Walter Muranyi*,
Udo Bahr*,
Martin Zeier* and
Fokko J. van der Woude
* Klinikum der Universität Heidelberg, Sektion Nephrologie, Heidelberg, Germany; and Medizinische Universitätsklinik (Nephrologie, Endokrinologie, Rheumatologie), Klinikum Mannheim, Mannheim, Germany
Address correspondence to: Dr. Martin Zeier, Klinikum der Universität Heidelberg, Sektion Nephrologie, Im Neuenheimer Feld 162, D-69120 Heidelberg, Germany. Phone: +49-6221-91120; Fax: +49-6221-9221-229; E-mail: martin_zeier{at}med.uni-heidelberg.de
Hantavirus has attracted more and more attention as an emergingpathogenic virus in the past decades. It causes two distincthuman diseases: Hemorrhagic fever with renal syndrome (HFRS)and human pulmonary syndrome (HPS).
Reports on clinical entities possibly caused by hantavirusesin China and England backdate into the first millennium andthe Middle Age, respectively (1,2). However, it lasted until1951 to 1953 during the Korean War before hantaviruses foundglobal attention. More than 3000 United Nations and US soldiersexperienced an acute febrile illness with acute renal failureand shock and a mortality rate of 7% close to a small rivercalled Hantaan (3,4; G. Schreiner, personal communication, Washington,1993). The causative agent, Hantaan virus (HTNV), was identifiedin 1978 by Lee et al. (5). Until now, 21 different hantavirusspecies have been described, and more than 30 genotypes arecharacterized and can be found all over the world (6,7).
Morphology
Hantaviruses comprise one of five genera of the virus familyBunyaviridae (8). They replicate in the cytoplasm of host cellsand are composed of a spherical lipid envelope; four viral proteins;and three single-stranded, negative-sensed RNA segments designatedS (small), M (medium), and L (large) that are coding for thenucleocapsid protein (NP), the surface envelope glycoproteinsG1 and G2, and the RNA-dependent RNA polymerase, respectively(Figure 1). Additional minor open reading frames are presentin the genomes of hantaviruses, but to date, no correspondingproteins were identified. NP, the main structural protein, iscomplexed with the viral RNA genome segments that form helicalnucleocapsids (9).
Figure 1. Schematic of hantavirus morphology. The (-)ssRNA segments S (small), M (medium), and L (large) are encoded for the nucleocapsid protein, the glycoproteins G1 and G2, and the RNA-dependent RNA polymerase, respectively.
Host Range
The main natural reservoir of hantaviruses is murid rodents(order Rodentia; family Muridae; subfamilies Murinae, Arvicolinae,and Sigmodontinae). Virus and host share a long period of co-evolutioncharacterized by the absence of any hantavirus-caused diseasein infected rodents (10,11). Originally, it was thought thatone rodent species is the predominant host for one hantavirusspecies, but recently more and more studies reveal that theremight be multiple rodent hosts for individual virus speciesand multiple viruses in a single host species (12–14).In addition, numerous studies have reported hantavirus infectionsto be present in animal species other than rodents, for example,in cattle, moose, cat, dog, etc. However, the question of whetherthese animals are infected accidentally or represent furthernatural reservoirs has not yet been answered (15). The distributionof single hantavirus species correlates with the geographicextension of their hosts (Table 1), and hantavirus genotypesof the same geographic area are phylogenetically related (15–17).
Table 1. Main natural reservoirs and geographic distribution of pathogenic hantavirusesa
Humans do not belong to the natural host range of hantaviruses,and infection occurs accidentally via virus-containing, aerosolizedrodent excretions such as urine, feces, or saliva. People wholive or work in close contact with infected rodents are at increasedinfection risk, and studies usually show higher percentagesof seropositive individuals in such groups as compared withcontrol subjects (18,19).
Old World and New World Hantaviruses
The genus Hantavirus is roughly composed of two main groups:Old World and New World hantaviruses. HFRS in humans is causedby pathogenic Old World hantaviruses that include Amur virus,Seoul virus, and HTNV, the epidemiologically most importantspecies, with lethality rates up to 15% in Asia, as well asDobrava virus (DOBV), Tula virus (TULV), and Puumala virus (PUUV)in Europe; the last one is the main hantavirus species in Europeand induces Nephropathia epidemica (NE), a milder variant ofHFRS, with mortality rates of 0.1% (10,20). HFRS affects approximately200,000 people each year predominantly in Asia. In 2004, 235cases were reported in Germany according to a recent epidemiologicbulletin of the Robert-Koch Institute.
