syndrome. Virology 206: 973–983.
- 27. Chu YK, Milligan B, Owen RD, Goodin DG, Jonsson CB (2006) Phylogenetic and geographical relationships of hantavirus strains in eastern and western Paraguay. Am J Trop Med Hyg 75: 1127–1134.
- 28. Nagai T, Tanishita O, Takahashi Y, Yamanouchi T, Domae K, et al. (1985) Isolation of hemorrhagic fever with renal syndrome virus from leukocytes of rats and virus- replication in cultures of rat and human macrophages. J Gen Virol 66: 1271–1278.
- 29. Easterbrook JD, Klein SL (2008) Seoul virus enhances regulatory and reduces proinflammatory responses in male Norway rats. J Med Virol 80: 1308–1318.
- 30. Raftery MJ, Kraus AA, Ulrich R, Kruger DH, Schonrich G (2002) Hantavirus infection of dendritic cells. J Virol 76: 10724–10733.
- 31. Blakqori G, Delhaye S, Habjan M, Blair CD, Sanchez-Vargas I, et al. (2007) La Crosse bunyavirus nonstructural protein NSs serves to suppress the type I interferon system of mammalian hosts. J Virol 81: 4991–4999.
- 32. Jaaskelainen KM, Kaukinen P, Minskaya ES, Plyusnina A, Vapalahti O, et al. (2007) Tula and Puumala hantavirus NSs ORFs are functional and the products inhibit activation of the interferon-beta promoter. J Med Virol 79: 1527–1536.
- 33. Jaaskelainen KM, Plyusnina A, Lundkvist A, Vaheri A, Plyusnin A (2008) Tula hantavirus isolate with the full-length ORF for nonstructural protein NSs survives for more consequent passages in interferon-competent cells than the isolate having truncated NSs ORF. Virol J 5: 3.
- 34. Geimonen E, LaMonica R, Springer K, Farooqui Y, Gavrilovskaya IN, Mackow ER (2003) Hantavirus pulmonary syndrome-associated hantaviruses contain conserved and functional ITAM signaling elements. J Virol 77: 1638–1643.
- 35. Mou DL, Wang YP, Jiang H, Xiao SY, Yu X, et al. (2007) Identification of a functional ITAM-like sequence within G1 cytoplasmic tail of Hantaan virus. Bing Du Xue Bao 23: 424–428.
- 36. Spiropoulou CF, Albarino CG, Ksiazek TG, Rollin PE (2007) Andes and Prospect Hill hantaviruses differ in early induction of interferon although both can downregulate interferon signaling. J Virol 81: 2769–2776.
- 37. Alff PJ, Gavrilovskaya IN, Gorbunova E, Endriss K, Chong Y, et al. (2006) The pathogenic NY-1 hantavirus G1 cytoplasmic tail inhibits RIG-I- and TBK-1-directed interferon responses. J Virol 80: 9676–9686.
- 38. Sundstrom JB, McMullan LK, Spiropoulou CF, Hooper WC, Ansari AA, et al. (2001) Hantavirus infection induces the expression of RANTES and IP-10 without causing increased permeability in human lung microvascular endothelial cells. J Virol 75: 6070–6085.
- 39. Geimonen E, Neff S, Raymond T, Kocer SS, Gavrilovskaya IN, et al. (2002) Pathogenic and nonpathogenic hantaviruses differentially regulate endothelial cell responses. Proc Natl Acad Sci U S A 99: 13837–13842.
- 40. Khaiboullina SF, Rizvanov AA, Deyde VM, St Jeor SC (2005) Andes virus stimulates interferon-inducible MxA protein expression in endothelial cells. J Med Virol 75: 267–275.
- 41. Kraus AA, Raftery MJ, Giese T, Ulrich R, Zawatzky R, et al. (2004) Differential antiviral response of endothelial cells after infection with pathogenic and nonpathogenic hantaviruses. J Virol 78: 6143–6150.
- 42. Easterbrook JD, Zink MC, Klein SL (2007) Regulatory T cells enhance persistence of the zoonotic pathogen Seoul virus in its reservoir host. Proc Natl Acad Sci U S A 104: 15502–15507.
- 43. Easterbrook JD, Kaplan JB, Vanasco NB, Reeves WK, Purcell RH, et al. (2007) A survey of zoonotic pathogens carried by Norway rats in Baltimore, Maryland, USA. Epidemiol Infect 135: 1192–1199.
- 44. Lehmer EM, Clay CA, Wilson E, St Jeor S, Dearing MD (2007) Differential resource allocation in deer mice exposed to Sin Nombre virus. Physiol Biochem Zool 80: 514–521.
