Expression Profile of Human Renal Mesangial Cells Is Altered by Infection with Pathogenic Puumala Orthohantavirus.

Acute kidney injury (AKI) with proteinuria is a hallmark of infections with Eurasian orthohantaviruses. Different kidney cells are identified as target cells of hantaviruses. Mesangial cells may play a central role in the pathogenesis of AKI by regulation of inflammatory mediators and signaling cascades. Therefore, we examined the characteristics of hantavirus infection on human renal mesangial cells (HRMCs). Receptor expression and infection with pathogenic Puumala virus (PUUV) and low-pathogenic Tula virus (TULV) were explored. To analyze changes in protein expression in infected mesangial cells, we performed a proteome profiler assay analyzing 38 markers of kidney damage. We compared the proteome profile of in vitro-infected HRMCs with the profile detected in urine samples of 11 patients with acute hantavirus infection. We observed effective productive infection of HRMCs with pathogenic PUUV, but only poor abortive infection for low-pathogenic TULV. PUUV infection resulted in the deregulation of proteases, adhesion proteins, and cytokines associated with renal damage. The urinary proteome profile of hantavirus patients demonstrated also massive changes, which in part correspond to the alterations observed in the in vitro infection of HRMCs. The direct infection of mesangial cells may induce a local environment of signal mediators that contributes to AKI in hantavirus infection.

Dissection of the NKG2C NK cell response against Puumala Orthohantavirus.

BACKGROUND: Infections with the Puumala orthohantavirus (PUUV) in humans may cause hemorrhagic fever with renal syndrome (HFRS), known as nephropathia epidemica (NE), which is associated with acute renal failure in severe cases. In response to PUUV-infections, a subset of potent antiviral NKG2C+ NK cells expand, whose role in virus defence and pathogenesis of NE is unclear. NKG2C+ NK cell proliferation is mediated by binding of NKG2C/CD94 to HLA-E on infected cells. The proliferation and activation of NKG2C+ NK cells via the NKG2C/HLA-E axis is affected by different NKG2C (NKG2Cwt/del) and HLA-E (HLA-E*0101/0103) alleles, which naturally occur in the human host. Homozygous (NKG2Cdel/del) and heterozygous (NKG2Cwt/del) deletions of the NKG2C receptor results in an impaired NKG2C/CD94 mediated proliferation and activation of NKG2C+ cells. We therefore analyzed the PUUV-mediated NKG2C+ NK cell responses and the impact of different NKG2C and HLA-E alleles in NE patients. METHODOLOGY/PRINCIPAL FINDINGS: NKG2C+ NK cell expansion and effector functions in PUUV-infected cells were investigated using flow cytometry and it was shown that PUUV-infected endothelial cells led to a NKG2C/CD94 mediated NKG2C+ NK cell activation and expansion, dependent on the HLA-G-mediated upregulation of HLA-E. Furthermore, the NKG2Cdel and HLA-E*0101/0103 alleles were determined in 130 NE patients and 130 matched controls, and it was shown that in NE patients the NKG2Cwt/del allele was significantly overrepresented, compared to the NKG2Cwt/wt variant (p = 0.01). In addition, in vitro analysis revealed that NKG2Cwt/del NK cells exhibited on overall a lower proliferation (p = 0.002) and lower IFNγ expression (p = 0.004) than NKG2Cwt/wt NK cells. CONCLUSIONS/SIGNIFICANCE: Our results corroborate the substantial impact of the NKG2C/HLA-E axis on PUUV-specific NK cell responses. A weak NKG2C+ NK cell response, as reflected by NKG2Cwt/del variant, may be associated with a higher risk for a severe hantavirus infections.

Cells of the human respiratory tract support the replication of pathogenic Old World orthohantavirus Puumala.