The first pathogenic New World hantavirus (Sin Nombre virus)was discovered in the early 1990s in the Four Corners regionof the United States (21). From this time on, numerous additionalpathogenic New World hantaviruses were identified and characterized(Table 1). New World hantaviruses are the causative agent ofapproximately 300 cases of HPS each year in North and SouthAmerica, with lethality rates up to 50%.
Human hantavirus infections are assumed to occur accidentally,and men represent a dead end for the hantavirus life cycle.Transfer of virus particles from infected to uninfected peoplenormally does not occur. One exception is the Andes hantavirusstrain Sout in Argentina, of which sporadical person-to-persontransmissions were reported (22,23). This finding reveals aworrying risk potential of hantaviruses for human health.
HFRS and HPS are partly overlapping clinical syndromes. In Europe,the hantavirus serotype Puumala causes NE, a milder variantof HFRS. Viremia occurs after initial infection of alveolarmacrophages and life-threatening acute-phase symptoms are inducedprimarily by infection of vascular endothelial cells of thelung and the kidneys with concomitant loss of barrier functionresulting in severely increased endothelial permeability.
NE
NE is characterized by a sudden onset with high fever, headache,backache, and abdominal pain. Transient thrombocytopenia isa typical finding in the early phase of the disease. The occurrenceof conjunctival hemorrhages, palatine petechiae, and a truncalpetechial rash after 3 or 4 d is possible. Approximately 1%of patients experience severe neurologic manifestations, e.g.,seizures or bladder paralysis. The hemorrhages are accompaniedby oliguria, azotemia, proteinuria, and hematuria. Within 3d, the rash disappears and the patients develop polyuria. Theconvalescent phase extends over several weeks, and sequelaeare rare. Severe courses of NE with acute renal failure andlethal outcome range between 0.1 and 1% (24).
HFRS
The incubation period of HFRS is 7 to 36 d. Only 10 to 15% ofcases have a severe course, with lethality rates between 6 to15%. HFRS is characterized by systemic involvement of capillariesand venules. It induces various hemorrhagic manifestations andcirculation disorders. Renal involvement is characterized byacute renal failure as a result of interstitial hemorrhage andinterstitial infiltrates (24,25).
The clinical course is subdivided into five phases: Febrile,hypotensive, oliguric, diuretic, and convalescent. The onsetof HFRS resembles NE with high fever, backache, abdominal pain,chills, myalgia, malaise, and bradycardia over 3 to 4 d. Photophobia,pharynx enanthema, and a diffuse reddening of the face are alsoobserved. On the third to fifth days, petechia develop initiallyon the palate. At the same time, conjunctival hemorrhages mayappear and a temporary impairment of the visual function isreported. The urinary sediment reveals hematuria and atypicalgross proteinuria (in some cases >3 g/24 h). The hypotensivephase ranges 3 to 6 d after onset of fever. Shock or hypotensionmay occur. Laboratory findings in this phase are leukocytosisand thrombocytopenia. Patients show a wide range of renal conditions,including acute tubulointerstitial nephritis, necrotizing glomerulonephritis,and IgA nephropathy. The oliguric phase starts at approximatelyday 8, and hemorrhagic manifestations become more prominent.The diuretic phase starts at approximately day 11, and the convalescentphase lasts approximately 3 wk to 6 mo.
Sequelae are rare but include chronic renal failure and hypertension.In a series of 46 patients in Tampere (Finland) who had NE 3to 7 y ago, the patients had higher GFR and filtration fraction,more proteinuria, and a higher ambulatory systolic BP comparedwith healthy control subjects (26). Furthermore, we studied42 patients after NE from several areas in Germany and foundhypertension or elevated serum creatinine (1.5 mg/dl) (27).
Extrarenal manifestations include acute impairment of visualfunction, acute myopia, CNS complications with seizures, sometimesmyocarditis, and severe gastrointestinal hemorrhages. In addition,thyroid, liver, and pancreas may be affected. Lung involvementbut to a lesser extent than in HPS is also observed during HFRS(24,28–30).
