Enhanced influenza A H1N1 T cell epitope recognition and cross-reactivity to protein-O-mannosyltransferase 1 in Pandemrix-associated narcolepsy type 1.

Narcolepsy type 1 (NT1) is a chronic neurological disorder having a strong association with HLA-DQB1*0602, thereby suggesting an immunological origin. Increased risk of NT1 has been reported among children or adolescents vaccinated with AS03 adjuvant-supplemented pandemic H1N1 influenza A vaccine, Pandemrix. Here we show that pediatric Pandemrix-associated NT1 patients have enhanced T-cell immunity against the viral epitopes, neuraminidase 175-189 (NA175-189) and nucleoprotein 214-228 (NP214-228), but also respond to a NA175-189-mimic, brain self-epitope, protein-O-mannosyltransferase 1 (POMT1675-689). A pathogenic role of influenza virus-specific T-cells and T-cell cross-reactivity in NT1 are supported by the up-regulation of IFN-γ, perforin 1 and granzyme B, and by the converging selection of T-cell receptor TRAV10/TRAJ17 and TRAV10/TRAJ24 clonotypes, in response to stimulation either with peptide NA175-189 or POMT1675-689. Moreover, anti-POMT1 serum autoantibodies are increased in Pandemrix-vaccinated children or adolescents. These results thus identify POMT1 as a potential autoantigen recognized by T- and B-cells in NT1.

Serological survey in the Finnish human population implies human-to-human transmission of Ljungan virus or antigenically related viruses.

Ljungan virus (LV) is a picornavirus related to human parechoviruses (HPeV). The virus has been found in bank voles (Myodes glareolus) and several other rodent species, and suggested to have zoonotic potential. Thus far, seroepidemiological data on LV infections in humans are scarce. In this study, we aimed to characterize the demographic and geographical distribution of LV-reactive antibodies in Finland, and to investigate its occurrence in patients suspected of having a rodent-borne disease, nephropathia epidemica (NE) caused by Puumala hantavirus (PUUV). Using an immunofluorescence assay (LV strain 145SLG), we screened human sera (n = 1378) and found LV-reactive antibodies in 36% of samples. The probability of possessing LV-reactive antibodies peaked at age of 14 years, suggesting that most infections occur in childhood. The prevalence of LV-reactive antibodies was significantly higher in the urbanized area surrounding Helsinki than in more rural Central Finland. These findings are uncharacteristic of a rodent-borne pathogen, and therefore we consider human-to-human transmission of one or several Ljungan-like viruses as a likely cause for most of the observed antibody responses.

Urine soluble urokinase-type plasminogen activator receptor levels correlate with proteinuria in Puumala hantavirus infection.

OBJECTIVES: Urokinase-type plasminogen activator receptor (uPAR) is upregulated during inflammation and known to bind to ß3 -integrins, receptors used by pathogenic hantaviruses to enter endothelial cells. It has been proposed that soluble uPAR (suPAR) is a circulating factor that causes focal segmental glomerulosclerosis and proteinuria by activating ß3 -integrin in kidney podocytes. Proteinuria is also a characteristic feature of hantavirus infections. The aim of this study was to evaluate the relation between urine suPAR levels and disease severity in acute Puumala hantavirus (PUUV) infection. DESIGN: A single-centre, prospective cohort study. SUBJECTS AND METHODS: Urinary suPAR levels were measured twice during the acute phase and once during convalescence in 36 patients with serologically confirmed PUUV infection. Fractional excretion of suPAR (FE suPAR) and of albumin (FE alb) was calculated. RESULTS: The FE suPAR was significantly elevated during the acute phase of PUUV infection compared to the convalescent phase (median 3.2%, range 0.8-52.0%, vs. median 1.9%, range 1.0-5.8%, P = 0.005). Maximum FE suPAR was correlated markedly with maximum FE alb (r = 0.812, P < 0.001) and with several other variables that reflect disease severity. There was a positive correlation with the length of hospitalization (r = 0.455, P = 0.009) and maximum plasma creatinine level (r = 0.780, P < 0.001) and an inverse correlation with minimum urinary output (r = -0.411, P = 0.030). There was no correlation between FE suPAR and plasma suPAR (r = 0.180, P = 0.324). CONCLUSION: Urinary suPAR is markedly increased during acute PUUV infection and is correlated with proteinuria. High urine suPAR level may reflect local production of suPAR in the kidney during the acute infection.