- 45. Ennis FA, Cruz J, Spiropoulou CF, Waite D, Peters CJ, et al. (1997) Hantavirus pulmonary syndrome: CD8+ and CD4+ cytotoxic T lymphocytes to epitopes on Sin Nombre virus nucleocapsid protein isolated during acute illness. Virology 238: 380–390.
- 46. Kilpatrick ED, Terajima M, Koster FT, Catalina MD, Cruz J, et al. (2004) Role of specific CD8+ T cells in the severity of a fulminant zoonotic viral hemorrhagic fever, hantavirus pulmonary syndrome. J Immunol 172: 3297–3304.
- 47. Dohmae K, Okabe M, Nishimune Y (1994) Experimental transmission of hantavirus infection in laboratory rats. J Infect Dis 170: 1589–1592.
- 48. Schountz T, Prescott J, Cogswell AC, Oko L, Mirowsky-Garcia K, et al. (2007) Regulatory T cell-like responses in deer mice persistently infected with Sin Nombre virus. Proc Natl Acad Sci U S A 104: 15496–15501.
- 49. Araki K, Yoshimatsu K, Lee BH, Kariwa H, Takashima I, et al. (2004) A new model of Hantaan virus persistence in mice: the balance between HTNV infection and CD8(+) T-cell responses. Virology 322: 318–327.
- 50. Araki K, Yoshimatsu K, Lee BH, Kariwa H, Takashima I, et al. (2003) Hantavirus-specific CD8(+)-T-cell responses in newborn mice persistently infected with Hantaan virus. J Virol 77: 8408–8417.
- 51. Belkaid Y (2007) Regulatory T cells and infection: a dangerous necessity. Nat Rev Immunol 7: 875–888.
- 52. Chen L, Yang W (1990) Abnormalities of T cell immunoregulation in hemorrhagic fever with renal syndrome. J Infect Dis 161: 1016–1019.
- 53. Zhang XK, Takashima I, Hashimoto N (1988) Role of maternal antibody in protection from hemorrhagic fever with renal syndrome virus infection in rats. Arch Virol 103: 253–265.
- 54. Kallio ER, Poikonen A, Vaheri A, Vapalahti O, Henttonen H, et al. (2006) Maternal antibodies postpone hantavirus infection and enhance individual breeding success. Proc Biol Soc 273: 2771–2776.
- 55. Borucki MK, Boone JD, Rowe JE, Bohlman MC, Kuhn EA, et al. (2000) Role of maternal antibody in natural infection of Peromyscus maniculatus with Sin Nombre virus. J Virol 74: 2426–2429.
- 56. Bernshtein AD, Apekina NS, Mikhailova TV, Myasnikov YA, Khlyap LA, et al. (1999) Dynamics of Puumala hantavirus infection in naturally infected bank voles (Clethrinomys glareolus). Arch Virol 144: 2415–2428.
- 57. Douglass RJ, Calisher CH, Wagoner KD, Mills JN (2007) Sin Nombre virus infection of deer mice in Montana: characteristics of newly infected mice, incidence, and temporal pattern of infection. J Wild Dis 43: 12–22.
- 58. Glass GE, Livingstone W, Mills JN, Hlady WG, Fine JB, et al. (1998) Black Creek Canal virus infection in Sigmodon hispidus in southern Florida. Am J Trop Med Hyg 59: 699–703.
- 59. Klein SL, Zink MC, Glass GE (2004) Seoul virus infection increases aggressive behaviour in male Norway rats. Animal Behaviour 67: 421–429.
- 60. Klein SL, Cernetich A, Hilmer S, Hoffman EP, Scott AL, et al. (2004) Differential expression of immunoregulatory genes in male and female Norway rats following infection with Seoul virus. J Med Virol 74: 180–190.
- 61. Easterbrook JD, Klein SL (2008) Corticosteroids modulate Seoul virus infection, regulatory T cell responses, and MMP-9 expression in male, but not female, Norway rats. J Gen Virol 89: 2723–2730.
- 62. Klingstrom J, Lindgren T, Ahlm C (2008) Sex-dependent differences in plasma cytokine responses to hantavirus infection. Clin Vaccine Immunol 15: 885–887.
- 63. Ahlm C, Linderholm P, Juto B, Stegmyr B, Settergen B (1994) Prevalence of serum IgG antibodies to Puumala virus (hemorrhagic fever with renal syndrome) in Northern Sweden. Epidemiol Infect 113: 129–136.
