Surveillance for European bat lyssavirus in Swiss bats.

Most countries in Western Europe are currently free of rabies in terrestrial mammals. Nevertheless, rabies remains a residual risk to public health due to the natural circulation of bat-specific viruses, such as European bat lyssaviruses (EBLVs). European bat lyssavirus types 1 and 2 (EBLV-1 and EBLV-2) are widely distributed throughout Europe, but little is known of their true prevalence and epidemiology. We report that only three out of 837 brains taken from bats submitted to the Swiss Rabies Centre between 1976 and 2009 were found by immunofluorescence (FAT) to be positive for EBLVs. All three positive cases were in Myotis daubentoni, from 1992, 1993 and 2002. In addition to this passive surveillance, we undertook a targeted survey in 2009, aimed at detecting lyssaviruses in live bats in Switzerland. A total of 237 bats of the species M. daubentoni, Myotis myotis, Eptesicus serotinus and Nyctalus noctula were captured at different sites in western Switzerland. Oropharyngeal swabs and blood from each individual were analysed by RT-PCR and rapid fluorescent focus inhibition test (RFFIT), respectively. RNA corresponding to EBLV-2 was detected from oropharyngeal swabs of a single M. daubentoni bat, but no infectious virus was found. Molecular phylogenetic analysis revealed that the corresponding sequence was closely related to the other EBLV-2 sequences identified in previous rabies isolates from Swiss bats (particularly to that found at Geneva in 2002). Three M. daubentoni bats were found to be seropositive by RFFIT. In conclusion, even though the prevalence is low in Switzerland, continuous management and surveillance are required to assess the potential risk to public health.

Seroprevalence of hepatitis e virus in domestic pigs and wild boars in Switzerland.

Hepatitis E is considered an emerging human viral disease in industrialized countries. Studies from Switzerland report a human seroprevalence of hepatitis E virus (HEV) of 2.6-21%, a range lower than in adjacent European countries. The aim of this study was to determine whether HEV seroprevalence in domestic pigs and wild boars is also lower in Switzerland and whether it is increasing and thus indicating that this zoonotic viral infection is emerging. Serum samples collected from 2,001 pigs in 2006 and 2011 and from 303 wild boars from 2008 to 2012 were analysed by ELISA for the presence of HEV-specific antibodies. Overall HEV seroprevalence was 58.1% in domestic pigs and 12.5% in wild boars. Prevalence in domestic pigs was significantly higher in 2006 than in 2011. In conclusion, HEV seroprevalence in domestic pigs and wild boars in Switzerland is comparable with the seroprevalence in other countries and not increasing. Therefore, prevalence of HEV in humans must be related to other factors than prevalence in pigs or wild boars.

Monocyte differentiation toward regulatory dendritic cells is not affected by respiratory syncytial virus-induced inflammatory mediators.

Airway epithelial cells were shown to drive the differentiation of monocytes into dendritic cells (DCs) with a suppressive phenotype. In this study, we investigated the impact of virus-induced inflammatory mediator production on the development of DCs. Monocyte differentiation into functional DCs, as reflected by the expression of CD11c, CD123, BDCA-4, and DC-SIGN and the capacity to activate T cells, was similar for respiratory syncytial virus (RSV)-infected and mock-infected BEAS-2B and A549 cells. RSV-conditioned culture media resulted in a partially mature DC phenotype, but failed to up-regulate CD80, CD83, CD86, and CCR7, and failed to release proinflammatory mediators upon Toll-like receptor (TLR) triggering. Nevertheless, these DCs were able to maintain an antiviral response by the release of Type I IFN. Collectively, these data indicate that the airway epithelium maintains an important suppressive DC phenotype under the inflammatory conditions induced by infection with RSV.

Expression of the alpha 1b-adrenergic receptor and G protein subunits in mammalian cell lines using the Semliki Forest virus expression system.

Semliki Forest virus (SFV) vectors have been efficiently used for rapid high level expression of several G protein-coupled receptors. Here we describe the use of SFV vectors to express the alpha 1b-adrenergic receptor (AR) alone or in the presence of the G protein alpha q and/or beta 2 and gamma 2 subunits. Infection of baby hamster kidney (BHK) cells with recombinant SFV-alpha 1b-AR particles resulted in high specific binding activity of the alpha 1b-AR (24 pmol receptor/mg protein). Time-course studies indicated that the highest level of receptor expression was obtained 30 hours post-infection. The stimulation of BHK cells, with epinephrine led to a 5-fold increase in inositol phosphate (IP) accumulation, confirming the functional coupling of the receptor to G protein-mediated activation of phospholipase C. The SFV expression system represents a rapid and reproducible system to study the pharmacological properties and interactions of G protein coupled receptors and of G protein subunits.