HPS
The onset of HPS is characterized by flu-like symptoms suchas high fever, myalgia, and headache. The patients develop acutenoncardiac pulmonary edema and hypotension within 2 to 15 d.Bilateral infiltrates develop rapidly, sometimes associatedwith pleural effusions. Neutrophilic leukocytosis, hemoconcentration,thrombocytopenia, and circulating immunoblasts are observed.Severe courses of HPS are associated with increased lactatelevels. The mortality rates of HPS are approximately 50%. Patientswho survive the acute phase of the disease recover normallywithin 5 to 7 d without any sequelae (24). Acute renal failureis secondary as a result of shock and respiratory failure.
The medical knowledge about HFRS and HPS has increased substantiallyin the past years, resulting in the conclusion that both syndromesare partly overlapping. The number of reports on HFRS caseswith lung involvement and HPS cases with renal involvement iscontinuously growing, and it is conceivable that the descriptionsof the clinical courses of both syndromes will further convergein the near future.
NE and HFRS
Immunohistochemistry analysis of hantavirus-infected renal tissuereveals interstitial infiltrates with immune cells and interstitialhemorrhage. The most common histopathologic lesion are acutetubulointerstitial nephritis. Tubular epithelial and luminalalterations are present. Intertubular capillaries are congested,and the interstitium is broadened by edema, indicative of ageneralized capillary damage. Occasionally, glomerular pathology,e.g., hypercellularity and expansion of the mesangium, are observed,and this is probably the underlying cause of gross proteinuria.Tubular, interstitial, and glomerular histologic damage areassociated with the clinical severity of renal failure (Figure 2).It is of note that urinary sediment contains tubular cellswith extremely enlarged nucleoli. These cells resemble uroepithelialtumor cells and spontaneously disappear after the disease hassubsided (29,31). Recent work has shown that these tubular cellscontain hantavirus antigen (32).
Figure 2. (A) Focal mononuclear interstitial infiltration, capillary congestion, and interstitial hemorrhage at the corticomedullary junction. (B) Normal glomeruli. Focal interstitial edema with a mild mononuclear infiltrate and prominent endothelial cells of peritubular capillaries. Magnification, x100 in A, Masson trichrome stain; x160 in B, PAS stain.
HPS
Immunohistochemistry analysis of HPS documents the distributionof viral antigens within the endothelium of capillaries throughoutvarious tissues. Infected endothelial cells lack any morphologicchanges and show no visible cytopathic effects (CPE). Accumulationsof hantaviral antigens are observed in the pulmonary microvasculatureand in dendritic cells within the lymphoid follicles of spleenand lymph nodes. In some autopsy cases, endothelial cells inthe capillaries of the myocardium and the endocardium bear hantavirusantigen, contributing substantially to severe courses of HPS.
Gross pathologic findings show that the lungs of patients withHPS are dense, rubbery, and heavy, usually weighing twice asmuch as the average lung. The pathologic lesions are primarilyvascular with variable degrees of generalized capillary dilationand edema. Frequently, the lungs reveal a mild to moderate interstitialpneumonitis with variable degrees of congestion, edema, andmononuclear cell infiltration (33).
The main factor that determines the course and the severityof HFRS and HPS is the degree of increased permeability of infectedendothelium that shows no histologic signs of damage and novisible CPE. At present, it is poorly understood how pathogenichantaviruses induce the capillary leakage during the acute phaseof the two syndromes and why some hantavirus species are nonpathogenic.
Genetic Predisposition
Patients with certain HLA antigens seem to have a genetic predispositionfor severe courses of HFRS and HPS. Patients who bear HLA-B8,DRB1*0301, C4A*Q0, or DQ2 alleles seem to have a significantlyhigher risk for a severe course of NE (34–36), and theHLA-B35 allele was associated with severe courses of HPS (37).The mechanisms that are involved in these genetic predispositionsare unknown.