BACKGROUND: Transmission of all known pathogenic orthohantaviruses (family Hantaviridae) usually occurs via inhalation of aerosols contaminated with viral particles derived from infected rodents and organ manifestation of infections is characterized by lung and kidney involvement. Orthohantaviruses found in Eurasia cause hemorrhagic fever with renal syndrome (HFRS) and New World orthohantaviruses cause hantavirus cardiopulmonary syndrome (HCPS). However, cases of infection with Old World orthohantaviruses with severe pulmonary manifestations have also been observed. Therefore, human airway cells may represent initial targets for orthohantavirus infection and may also play a role in the pathogenesis of infections with Eurasian orthohantaviruses. METHODS: We analyzed the permissiveness of primary endothelial cells of the human pulmonary microvasculature and of primary human epithelial cells derived from bronchi, bronchioles and alveoli for Old World orthohantavirus Puumala virus (PUUV) in vitro. In addition, we examined the expression of orthohantaviral receptors in these cell types. To minimize donor-specific effects, cells from two different donors were tested for each cell type. RESULTS: Productive infection with PUUV was observed for endothelial cells of the microvasculature and for the three tested epithelial cell types derived from different sites of the respiratory tract. Interestingly, infection and particle release were also detected in bronchial and bronchiolar epithelial cells although expression of the orthohantaviral receptor integrin ß3 was not detectable in these cell types. In addition, replication kinetics and viral release demonstrate enormous donor-specific variations. CONCLUSIONS: The human respiratory epithelium is among the first targets of orthohantaviral infection and may contribute to virus replication, dissemination and pathogenesis of HFRS-causing orthohantaviruses. Differences in initial pulmonary infection due to donor-specific factors may play a role in the observed broad variance of severity and symptoms of orthohantavirus disease in patients. The absence of detectable levels of integrin αVß3 surface expression on bronchial and small airway epithelial cells indicates an alternate mode of orthohantaviral entry in these cells that is independent from integrin ß3.

Inhibition of interferon I induction by non-structural protein NSs of Puumala virus and other vole-associated orthohantaviruses: phenotypic plasticity of the protein and potential functional domains.

The orthohantavirus Puumala virus (PUUV), which is transmitted by bank voles (Clethrionomys glareolus), and other vole-borne hantaviruses contain in their small (S) genome segment two overlapping open reading frames, coding for the nucleocapsid protein and the non-structural protein NSs, a putative type I interferon (IFN-I) antagonist. To investigate the role of NSs of PUUV and other orthohantaviruses, the expression pattern of recombinant NSs constructs and their ability to inhibit human IFN-I promoter activity were investigated. The NSs proteins of PUUV and related cricetid-borne orthohantaviruses showed strong inhibition of IFN-I promoter induction. We identified protein products originating from three and two methionine initiation codons in the NSs ORF of PUUV during transfection and infection, respectively. The three putative start codons are conserved in all PUUV strains analysed. Translation initiation at these start codons influenced the inhibitory activity of the NSs products, with the wild-type (wt) construct expressing two proteins starting at the first and second methionine and showing strong inhibition activity. Analysis of in vitro-generated variants and naturally occurring PUUV NSs proteins indicated that amino acid variation in the NSs protein is well tolerated, suggesting its phenotypic plasticity. The N-terminal 20-amino-acid region of the NSs protein was found to be associated with strong inhibition and to be highly vulnerable to amino acid exchanges and tag fusions. Infection studies using human, bank vole, and Vero E6 cells did not show obvious differences in the replication capacity of PUUV Sotkamo wt and a strain with a truncated NSs protein (NSs21Stop), showing that the lack of a full-length NSs might be compensated by its N-terminal peptide, as seen in transfection experiments. These results contribute to our understanding of virus-host interactions and highlight the importance of future innate immunity studies in reservoir hosts.

Puumala and Andes Orthohantaviruses Cause Transient Protein Kinase R-Dependent Formation of Stress Granules.

Virus infection frequently triggers host cell stress signaling resulting in translational arrest; as a consequence, many viruses employ means to modulate the host stress response. Hantaviruses are negative-sense, single-stranded RNA viruses known to inhibit host innate immune responses and apoptosis, but their impact on host cell stress signaling remains largely unknown. In this study, we investigated activation of host cell stress responses during hantavirus infection. We show that hantavirus infection causes transient formation of stress granules (SGs) but does so in only a limited proportion of infected cells. Our data indicate some cell type-specific and hantavirus species-specific variability in SG prevalence and show SG formation to be dependent on the activation of protein kinase R (PKR). Hantavirus infection inhibited PKR-dependent SG formation, which could account for the transient nature and low prevalence of SG formation observed during hantavirus infection. In addition, we report only limited colocalization of hantaviral proteins or RNA with SGs and show evidence indicating hantavirus-mediated inhibition of PKR-like endoplasmic reticulum (ER) kinase (PERK).IMPORTANCE Our work presents the first report on stress granule formation during hantavirus infection. We show that hantavirus infection actively inhibits stress granule formation, thereby escaping the detrimental effects on global translation imposed by host stress signaling. Our results highlight a previously uncharacterized aspect of hantavirus-host interactions with possible implications for how hantaviruses are able to cause persistent infection in natural hosts and for pathogenesis.