Altered expression of ezrin, E-Cadherin and ß-Catenin in cervical neoplasia.

High-grade cervical squamous intraepithelial lesions (CIN) as well as squamous cell carcinoma and adenocarcinoma of the cervix are associated with persistent high-risk human papillomavirus (HPV) infection. A number of cellular events play a role in HPV pathogenesis and in the development of cervical lesions, including alterations in cell adhesion and motility. The crucial plasma membrane - cytoskeleton linker protein ezrin of the Ezrin-Radixin-Moesin (ERM) protein family is involved in the regulation of cell morphology, cell adhesion and invasion. Based on our previous work on ERM proteins we sought out to study the expression of ezrin in cervical premalignant lesions. We also studied the expression of E-cadherin and ß-catenin, which play an important role in epithelial cell adhesion. We observed intensifying expression of ezrin along with progressing grade of neoplasia. Ezrin staining was found to colocalize with p16 staining in high-risk HPV associated lesions. Expression of E-cadherin and ß-catenin was found to be altered along with the severity of the lesion, similar to ezrin. Enhanced expression of ezrin in cervical HPV associated lesions suggests a role in the development of cervical neoplasia and cancer. Further clinical evaluation should reveal the feasibility of ezrin as a biomarker for the progression of cervical lesions.

High pentraxin-3 plasma levels associate with thrombocytopenia in acute Puumala hantavirus-induced nephropathia epidemica.

Our aim was to investigate whether plasma levels of the long pentraxin-3 (PTX3) associate with the severity of Puumala hantavirus-induced nephropathia epidemica (NE). Sixty-one prospectively identified consecutively hospitalized NE patients were examined. Plasma PTX3, interleukin (IL)-6, terminal complement complex SC5b-9, complement component C3, C-reactive protein (CRP), creatinine, sodium, kynurenine, and tryptophan levels, as well as the blood cell count, were determined for up to five consecutive days after hospitalization. Receiver operating characteristic (ROC) analysis revealed that the maximum PTX3 level >101.6 ng/ml (high PTX3) showed a sensitivity of 71% and a specificity of 89% for detecting platelet level <50 × 10(9)/l, with an area under the curve (AUC) value of 0.78 (95% confidence interval [CI] 0.63-0.94). High PTX3 level was also associated with several other variables reflecting the severity of the disease: patients with high PTX3 level had higher maximum blood leukocyte (16.1 vs. 9.7 × 10(9)/l, p < 0.001), plasma IL-6 (16.9 vs. 9.0 pg/ml, p = 0.007), and creatinine (282 vs. 124 µmol/l, p = 0.007) levels than patients with low maximum PTX3 level. They also had longer hospital stays (8 vs. 5 days, p = 0.015) compared to patients with low PTX3 level. High plasma PTX3 levels are associated with thrombocytopenia and the overall severity of NE.

A five-year perspective on the situation of haemorrhagic fever with renal syndrome and status of the hantavirus reservoirs in Europe, 2005-2010.

Hantavirus infections are reported from many countries in Europe and with highly variable annual case numbers. In 2010, more than 2,000 human cases were reported in Germany, and numbers above the baseline have also been registered in other European countries. Depending on the virus type human infections are characterised by mild to severe forms of haemorrhagic fever with renal syndrome. The member laboratories of the European Network for diagnostics of Imported Viral Diseases present here an overview of the progression of human cases in the period from 2005 to 2010. Further we provide an update on the available diagnostic methods and endemic regions in their countries, with an emphasis on occurring virus types and reservoirs.

Fibroblast activation in vascular inflammation.