- 64. Vapalahti O, Mustonen J, Lundkvist A, Henttonen H, Plyusnin A, et al. (2003) Hantavirus infections in Europe. Lancet Infect Dis 3: 653–661.
- 65. Seitsonen E, Hynninen M, Kolho E, Kallio-Kokko H, Pettila V (2006) Corticosteroids combined with continuous veno-venous hemodiafiltration for treatment of hantavirus pulmonary syndrome caused by Puumala virus infection. Eur J Clin Microbiol Infect Dis 25: 261–266.
- 66. Dunst R, Mettang T, Kuhlmann U (1998) Severe thrombocytopenia and response to corticosteroids in a case of nephropathia epidemica. Am J Kidney Dis 31: 116–120.
- 67. Hautala T, Sironen T, Vapalahti O, Paakko E, Sarkioja T, et al. (2002) Hypophyseal hemorrhage and panhypopituitarism during Puumala virus infection: magnetic resonance imaging and detection of viral antigen in the hypophysis. Clin Infect Dis 35: 96–101.
- 68. Steer A (1955) Pathology of hemorrhagic fever: A comparison of the findings-1951 and 1952. Am J Path 31: 201–221.
- 69. Lafrenie RM, Wahl LM, Epstein JS, Hewlett IK, Yamada KM, et al. (1996) HIV-1-Tat modulates the function of monocytes and alters their interactions with microvessel endothelial cells. A mechanism of HIV pathogenesis. J Immunol 156: 1638–1645.
- 70. Maes P, Clement J, Groeneveld PH, Colson P, Huizinga TW, et al. (2006) Tumor necrosis factor-alpha genetic predisposing factors can influence clinical severity in nephropathia epidemica. Viral Immunol 19: 558–564.
- 71. Kanerva M, Vaheri A, Mustonen J, Partanen J (1998) High-producer allele of tumour necrosis factor-alpha is part of the susceptibility MHC haplotype in severe puumala virus-induced nephropathia epidemica. Scan J Infect Dis 30: 532–534.
- 72. Makela S, Mustonen J, Ala-Houhala I, Hurme M, Partanen J, et al. (2002) Human leukocyte antigen-B8-DR3 is a more important risk factor for severe Puumala hantavirus infection than the tumor necrosis factor-alpha(-308) G/A polymorphism. J Infect Dis 186: 843–846.
- 73. Mustonen J, Partanen J, Kanerva M, Pietila K, Vapalahti O, et al. (1996) Genetic susceptibility to severe course of nephropathia epidemica caused by Puumala hantavirus. Kidney Int 49: 217–221.
- 74. Deter J, Bryja J, Chaval Y, Galan M, Henttonen H, et al. (2008) Association between the DQA MHC class II gene and Puumala virus infection in Myodes glareolus, the bank vole. Infect Genet Evol 8: 450–458.
- 75. Mackow ER, Gavrilovskaya IN (2001) Cellular receptors and hantavirus pathogenesis. Curr Top Microbiol Immunol 256: 91–115.
- 76. Medina RA, Mirowsky-Garcia K, Hutt J, Hjelle B (2007) Ribavirin, human convalescent plasma and anti-beta3 integrin antibody inhibit infection by Sin Nombre virus in the deer mouse model. J Gen Virol 88: 493–505.
- 77. Hooper JW, Ferro AM, Wahl-Jensen V (2008) Immune serum produced by DNA vaccination protects hamsters against lethal respiratory challenge with Andes virus. J Virol 82: 1332–1338.
- 78. Hjelle B (2002) Vaccines against hantaviruses. Expert Rev Vaccines 1: 373–384.
- 79. Prescott J, Ye C, Sen G, Hjelle B (2005) Induction of innate immune response genes by Sin Nombre hantavirus does not require viral replication. J Virol 79: 15007–15015.
- 80. Pensiero MN, Sharefkin JB, Dieffenbach CW, Hay J (1992) Hantaan virus infection of human endothelial cells. J Virol 66: 5929–5936.
- 81. Krakauer T, Leduc JW, Morrill JC, Anderson AO, Krakauer H (1994) Serum levels of alpha and gamma interferons in hemorrhagic fever with renal syndrome. Viral Immunol 7: 97–101.
- 82. Takala A, Lahdevirta J, Jansson SE, Vapalahti O, Orpana A, et al. (2000) Systemic inflammation in hemorrhagic fever with renal syndrome correlates with hypotension and thrombocytopenia but not with renal injury. J Infect Dis 181: 1964–1970.