Puumala and Tula Virus Differ in Replication Kinetics and Innate Immune Stimulation in Human Endothelial Cells and Macrophages.

Old world hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) upon zoonotic transmission to humans. In Europe, the Puumala virus (PUUV) is the main causative agent of HFRS. Tula virus (TULV) is also widely distributed in Europe, but there is little knowledge about the pathogenicity of TULV for humans, as reported cases are rare. We studied the replication of TULV in different cell types in comparison to the pathogenic PUUV and analyzed differences in stimulation of innate immunity. While both viruses replicated to a similar extent in interferon (IFN)-deficient Vero E6 cells, TULV replication in human lung epithelial (A549) cells was slower and less efficient when compared to PUUV. In contrast to PUUV, no replication of TULV could be detected in human microvascular endothelial cells and in macrophages. While a strong innate immune response towards PUUV infection was evident at 48 h post infection, TULV infection triggered only a weak IFN response late after infection of A549 cells. Using appropriate in vitro cell culture models for the orthohantavirus infection, we could demonstrate major differences in host cell tropism, replication kinetics, and innate immune induction between pathogenic PUUV and the presumably non- or low-pathogenic TULV that are not observed in Vero E6 cells and may contribute to differences in virulence.

Spatial dynamics of a zoonotic orthohantavirus disease through heterogenous data on rodents, rodent infections, and human disease.

Zoonotic diseases are challenging to study from the ecological point of view as, broadly speaking, datasets tend to be either detailed on a small spatial extent, or coarse on a large spatial extent. Also, there are many ways to assess zoonotic disease transmission systems, from pathogens to hosts to humans. We explore the complementarity of datasets considering the pathogen in its host, the host and human cases in the context of Puumala orthohantavirus infection in Germany. We selected relevant environmental predictors using a conceptual framework based on resource-based habitats. This framework assesses the functions, and associated environmental resources of the pathogen and associated host. A resource-based habitat framework supports variable selection and result interpretation. Multiplying 'keyholes' to view a zoonotic disease transmission system is valuable, but requires a strong conceptual framework to select and interpret environmental explanatory variables. This study highlights the usefulness of a structured, ecology-based approach to study drivers of zoonotic diseases at the level of virus, host, and human - not only for PUUV but also for other zoonotic pathogens. Our results show that human disease cases are best explained by a combination of variables related to zoonotic pathogen circulation and human exposure.

Self-association and subcellular localization of Puumala hantavirus envelope proteins.

Hantavirus assembly and budding are governed by the surface glycoproteins Gn and Gc. In this study, we investigated the glycoproteins of Puumala, the most abundant Hantavirus species in Europe, using fluorescently labeled wild-type constructs and cytoplasmic tail (CT) mutants. We analyzed their intracellular distribution, co-localization and oligomerization, applying comprehensive live, single-cell fluorescence techniques, including confocal microscopy, imaging flow cytometry, anisotropy imaging and Number&Brightness analysis. We demonstrate that Gc is significantly enriched in the Golgi apparatus in absence of other viral components, while Gn is mainly restricted to the endoplasmic reticulum (ER). Importantly, upon co-expression both glycoproteins were found in the Golgi apparatus. Furthermore, we show that an intact CT of Gc is necessary for efficient Golgi localization, while the CT of Gn influences protein stability. Finally, we found that Gn assembles into higher-order homo-oligomers, mainly dimers and tetramers, in the ER while Gc was present as mixture of monomers and dimers within the Golgi apparatus. Our findings suggest that PUUV Gc is the driving factor of the targeting of Gc and Gn to the Golgi region, while Gn possesses a significantly stronger self-association potential.

Motility of human renal cells is disturbed by infection with pathogenic hantaviruses.