Vascular inflammation is implicated in both local and systemic inflammatory conditions. Endothelial activation and leukocyte extravasation are key events in vascular inflammation. Lately, the role of the stromal microenvironment as a source of proinflammatory stimuli has become increasingly appreciated. Stromal fibroblasts produce cytokines, growth factors and proteases that trigger and maintain acute and chronic inflammatory conditions. Fibroblasts have been associated with connective tissue pathologies such as scar formation and fibrosis, but recent research has also connected them with vascular dysfunctions. Fibroblasts are able to modulate endothelial cell functions in a paracrine manner, including proinflammatory activation and promotion of angiogenesis. They are also able to activate and attract leukocytes. Stromal fibroblasts can thus cause a proinflammatory switch in endothelial cells, and promote leukocyte infiltration into tissues. New insights in the role of adventitial fibroblasts have further strengthened the link between stromal fibroblasts and proinflammatory vascular functions. This review focuses on the role of fibroblasts in inducing and maintaining vascular inflammation, and describes recent findings and concepts in the field, along with examples of pathologic implications.

Cytoplasmic tails of hantavirus glycoproteins interact with the nucleocapsid protein.

Here we characterize the interaction between the glycoproteins (Gn and Gc) and the ribonucleoprotein (RNP) of Puumala virus (PUUV; genus Hantavirus, family Bunyaviridae). The interaction was initially established with native proteins by co-immunoprecipitating PUUV nucleocapsid (N) protein with the glycoprotein complex. Mapping of the interaction sites revealed that the N protein has multiple binding sites in the cytoplasmic tail (CT) of Gn and is also able to bind to the predicted CT of Gc. The importance of Gn- and Gc-CTs to the recognition of RNP was further verified in pull-down assays using soluble peptides with binding capacity to both recombinant N protein and the RNPs of PUUV and Tula virus. Additionally, the N protein of PUUV was demonstrated to interact with peptides of Gn and Gc from a variety of hantavirus species, suggesting a conserved RNP-recognition mechanism within the genus. Based on these and our previous results, we suggest that the complete hetero-oligomeric (Gn-Gc)(4) spike complex of hantaviruses mediates the packaging of RNP into virions.

Hormonal deficiencies during and after Puumala hantavirus infection.

Previous reports have described panhypopituitarism associated with severe cases of hemorrhagic fever with renal syndrome (HFRS), but the prevalence of hormonal deficiencies after nephropathia epidemica (NE), a milder form of HFRS, has not been studied. This study was conducted in order to determine the prevalence of hormonal defects in patients with acute NE and during long-term follow-up. Fifty-four patients with serologically confirmed acute NE were examined by serum hormonal measurements during the acute NE, after 3 months, and after 1 to 10 (median 5) years. Thirty out of 54 (56%) patients had abnormalities of the gonadal and/or thyroid axis during the acute NE. After a median follow-up of 5 years, 9 (17%) patients were diagnosed with a chronic, overt hormonal deficit: hypopituitarism was found in five patients and primary hypothyroidism in five patients. In addition, chronic subclinical testicular failure was found in five men. High creatinine levels and inflammatory markers during NE were associated with the acute central hormone deficiencies, but not with the chronic deficiencies. Hormonal defects are common during acute NE and, surprisingly, many patients develop chronic hormonal deficiencies after NE. The occurrence of long-term hormonal defects cannot be predicted by the severity of acute NE.

Case-control study on Puumala virus infection: smoking is a risk factor.

Puumala hantavirus (PUUV) is apparently transmitted to humans by inhalation of aerosolized secretions of carrier rodents (bank voles). The means of transmission and the associated risk factors are poorly defined. An epidemiological study during the peak of an epidemic season in Finland was conducted based on 282 acute clinical PUUV infections and 204 controls without PUUV infection or immunity. The main risk factors adjusted by age, sex and living environment were cigarette smoking [odds ratio (OR) 3.6, 95% confidence interval (CI) 2.1-5.9, P<0.0001] and buildings with holes allowing rodents to enter (OR 3.3, 95% CI 2.0-5.6); these results were similar in two subsets. Further, use of rodent traps (OR3.5, 95% CI 2.2-5.7) and handling firewood (OR 2.7, 95% CI 1.6-4.4) were associated with a risk. The risk attributed to smoking also remained high using simulated population controls with average smoking habits. The results suggest that hantavirus transmission occurs by inhalation mainly indoors and is dependent on the condition of the respiratory tract.

Situation of hantavirus infections and haemorrhagic fever with renal syndrome in European countries as of December 2006.