Hantavirus Replication Cycle
Hantavirus replication takes place in macrophages and vascularendothelial cells, especially in the lung and the kidney (10,38).For pathogenic hantaviruses, the entry into host cells occursby attachment to V3 integrin on the cellular surface and subsequentendocytosis (39,40). The virion envelope fuses with the endosomemembrane in a pH-dependent way, and nucleocapsids are releasedinto the cytoplasm. Thereafter, the viral RNA-dependent RNApolymerase directs transcription of viral genes and replicationof the viral RNA genome segments. The viral NP and RNA polymerasemRNA are translated at free ribosomes, whereas the glycoproteinmRNA is translated into the endoplasmic reticulum. G1 and G2glycoproteins are transported to the Golgi complex for finalglycosylation. Large intracellular inclusion bodies, probablycomposed of NP, are formed in the cytoplasm. It is assumed thathantavirions are formed at the membranes of the Golgi complex,followed by budding into the Golgi cisternae, migration in secretoryvesicles to the plasma membrane, and release by exocytosis.Several in vitro studies have shown that this hantaviral lifecycle does not induce any visible CPE in endothelial cells.Host cells are not lysed by infection with pathogenic hantaviruses,and no increased permeability is induced in endothelial cellcultures (41,42). Apoptosis and expression of apoptosis-relatedgenes in cells that were infected with pathogenic hantaviruseswas reported for cultured VeroE6 and human embryonic kidneycells; however, in vivo, there is no evidence for programmedcell death in infected endothelial cells (43–46). Thesedata indicate that increased endothelial permeability duringHFRS and HPS might be the result of the infection with pathogenichantaviruses in combination with additional factors that arespecific for the in vivo situation and that are not presentin in vitro cell cultures. In this context, it is assumed thatantiviral processes in the infected cells and immune mechanismsmay play a key role in the development of vascular dysfunction(10,47).
Innate Antiviral Immune Responses
Infection with hantaviruses induces innate immune responsesin host cells, whereas pathogenic hantaviruses seem to be ableto evade these responses to a certain degree. Different typesof interferons are expressed, and IFN-inducible genes are activated.The expression of the IFN-inducible MxA protein is delayed incells that are infected with pathogenic hantaviruses in comparisonwith nonpathogenic hantaviruses. Similarly, levels of antigen-presentingmolecules, e.g., HLA class I, are elevated; however, the upregulationproceeds more slowly after infection with pathogenic than withnonpathogenic hantaviruses (48–51). Other innate antiviralmechanisms that are induced during hantavirus infection includeactivation of the complement system of the classical and alternativeroute, e.g., with elevated titers of soluble terminal complexSC5b-9 and higher C4d/C4 ratios during NE caused by PUUV (52).Natural killer cells, known as effector and regulatory cellsin innate and adaptive immunity in terms of production of cytotoxicmolecules and secretion of cytokines and chemokines (10), areassumed to migrate into hantavirus-infected tissues (53,54).
Humoral Immune Response
The adaptive immune system counters a hantavirus infection viaa humoral and a cellular response. In the course of the humoralimmune response, all types of Ig are expressed during HFRS andHPS. Elevated titers of total serum and virus-specific IgA,the main immunologic component of the mucosa, were detectedduring the acute phase of both syndromes (55,56). Total andvirus-specific IgE titers were found to be increased beforeand during the acute phase of HFRS. It is conceivable that IgEparticipates in hantavirus pathophysiology by activation ofIL-1 and TNF- secretion that could influence permeability ofinfected endothelium; however, it was not possible to find acorrelation between IgE levels and HFRS severity (10,57,58).High titers of virus-specific IgM against viral NP, G1, andG2 are produced during and after the acute phase of HFRS andHPS, whereas the hantavirus NP is regarded as the major viralantigen (10,55,56,59,60). Virus-specific IgG, the most abundantantibody of total Ig against hantaviruses, is also predominantlydirected against viral NP and appears during the acute phaseof HFRS and HPS, whereas further increasing titers can be observedduring the early convalescent phase (10,56,61).
Cellular Immune Response
Cytotoxic CD8+ T cells (CTL) are the predominant lymphocytesin the course of the cellular immune response to a hantavirusinfection and are assumed to play important roles in virus clearingand HFRS/HPS pathogenicity. Increased numbers of CTL were observedat the onset of HFRS and HPS and were also found in the lungsof patients who died from HPS. The severity of disease generallycorrelates with the number of CTL (10,37,51). CTL epitopes wereidentified in all three viral structural proteins, whereas NPagain seems to be the predominant immunogenic protein (62,63).