BACKGROUND: Hemorrhagic fever with renal syndrome (HFRS) caused by pathogenic hantaviruses in Europe and Asia is often characterized by acute kidney injury (AKI) with massive proteinuria. Renal filtration depends on the integrity of epithelial and endothelial monolayers in the tubular and glomerular apparatus. Tubular and glomerular cells represent target cells of hantavirus infection. However, the detailed mechanisms of renal impairment induced by hantaviruses are not well understood. METHODS: We analyzed the cellular consequences of hantavirus infection by measuring adhesion and migration capacity of human renal cells infected with Puumala (PUUV) or Hantaan (HTNV) virus. The impact of hantaviral nucleocapsid proteins (N proteins) on motility was examined by transfection of podocytes. RESULTS: Infection of kidney cells with hantavirus species PUUV and HTNV causes a significant reduction of migration capacity. The impaired motility depends on viral replication and transfection of podocytes with N protein of PUUV or HTNV reveals that the expression of N protein alone is sufficient to deteriorate podocyte function. The cellular effects are more pronounced for the more pathogenic HTNV than for PUUV that causes a milder form of HFRS. CONCLUSIONS: The direct impairment of migration capacity of renal cells by hantaviral N proteins may contribute substantially to proteinuria observed in the clinical picture of hantavirus infection.

High plasma resistin associates with severe acute kidney injury in Puumala hantavirus infection.

BACKGROUND: Puumala hantavirus (PUUV) infected patients typically suffer from acute kidney injury (AKI). Adipokines have inflammation modulating functions in acute diseases including AKI. We examined plasma levels of three adipokines (resistin, leptin, and adiponectin) in acute PUUV infection and their associations with disease severity. METHODS: This study included 79 patients hospitalized due to acute PUUV infection. Plasma resistin, leptin, adiponectin, as well as IL-6 and CRP, were measured at the acute phase, recovery phase and one year after hospitalization. RESULTS: Plasma resistin levels were significantly higher in the acute phase compared to the recovery phase and one year after (median resistin 28 pg/mL (11-107) vs. 17 pg/mL (7-36) vs. 14 pg/mL (7-31), p<0.001). Maximum resistin concentration correlated with maximum plasma creatinine levels (r = 0.63; p<0.001). The higher the amount of albuminuria in the urine dipstick test (0-1+, 2+ or 3+) at admission, the higher the median of maximum resistin (24.7 pg/mL, 25.4 pg/mL and 39.6 pg/mL, respectively, p = 0.002). High resistin was also an independent risk factor for severe AKI (creatinine ≥353.6µmol/L) (OR 1.08, 95% CI 1.02-1.14). Neither plasma leptin nor adiponectin level had any correlation with creatinine concentration or the amount of albuminuria. CONCLUSIONS: Plasma resistin independently associates with the severity of AKI in acute PUUV infection. The association of resistin with the amount of albuminuria suggests that the level of plasma resistin is not only influenced by renal clearance but could have some role in the pathogenesis of AKI during PUUV infection.

Population-based seroprevalence of Puumala hantavirus in Finland: smoking as a risk factor.

Puumala hantavirus (PUUV) causes hemorrhagic fever with renal syndrome in humans, that is an endemic disease in Finland. We estimated the seroprevalence of PUUV in Finland and explored risk factors and disease associations by using unique survey data with health register linkage. A total of 2000 sera from a nationwide health survey from 2011, representative of the adult population, were screened for PUUV IgG by immunofluorescence assay. We performed statistical analysis adjusting for stratified cluster design and taking into account sampling weights. In total, 254 sera among 2000 tested were PUUV-IgG-positive resulting in a weighted seroprevalence of 12.5%, (95% CI 10.9-14.4), mirroring known age and regional variation in reported incidence. No associations between PUUV-seropositivity and chronic diseases including cardiovascular (including hypertension), pulmonary, kidney disease and cancer were observed. Smoking was significantly associated with seropositivity (adjusted OR 1.54; 95% CI 1.16-2.04). In addition, significant dose-response relations were found for the number of cigarettes smoked daily (OR 1.14; 95% CI 1.12-1.28). The results are important for disease burden assessment and guide intervention strategies, highlighting also the role of smoking prevention.

Puumala hantavirus infections in bank vole populations: host and virus dynamics in Central Europe.