Hantavirus infections are widely distributed in Europe with the exception of the far north and the Mediterranean regions. The underlying causes of varying epidemiological patterns differ among regions: in western and central Europe epidemics of haemorrhagic fever with renal syndrome (HFRS) caused by hantavirus infections follow mast years with increased seed production by oak and beech trees followed by increased rodent reproduction. In the northern regions, hantavirus infections and HFRS epidemics occur in three to four year cycles and are thought to be driven by prey - predator interactions. Hantavirus infections and HFRS seem to be on the increase in Europe, partly because of better diagnostics, partly perhaps due to environmental changes. Unfortunately, hantavirus infections are still heavily under-diagnosed in many European countries. Here we report the results of a survey conducted in 2007 amongst the member laboratories of the European Network for diagnostics of Imported Viral Diseases (ENIVD).

Analysis of the surveillance situation for viral encephalitis and meningitis in Europe.

Infective processes in the brain, spinal cord and meninges are considered to be the main causes of encephalitis, myelitis and meningitis. However, most cases remain unexplained. The incidence of different viral aetiologies (zoonotic and non-zoonotic) is especially poorly estimated, due to the lack of a standard case definition and of agreed diagnostic algorithms, including harmonised diagnostic methods and sample collection. It is important to clarify the incidence of viral encephalitis/meningitis and to optimise the diagnosis of infectious neurological illness, particularly to ensure early recognition of outbreaks or emerging infectious such a West Nile encephalitis. The European Network for Diagnostics of 'Imported' Viral Diseases (ENIVD) has analysed the present surveillance situation for viral encephalitis/meningitis in Europe. Here we give an overview of the existing epidemiological sources of information in European Union (EU) Member States, mapping the laboratory capacity and identifying key requirements for a possible future surveillance study at European level. The data presented will help design a harmonised/standardised Europe-wide surveillance study investigating patients with encephalitis and/or meningitis in order to obtain more information on the role of infections in these rarely analysed syndromes, both from a clinical and an epidemiological perspective.

Evidence for human herpesvirus 6 variant A antibodies in multiple sclerosis: diagnostic and therapeutic implications.

Human herpesvirus 6 (HHV-6) has been linked to the pathogenesis of multiple sclerosis (MS). HHV-6 antibodies in serum and cerebrospinal fluid (CSF) of 27 patients with clinically definite MS (CDMS) were compared with age- and sex-matched controls, including various other neurological diseases and symptoms (OND). In addition, we studied a series of 19 patients with clinically or laboratory supported possible MS (CPMS). Seroprevalence to HHV-6A was 100% in patients with MS, both in CDMS and CPMS, compared to 69.2% in patients with OND (P = .001 and .007). The mean immunoglobulin G (IgG) titers were significantly higher in patients with CDMS and CPMS than in controls (P = .005 and .00002). The proportion of acute primary infections without CSF involvement was similar in all groups; however, primary infections with intrathecal HHV-6 antibody production were more frequent in MS. In CSF, HHV-6A-specific antibodies were present in three (11.5%) and four (21.1%) patients with CDMS and CPMS, compared to none with OND (P = .06 and .01, respectively). Serological suggestions to HHV-6A infection occurred more often in both CDMS and CPMS than in OND (14.8% versus 21.1% versus 3.8%). We conclude that a subpopulation of MS patients, and even a greater proportion of possible MS subjects, has serological evidence of HHV-6A infection, which might provide new markers for diagnosis and therapy.

Ex vivo stability of the rodent-borne Hantaan virus in comparison to that of arthropod-borne members of the Bunyaviridae family.