Secretion of Cytokines and Chemokines
Various types of chemokines and cytokines are secreted in variableamounts to regulate the immune response during a hantavirusinfection. It is assumed that cytokines/chemokines play an importantrole in vascular dysfunction during HFRS and HPS. Many cytokines/chemokines,such as TNF-, are known to increase endothelial permeabilityin the course of natural immune response mechanisms, e.g., duringlymphocyte migration through the vascular walls. Significantlyelevated plasma levels of IFN-, TNF-, IL-2, and IL-6 were detectedat the onset of the acute phase of HFRS and HPS (51,64,65).Increased titers of TNF- seem to correlate with a severe courseof NE (66). Increased expression of cytokines, especially ofTNF- in the peritubular area of the distal nephron, was reportedduring HFRS (67,68), and in the lungs of patients with HPS,increased numbers of IFN-–, IL-1–, IL-1–,IL-2–, IL-4–, IL-6–, and TNF-/–producingcells were observed (37). Hantaviruses are also able to infectdendritic cells, resulting in secretion of proinflammatory cytokines,e.g., IFN- and TNF- that could also contribute to increasedendothelial permeability (65,69). In vitro infection of humanlung microvascular endothelial cells with HTNV or Sin Nombrevirus generated increased amounts of RANTES and 10-kD IFN-inducibleprotein (42). A recent in vitro study by Niikura et al. (70)showed that TNF-–induced increased permeability of endothelialcells is significantly prolonged in HTNV-infected cells in comparisonwith uninfected cells.
Cellular Target Proteins
The cumulative data about hantavirus pathophysiology so farindicate that a hantavirus infection interferes in a thus farunknown way with vascular permeability regulation during inflammation,resulting in endothelial dysfunction. It is conceivable thatthis interference is mediated by interactions between viraland cellular proteins that participate in permeability regulation.Several studies identified associations of hantavirus NP withsmall ubiquitin-like modifier-1, with small ubiquitin-like modifier-1–interactingproteins and with the Fas-mediated apoptosis enhancer Daxx (71–74);however, none of these proteins is involved in permeabilityregulation in endothelial cells.
A much more promising candidate is V3-integrin, the cellularsurface receptor for pathogenic hantaviruses. V3 Integrin participatesin the regulation of cell-to-cell adhesion, platelet aggregation,and maintenance of vascular barrier function. The binding ofhantaviruses to V3 integrin inhibits 3 integrin–directedendothelial cell migration. Furthermore, it was shown that hantavirusesbind to so-called plexin-semaphorin-integrin domains that arepresent on the surface of inactive V3 integrin molecules. Theseinteractions are assumed to inhibit regular V3 integrin functionsand probably interfere with endothelial permeability regulation(39,40,74–76). A further study identified immunoreceptortyrosine-based activation motifs within the G1 cytoplasmic tailof all HPS-causing hantaviruses. These G1 immunoreceptor tyrosine-basedactivation motifs bind key cellular kinases that regulate immuneand endothelial cell functions. The implications of these interactionsare not clear, but an influence on permeability regulation ispossible (77).
Diagnosis of hantaviruses is usually made on the basis of clinicaland serologic findings. Hantavirus should be performed in apatient with fever, lumbago, renal failure, and recent outdooractivities. In the early course of the disease, thrombocytopeniais detectable. An ELISA-based detection of NP-specific IgM antibodiesis usually performed for laboratory diagnosis of an acute hantavirusinfection (78). The highest titers are demonstrable between8 and 25 d after onset of disease. It is important to note forthe differential diagnosis of Puumala and Hantaan virus infectionsthat PUUV NP-specific ELISA cross-reacts with HTNV NP, whereasHTNV NP-specific ELISA shows virtually no cross-reaction withPUUV NP (29). In addition, immunochromatographic assays (79)and reverse transcriptase–PCR have been used increasinglyin recent years, but they have not yet become widely acceptedas standard clinical laboratory tests (80,81).
At present, there are no antiviral drugs that are applicableto cure hantavirus infections. The treatment of patients withHFRS or HPS is restricted to supportive procedures to keep undercontrol the symptoms, which may be life-threatening. Patientsare normally supervised in an emergency department or intensivecare unit for close monitoring and care until the patient’simmune system has cleared the virus and the convalescent phasebegins.
Ribavirin (1--D-ribofuranosyl-1,2,4-triazole-3-carboxamide),a guanosine-analog, was shown to possess anti-hantaviral activity.Controlled trials in the early 1990s reported on decreased virustiters, higher survival rates, and decreased morbidity bothin murine models and in patients with HFRS (82,83). However,newer ribavirin trials in patients with HPS did not confirmthe promising results (10,84,85). Recently, it was shown thatribavirin inhibits the production of hantavirus progeny in vitro.The antiviral activity was due to incorporation of ribavirininto nascent RNA, resulting in high mutation frequencies (9.5/1000nucleotides) and, hence, in the synthesis of transcriptionallydefect viral RNA (86). The study showed that hantavirus RNA-dependentRNA polymerase is susceptible to drugs that lead to error catastrophesduring the viral replication cycle. These insights allow newstrategies for the development of therapeutic procedures thatinclude the incorporation of lethal mutations during hantavirusreplication (87).