BACKGROUND: In Europe, bank voles (Myodes glareolus) are widely distributed and can transmit Puumala virus (PUUV) to humans, which causes a mild to moderate form of haemorrhagic fever with renal syndrome, called nephropathia epidemica. Uncovering the link between host and virus dynamics can help to prevent human PUUV infections in the future. Bank voles were live trapped three times a year in 2010-2013 in three woodland plots in each of four regions in Germany. Bank vole population density was estimated and blood samples collected to detect PUUV specific antibodies. RESULTS: We demonstrated that fluctuation of PUUV seroprevalence is dependent not only on multi-annual but also on seasonal dynamics of rodent host abundance. Moreover, PUUV infection might affect host fitness, because seropositive individuals survived better from spring to summer than uninfected bank voles. Individual space use was independent of PUUV infections. CONCLUSIONS: Our study provides robust estimations of relevant patterns and processes of the dynamics of PUUV and its rodent host in Central Europe, which are highly important for the future development of predictive models for human hantavirus infection risk.

Expansion of spatial and host range of Puumala virus in Sweden: an increasing threat for humans?

Hantaviruses are globally distributed and cause severe human disease. Puumala hantavirus (PUUV) is the most common species in Northern Europe, and the only hantavirus confirmed to circulate in Sweden, restricted to the northern regions of the country. In this study, we aimed to further add to the natural ecology of PUUV in Sweden by investigating prevalence, and spatial and host species infection patterns. Specifically, we wanted to ascertain whether PUUV was present in the natural reservoir, the bank vole (Myodes glareolus) further south than Dalälven river, in south-central Sweden, and whether PUUV can be detected in other rodent species in addition to the natural reservoir. In total, 559 animals were collected at Grimsö (59°43'N; 15°28'E), Sala (59°55'N; 16°36'E) and Bogesund (59°24'N; 18°14'E) in south-central Sweden between May 2013 and November 2014. PUUV ELISA-reactive antibodies were found both in 2013 (22/295) and in 2014 (18/264), and nine samples were confirmed as PUUV-specific by focus reduction neutralization test. Most of the PUUV-specific samples were from the natural host, the bank vole, but also from other rodent hosts, indicating viral spill-over. Finally, we showed that PUUV is present in more highly populated central Sweden.

Spatial and Temporal Epidemiology of Nephropathia Epidemica Incidence and Hantavirus Seroprevalence in Rodent Hosts: Identification of the Main Environmental Factors in Europe.

In Europe, the increasing number of nephropathia epidemica (NE) infections in humans, caused by Puumala virus carried by bank voles (Myodes glareolus), has triggered studies of environmental factors driving these infections. NE infections have been shown to occur in specific geographical areas characterized by environmental factors that influence the distribution and dynamics of host populations and virus persistence in the soil. Here, we review the influence of environmental conditions (including climate factors, food availability and habitat conditions) with respect to incidence in humans and seroprevalence in rodents, considering both direct and indirect transmission pathways. For each type of environmental factor, results and discrepancies between studies are presented and examined in the light of biological hypotheses. Overall, food availability and temperature appear to be the main drivers of host seroprevalence and NE incidence, but data quality and statistical approaches varied greatly among studies. We highlight the issues that now need to be addressed and suggest improvements for study design in regard to the current knowledge on hantavirus epidemiology.

Host-Associated Absence of Human Puumala Virus Infections in Northern and Eastern Germany.

Human hantavirus disease cases, caused by Puumala virus (PUUV), are mainly recorded in western and southern areas of Germany. This bank vole reservoir survey confirmed PUUV presence in these regions but its absence in northern and eastern regions. PUUV occurrence is associated with the presence of the Western bank vole phylogroup.

Regional differences in long-term cycles and seasonality of Puumala virus infections, Finland, 1995-2014.

Puumala hantavirus (PUUV) causes haemorrhagic fever with renal syndrome in humans, an endemic disease in Finland. We aimed to study recent trends in PUUV infections in Finland, to evaluate whether there are regional differences in seasonality and long-term cycles and whether the patterns have changed over time. We analysed serologically confirmed acute PUUV infections reported to the National Infectious Disease Register from 1 April 1995 to 31 March 2014. A total of 30 942 cases of PUUV infections were identified during the study period. The average annual incidence was 31 cases/100 000 person-years with the highest in Eastern Finland and the lowest in Southwestern Finland. Throughout Finland there was not an increasing trend in incidence but changes in incidence, seasonality and long-term cycles differed regionally. Long-term cycles supported by high Bayesian posterior probabilities (73-100%) differed between the south and the north, shifting from 3 to 4 years, respectively. Temporal changes in seasonality were most prominent in Southwestern Finland. The pattern of human PUUV infection epidemiology probably primarily reflects the spatio-temporal interaction between bank-vole population dynamics and climate.