The possible effect of virus adaptation to different transmission routes on virus stability in the environment is not well known. In this study we have compared the stabilities of three viruses within the Bunyaviridae family: the rodent-borne Hantavirus Hantaan virus (HTNV), the sand fly-borne Phlebovirus sandfly fever Sicilian virus (SFSV), and the tick-borne Nairovirus Crimean-Congo hemorrhagic fever virus (CCHFV). These viruses differ in their transmission routes: SFSV and CCHFV are vector borne, whereas HTNV is spread directly between its hosts, and to humans, via the environment. We studied whether these viruses differed regarding stability when kept outside of the host. Viral survival was analyzed at different time points upon exposure to different temperatures (4 degrees C, 20 degrees C, and 37 degrees C) and drying at 20 degrees C. We observed clearly different stabilities under wet conditions, particularly at 4 degrees C, where infectious SFSV, HTNV, and CCHFV were detectable after 528, 96, and 15 days, respectively. All three viruses were equally sensitive to drying, as shown by drying on aluminum discs. Furthermore, HTNV and SFSV partially survived for 2 min in 30% ethanol, whereas CCHFV did not. Electron microscopy images of HTNV, SSFSV, and CCHFV stored at 37 degrees C until infectivity was lost still showed the occurrence of virions, but with abnormal shapes and densities compared to those of the nonincubated samples. In conclusion, our study points out important differences in ex vivo stability among viruses within the Bunyaviridae family.

Confirmed primary HHV-6 infection in children with suspected encephalitis.

HHV-6 infection has been associated with neurological symptoms in children. Two variants of human herpes virus 6, HHV-6A and HHV-6B, have been identified. Their role in neurological infections is poorly understood. We studied 53 children with suspected encephalitis for HHV-6A (strain GS) and HHV-6B (strain Z29) antibodies using an indirect immunofluorescence test. Primary infection was separated from past infection by an IgG-avidity test. The identified primary infections were studied for HHV-6 specific DNA by PCR. Forty-one children of 53 had IgG antibodies to HHV-6. Six children had low avidity of HHV-6 IgG antibodies indicating acute primary infection; four to type A, one to B, and one to both types. By serology, HHV-6 viral etiology was suggested in 6/53 (11.3%) of cases. One of the six patients with primary infection had HHV-6 DNA in serum and two in CSF. The children with primary HHV-6 infection were significantly younger than the whole series, 2.3 years vs. 6.4 years. We conclude that primary HHV-6 infection appears to be an important associated or causative agent in neurological infections of young children, and it can be confirmed from a single serum specimen using the IgG-avidity test.

Serological survey for viral pathogens in Turkish rodents.

Wild rodents (n = 330) were trapped around the villages of Altindere and Cosandere (Maçka, Trabzon Province), Ayder, Ortan, and Yolkiyi (Camlihemsin, Rize Province), and Bozdag (Odemis, Izmir Province) in northeastern and western Turkey during April 2004. Samples were tested for arenavirus, hantavirus, and cowpox virus (family Poxviridae, genus Orthopoxvirus, CPXV) antibodies by using immunofluorescence assays (IFAs). Antibodies against arenaviruses were found in eight of 330 (2.4%) rodents. Arenavirus sero-positive animals were found from all study sites. Antibodies to Puumala virus (family Bunyaviridae, genus Hantavirus, PUUV) were detected in four of 65 Microtus voles tested. Of the PUUV-IFA-positive voles, one Microtus guentheri lydius was caught from Izmir, and one Microtus roberti and two Microtus rossiaemeridionalis were captured near Trabzon. All 264 Apodemus spp. mice tested negative for antibodies to Saaremaa virus (family Bunyaviridae, genus Hantavirus, SAAV); the single Dryomys nitedula tested negative for both PUUV and SAAV antibodies. Only one (0.3%) of the rodents, an Apodemus sylvaticus from Trabzon area, tested seropositive to CPXV. This is the first serologic survey for rodent-borne viruses in their natural hosts in Turkey. Although these preliminary results support presence of several virus groups with zoonotic potential, additional studies are needed to identify the specific viruses that are present in these populations.

Ten-year prognosis of Puumala hantavirus-induced acute interstitial nephritis.