Normally, a viral infection induces specific antiviral processesin target cells; among them is the expression of interferonsand IFN-inducible genes. In VeroE6 cells, it was shown thatpretreatment with human IFN-, -, and - leads to an inhibitionof HTNV, PUUV, and TULV replication (88). The IFN-induciblehuman MxA protein, an intracytoplasmic GTPase of the dynaminsuperfamily, shows antiviral activity against a wide range ofRNA viruses, including hantaviruses. Viral replication inducesthe expression of the MxA protein that was shown to interferewith the replication cycle of hantaviruses (46,89,90). However,up to now, no study has indicated how interferons and IFN-inducibleproteins could serve as therapeutic agents during hantavirusinfections.
Recently, Klingstrom et al. (91) passively immunized cynomolgusmacaques with neutralizing mAb and subsequently challenged themwith wild-type PUUV. A delayed onset of viremia and seroconversionwas observed, and one of the immunized monkeys showed neithersymptoms nor elevated levels of IL-6, IL-10, and TNF-. The efficiencyof passive immunization was also confirmed in an earlier studyin a Syrian hamster model for lethal HPS using antibodies againstAndes virus glycoproteins that were induced by DNA vaccines(92). Future clinical trials have to show whether passive immunizationcould represent a therapeutic instrument for the treatment ofacute HFRS or HPS in humans.
In a recent Chinese study, it was reported that intracellularlyapplied single-chain Fv of mAb against HTNV NP was able to bindto the hantavirus nucleocapsid protein in the cytoplasm of infectedcells. The method could represent a new therapeutic approachin the future (93).
Unusual approaches such as the therapeutic assessment of plantcompounds directed against phleboviruses (plant viruses of thefamily Bunyaviridae) (94) or the application of integrated traditionalChinese medicine (95) were pursued to identify a treatment forhantavirus infections; however, up to now, none of these studieshas provided a crucial breakthrough in HFRS or HPS therapy.It is conceivable that the elucidation of the molecular mechanismsof hantavirus pathophysiology or, more precise, the clarificationof cellular processes that participate in endothelial dysfunctionduring HFRS and HPS in the future will provide new targets foreffective therapeutic strategies.
Because infection with some hantavirus species results in highmorbidity and mortality rates and in view of the present situationof missing effective antiviral drugs, it is of particular importanceto try to prevent an infection. On the one hand, it is indicatedto avoid places where murid rodents live in large quantitiesto avert contact with virus-containing rodent excretions. Thisincludes keeping homes and the near surrounding area rodent-free,for example, by eliminating crawl spaces and debris and removingfood sources to make homes and work areas unattractive for rodents.On the other hand, many research efforts have been made in thepast years to develop an effective and safe vaccine againsthantaviruses applying vaccination techniques varying from killedvirus to recombinant DNA technology. In Korea, a formalin-inactivatedHTNV vaccine, Hantavax (Korea Green Cross, Seoul, Korea), thatis produced from rodent brain–derived virus, is commerciallyavailable. Hantavax was shown to induce high titers of IgG-specificantibodies in almost 100% of human volunteers after three vaccinationsaccompanied by the production of neutralizing antibodies inapproximately 80% of test individuals; however, the antibodytiters declined very rapidly within months, and boosters yieldedno satisfactory protection rates (96–99). Further studiesconfirmed that Hantavax elicited only protection rates between30 and 50% for longer time periods (100). In another study,a VeroE6 cell culture–derived, formaldehyde-inactivatedHTNV vaccine showed significantly higher antibody titer andprotection rates in Balb/c mice in comparison with Hantavax;however, protection rates in humans were also very low (101).To this day, there are no hantavirus vaccines that are basedon inactivated viruses and that elicit satisfactory protectionrates in humans (10,98,102).