Long-term hormonal follow-up after human Puumala hantavirus infection.

OBJECTIVE: Nephropathia epidemica (NE) is a haemorrhagic fever with renal syndrome (HFRS) caused by Puumala hantavirus (PUUV). Pituitary haemorrhage and hypopituitarism may complicate recovery from acute NE. DESIGN: Forty-seven of our recent cohort of 58 NE patients volunteered to be re-examined in order to estimate the burden of hormonal deficiency 4 to 8 years after the acute illness. Two patients had suffered from pituitary haemorrhage, but many others exhibited pituitary oedema during their acute infection. In this study, we searched for symptoms of hormonal deficiency, performed hormonal laboratory screening, and most patients underwent pituitary MRI examination. RESULTS: The pituitary size had diminished in all patients in whom MRI was performed (P < 0·001). One patient with acute phase haemorrhage had made a complete recovery while the other continued to require hormonal substitution. In addition, hormonal laboratory abnormalities were observed in nine other patients; these being attributable to several reasons, for example independent peripheral hormonal diseases, side effects of medication or other secondary causes such as obesity. None of them had signs of late-onset pituitary insufficiency caused by their previous NE. Health-related quality of life (mean and median 15D score) of patients was comparable to that of age-standardized general population. CONCLUSIONS: None of our patients had developed obvious late-onset hypopituitarism despite of the fact that pituitary gland can be affected during acute NE. We recommend requesting a history of hantavirus infection whenever the possibility of pituitary dysfunction is suspected at least in patients originating from regions with high NE infection rate.

Age-related effects of chronic hantavirus infection on female host fecundity.

1. Pathogens often cause detrimental effects to their hosts and, consequently, may influence host population dynamics that may, in turn, feed back to pathogen transmission dynamics. Understanding fitness effects of pathogens upon animal host populations can help to predict the risks that zoonotic pathogens pose to humans. 2. Here we determine whether chronic infection by Puumala hantavirus (PUUV) affects important fitness-related traits, namely the probability of breeding, reproductive effort and mother and offspring condition, in the bank vole (Myodes glareolus). Using 9 years empirical data in a PUUV endemic area in Central Finland, we found differences between reproductive characteristics of PUUV-infected and uninfected female bank voles. 3. Young infected females had a significantly higher, and old individuals lower, likelihood of reproducing than uninfected animals during the middle of the breeding season. The implication is that PUUV infection may have long-term deleterious effects that are observed at old age, while in young individuals, the infection may enhance breeding probability by directing resources towards current breeding. 4. Moreover, PUUV infection was related with the mother's body condition. Infected mothers were in poorer condition than uninfected mothers in the early breeding season, but were in better condition than uninfected mothers during the middle of the breeding season. Offspring body condition was positively associated with mother's body condition, which, in turn, was related to the PUUV infection status of the mother. 5. Our findings indicate that chronic infection may affect the reproduction of female hosts, but the effect is dependent on the host age. The effect of chronic hantavirus infection was small and density-independent and hence unlikely to contribute to the cyclic population dynamics of the host. However, the effects on a female's reproductive output might affect the abundance of young susceptible individuals in the population and hence influence the transmission and persistence of the pathogen. Although experimental and long-term capture-mark-recapture studies are required to further clarify the fitness effects of hantavirus infection and their consequences for pathogen dynamics, this study shows that the infection may have complex effects that are dependent on the age of the individual and the time of the breeding season.

Pathophysiology of a severe case of Puumala hantavirus infection successfully treated with bradykinin receptor antagonist icatibant.

We recently described a patient with very severe Puumala hantavirus infection manifested by capillary leakage syndrome and shock. He was successfully treated with the bradykinin receptor antagonist, icatibant (Antonen et al., 2013). Here we report analysis of the pathophysiology which indicated pronounced complement activation, prolonged leukocytosis, extensive fibrinolysis, circulating histones, and defects in liver function. The patient had an uncommon HLA-phenotype, which may have contributed to the severe course of the disease.