Nephropathia epidemica (NE) is a hemorrhagic fever with renal syndrome caused by Puumala hantavirus. Its long-term prognosis is considered favorable. There are, however, some reports about subsequent hypertension, glomerular hyperfiltration, and proteinuria after previous hantavirus infection. Therefore, we studied 36 patients 5 and 10 years after acute NE, with 29 seronegative controls. Office blood pressure, ambulatory 24-h blood pressure (ABP), glomerular filtration rate (GFR), and proteinuria were examined. Hypertensive subjects were defined as those patients having increased ambulatory or office blood pressure, or receiving antihypertensive therapy. Office blood pressure was used to define hypertension only if ABP was not determined. At 5 years, the prevalence of hypertension was higher among NE patients than in controls (50 vs 21%, P=0.020). At 10 years, the difference between the groups was no more significant (39 vs 17%, P=0.098). Five years after NE, patients showed higher GFR (121+/-19 vs 109+/-16 ml/min/1.73 m(2), P=0.012) and urinary protein excretion (0.19 g/day, range 0.12-0.38 vs 0.14 g/day, range 0.09-0.24, P=<0.001) than controls. At 10 years, there were no more differences in GFR or protein excretion between the groups (GFR: 113+/-20 vs 108+/-17 ml/min/1.73 m(2), P=0.370; proteinuria: 0.14 g/day, range 0.07-0.24 vs 0.13 g/day, range 0.06-0.31, P=0.610). In conclusion, the 10-year prognosis of NE is favorable, as glomerular hyperfiltration and slight proteinuria detected at 5 years disappeared during the longer follow-up. However, the possibility exists that NE may predispose some patients to the development of hypertension.

HLA-DRB1, -DQB1 alleles in head and neck carcinoma patients.

Certain HLA class II alleles have been reported to play a role in development or prevention of cervical carcinoma, an epithelial malignancy linked to human papillomavirus (HPV). In head and neck carcinomas, of which a subset is also HPV associated, the impact of HLA genes remains unknown. HLA-DRB1, -DQB1 alleles were determined in a comprehensive series of 162 head and neck carcinoma patients, for which 83 consecutive cadaveric organ donors of Finnish origin served as controls. DRB1*03 was associated with node-negative disease and DRB1*08 and 13 with small tumors; DRB1*04 was protective against disease relapse. Most alleles of borderline significance in this study act similarly in cervical carcinomas.

Is there an association of Pneumocystis infection with the presence of arena-, hanta-, and poxvirus antibodies in wild mice and shrews in Finland?

As part of studies on the nature of the endemic virus infections in natural rodent hosts, the possible association of cyst forms of Pneumocystis spp. with the presence of hanta-, cowpox-, and arenavirus antibodies in wild mice (Apodemus flavicollis, N=105; Apodemus agrarius, N=63; Micromys minutus, N=50) and the common shrew (Sorex araneus, N=101) was studied in south-central Finland. One hantavirus (Saaremaa virus, SAAV) seropositive A. agrarius, and 2 cowpoxvirus (CPXV) seropositive S. araneus were detected, and antibodies against an arenavirus (Lymphocytic choriomeningitis virus, LCMV) were found in all 3 mouse species but not in shrews. Cyst forms of Pneumocystis spp. were detected in all species except A. agrarius. There was no significant association between virus antibodies (LCMV in mice, and CPXV in shrews) and cyst forms of Pneumocystis in any of the species. Concurrent presence of virus antibodies (LCMV) and cyst forms of Pneumocystis were detected only in 1 M. minutus. In conclusion, we found no evidence of any association between Pneumocystis and antibodies to any of the viruses tested.

Hantavirus and arenavirus antibody prevalence in rodents and humans in Trentino, Northern Italy.

The spatial and temporal distribution of hantavirus and arenavirus antibody-positive wild rodents in Trentino, Italy, was studied using immunofluorescence assays (IFA) in two long-term sites trapped in 2000-2003, and six other sites trapped in 2002. The overall hantavirus seroprevalence in the bank voles, Clethrionomys glareolus (n=229) screened for Puumala virus (PUUV) antibodies was 0.4%, and that for Apodemus flavicollis mice (n=1416) screened for Dobrava virus (DOBV) antibodies was 0.2%. Antibodies against lymphocytic choriomeningitis virus (LCMV) were found in 82 (5.6%) of the 1472 tested rodents; the seroprevalence being 6.1% in A. flavicollis (n=1181), 3.3% in C. glareolus (n=276), and 14.3% in Microtus arvalis (n=7). Of the serum samples of 488 forestry workers studied by IFA, 12 were LCMV-IgG positive (2.5%) and one DOBV-IgG positive (0.2%), however, the latter could not be confirmed DOBV-specific with a neutralization assay. Our results show a widespread distribution but low prevalence of DOBV in Trentino, and demonstrate that the arenavirus antibodies are a common finding in several other rodent species besides the house mouse.