In addition to inactivated whole-virus particles, single viralcomponents (the viral structural proteins NP, G1, and G2) wereobtained with recombinant DNA technology, expressed in severalcell culture systems and organisms, and tested for their immunogenicityand protective potential. For example, recombinant PUUV NP expressedin yeast induced protective immunity in bank voles (103), andrecombinant NP of DOBV expressed in yeast induced high antibodytiters in Balb/c and C57BL/6 mice (104). PUUV NP was expressedsuccessfully in transgenic tobacco and potato plants by ourgroup but failed to induce an antibody response in mice whenadministered as an oral vaccine (105,106). Recently, recombinantNP of DOBV was tested in combination with various adjuvantsfor immunogenicity and protective efficacy in C57/BL6 mice.The study identified Freund’s adjuvant as the additiveof choice because mice that were vaccinated with this adjuvantin combination with the DOBV NP showed a protection rate fromchallenge of 75%, whereas the usage of other adjuvants suchas Alum, which induces strong Th2-type immune responses, didnot result in protective immunity (107).
Furthermore, known immunogenic epitopes of PUUV, DOBV, and HTNVNP were incorporated into chimeric hepatitis B virus core particlesand elicited high antibody titers and protective immunity inbank voles (108,109). In addition, life recombinant virusesthat express and carry hantavirus structural proteins were constructed.For example, HTNV NP, G1, and G2 expressed with baculovirusand vaccinia virus vectors were shown to induce protection aftera Hantaan virus challenge in hamster and mouse models (110–112).A vaccinia-vectored Hantaan virus vaccine was tested in a PhaseII, double-blinded, placebo-controlled clinical trial among142 volunteers. Neutralizing antibodies to Hantaan virus weredetected in 72% of the test individuals (113).
Finally, plasmid-based DNA vaccines, which express hantavirusstructural proteins, were tested for their immunogenic and protectivepotential. Many groups introduced the coding sequences of thestructural proteins of various pathogenic hantaviruses intousually CMV-based eukaryotic expression vectors and tested theimmunogenic potential of these DNA vaccines in mouse, hamster,and Rhesus macaque models. The DNA vaccines always induced highantibody titers often of the neutralizing type (10,92,114–117).Despite the extensive work of many research groups on the fieldof hantavirus vaccine development and the presence of promisingdata in animal models, there is still no worldwide approvedand commercially available vaccine against hantaviruses, andit seems unlikely that this situation will change in the nearfuture.
Since the discovery of HTNV as the causative agent of HFRS,much knowledge about the various hantaviruses and their manifestationsin animals and humans has been gathered. NE, HFRS, and HPS arehuman diseases, caused by hantaviruses, which may be encounteredby clinical nephrologists. The diagnosis rests on serologicevidence. Supportive therapy is dependent on the Hantavirusstrain and clinical symptoms, especially important in HFRS andHPS, for which correction of bleeding, maintenance of BP, andtreatment of renal or respiratory insufficiency may be indicated.It is hoped that a better understanding of viral biology andpathophysiology will lead to more effective and specific therapeuticmodalities in the future.
Acknowledgments
We are deeply grateful to Prof. Konrad Andrassy and Prof. GholamrezaDarai for generous support and helpful critical comments.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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V
3 integrin on the cellular surface and subsequent endocytosis (39,40). The virion envelope fuses with the endosome membrane in a pH-dependent way, and nucleocapsids are released into the cytoplasm. Thereafter, the viral RNA-dependent RNA polymerase directs transcription of viral genes and replication of the viral RNA genome segments. The viral NP and RNA polymerase mRNA are translated at free ribosomes, whereas the glycoprotein mRNA is translated into the endoplasmic reticulum. G1 and G2 glycoproteins are transported to the Golgi complex for final glycosylation. Large intracellular inclusion bodies, probably composed of NP, are formed in the cytoplasm. It is assumed that hantavirions are formed at the membranes of the Golgi complex, followed by budding into the Golgi cisternae, migration in secretory vesicles to the plasma membrane, and release by exocytosis. Several in vitro studies have shown that this hantaviral life cycle does not induce any visible CPE in endothelial cells. Host cells are not lysed by infection with pathogenic hantaviruses, and no increased permeability is induced in endothelial cell cultures (41,42). Apoptosis and expression of apoptosis-related genes in cells that were infected with pathogenic hantaviruses was reported for cultured VeroE6 and human embryonic kidney cells; however, in vivo, there is no evidence for programmed cell death in infected endothelial cells (43–46). These data indicate that increased endothelial permeability during HFRS and HPS might be the result of the infection with pathogenic hantaviruses in combination with additional factors that are specific for the in vivo situation and that are not present in in vitro cell cultures. In this context, it is assumed that antiviral processes in the infected cells and immune mechanisms may play a key role in the development of vascular dysfunction (10,47). 
, TNF-
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