- 83. Markotic A, Hensley L, Daddario K, Spik K, Anderson K, et al. (2007) Pathogenic hantaviruses elicit different immunoreactions in THP-1 cells and primary monocytes and induce differentiation of human monocytes to dendritic-like cells. Coll Antropol 31: 1159–1167.
- 84. Linderholm M, Bjermer L, Juto P, Roos G, Sandstrom T, et al. (1993) Local host response in the lower respiratory tract in nephropathia epidemica. Scand J Infect Dis 25: 639–646.
- 85. Mori M, Rothman AL, Kurane I, Montoya JM, Nolte KB, et al. (1999) High levels of cytokine-producing cells in the lung tissues of patients with fatal hantavirus pulmonary syndrome. J Infect Dis 179: 295–302.
- 86. Krakauer T, Leduc JW, Krakauer H (1995) Serum levels of tumor necrosis factor-alpha, interleukin-1, and interleukin-6 in hemorrhagic fever with renal syndrome. Viral Immunol 8: 75–79.
- 87. Markotic A, Gagro A, Dasic G, Kuzman I, Lukas D, et al. (2002) Immune parameters in hemorrhagic fever with renal syndrome during the incubation and acute disease: Case report. Croatian Med J 43: 587–590.
- 88. Linderholm M, Ahlm C, Settergren B, Waage A, Tarnvik A (1996) Elevated plasma levels of tumor necrosis factor (TNF)-alpha, soluble TNF receptors, interleukin (IL)-6, and IL-10 in patients with hemorrhagic fever with renal syndrome. J Infect Dis 173: 38–43.
- 89. Temonen M, Mustonen J, Helin H, Pasternack A, Vaheri A, et al. (1996) Cytokines, adhesion molecules, and cellular infiltration in nephropathia epidemica kidneys: An immunohistochemical study. Clin Immunol Immunopathol 78: 47–55.
- 90. Khaiboullina SF, Rizvanov AA, Otteson E, Miyazato A, Maciejewski J, et al. (2004) Regulation of cellular gene expression in endothelial cells by Sin Nombre and Prospect Hill viruses. Viral Immunol 17: 234–251.
- 91. Cebalo L, Markotić A (2007) Chemokine production predominates in human monocytes infected with Tula virus. Viral Immunol 20: 206–213.
- 92. Groeneveld PH, Colson P, Kwappenberg KM, Clement J (1995) Increased production of nitric oxide in patients infected with the European variant of hantavirus. Scand J Infect Dis 27: 453–456.
- 93. Markotic A, Dasic G, Gagro A, Sabioncello A, Rabatic S, et al. (1999) Role of peripheral blood mononuclear cell (PBMC) phenotype changes in the pathogenesis of haemorrhagic fever with renal syndrome (HFRS). Clin Exp Immunol 115: 329–334.
- 94. Tuuminen T, Kekalainen E, Makela S, Ala-Houhala I, Ennis FA, et al. (2007) Human CD8+ T cell memory generation in puumala hantavirus infection occurs after the acute phase and is associated with boosting of EBV-specific CD8+ memory T cells. J Immunol 179: 1988–1995.
- 95. Klingstrom J, Hardestam J, Stoltz M, Zuber B, Lundkvist A, et al. (2006) Loss of cell membrane integrity in Puumala hantavirus-infected patients correlates with levels of epithelial cell apoptosis and perforin. J Virol 80: 8279–8282.
- 96. Imaizumi T, Hatakeyama M, Yamashita K, Ishikawa A, Yoshida H, et al. (2005) Double-stranded RNA induces the synthesis of retinoic acid-inducible gene-I in vascular endothelial cells. Endothelium 12: 133–137.
- 97. Jin HK, Yoshimatsu K, Takada A, Ogino M, Asano A, et al. (2001) Mouse Mx2 protein inhibits hantavirus but not influenza virus replication. Arch Virol 146: 41–49.
- 98. Plekhova NG, Somova LM, Slonova RA, Companets GG, Luk'yanova VV, et al. (2005) Metabolic activity of macrophages infected with hantavirus, an agent of hemorrhagic fever with renal syndrome. Biochem 70: 990–997.
- 99. Taruishi M, Yoshimatsu K, Araki K, Okumura M, Nakamura I, et al. (2007) Analysis of the immune response of Hantaan virus nucleocapsid protein-specific CD8(+) T cells in mice. Virology 365: 292–301.
- 100. Asada H, Tamura M, Kondo K, Okuno Y, Takahashi Y, et al. (1986) Role of T lymphocyte subsets in protection and recovery from Hantaan virus infection in mice. J Gen Virol 68: 1961–1969.