Thymoma, immunodeficiency, and herpes simplex virus infections.

Hypogammaglobulinemia develops in 3 to 6% of patients with thymoma and this association is commonly referred to as thymoma with immunodeficiency (formerly Good syndrome). Recurrent infections with encapsulated bacteria and opportunistic infections associated with disorders of both humoral and cell mediated immunity frequently occur in this rare primary, adult-onset immunodeficiency. We report a case of thymoma with immunodeficiency complicated by disseminated herpes simplex virus (HSV) infection and review five additional cases of HSV-related infections reported since 1966 in patients presenting with thymoma with immunodeficiency. Patients presented with epiglottitis, keratitis, recurrent genital herpes, ulcerative dermatitis, and acute hepatitis. Four of the six cases had a fatal outcome, two of which were directly attributable to HSV infection. Since the risk of invasive opportunistic infections is high and the presentation atypical, lymphocyte count and total serum immunoglobulin should be measured regularly in all patients presenting with thymoma with immunodeficiency.

Nasal immunization of mice with human papillomavirus type 16 virus-like particles elicits neutralizing antibodies in mucosal secretions.

To specifically induce a mucosal antibody response to purified human papillomavirus type 16 (HPV16) virus-like particles (VLP), we immunized female BALB/c mice orally, intranasally, and/or parenterally and evaluated cholera toxin (CT) as a mucosal adjuvant. Anti-HPV16 VLP immunoglobulin G (IgG) and IgA titers in serum, saliva, and genital secretions were measured by enzyme-linked immunosorbent assay (ELISA). Systemic immunizations alone induced HPV16 VLP-specific IgG in serum and, to a lesser extent, in genital secretions but no secretory IgA. Oral immunization, even in the presence of CT, was inefficient. However, three nasal immunizations with 5 microgram of VLP given at weekly intervals to anesthetized mice induced high (>10(4)) and long-lasting (>15 weeks) titers of anti-HPV16 VLP antibodies in all samples, including IgA and IgG in saliva and genital secretions. CT enhanced the VLP-specific antibody response 10-fold in serum and to a lesser extent in saliva and genital secretions. Nasal immunization of conscious mice compared to anesthetized mice was inefficient and correlated with the absence of uptake of a marker into the lung. However, a 1-microgram VLP systemic priming followed by two 5-microgram VLP intranasal boosts in conscious mice induced both HPV16 VLP-specific IgG and IgA in secretions, although the titers were lower than in anesthetized mice given three intranasal immunizations. Antibodies in serum, saliva, and genital secretions of immunized mice were strongly neutralizing in vitro (50% neutralization with ELISA titers of 65 to 125). The mucosal and systemic/mucosal HPV16 VLP immunization protocols that induced significant titers of neutralizing IgG and secretory IgA in mucosal secretions in mice may be relevant to genital HPV VLP-based human vaccine trials.

Epidemiological traits of the malaria-like parasite Polychromophilus murinus in the Daubenton¿s bat Myotis daubentonii.

BackgroundThe great diversity of bat haemosporidians is being uncovered with the help of molecular tools. Yet most of these studies provide only snapshots in time of the parasites discovered. Polychromophilus murinus, a malaria-like blood parasite, specialised on temperate-zone bats is a species that is being `rediscovered¿. This study describes the infection dynamics over time and between host sex and age classes.MethodsFor three years we followed the members of three breeding colonies of Myotis daubentonii in Western Switzerland and screened them for the prevalence and parasitemia of P. murinus using both molecular tools and traditional microscopy. In order to identify more susceptible classes of hosts, we measured, sexed and aged all individuals. During one year, we additionally measured body temperature and haematocrit values.ResultsJuvenile bats demonstrated much higher parasitemia than any other age class sampled, suggesting that first exposure to the parasite is very early in life during which infections are also at their most intense. Moreover, in subadults there was a clear negative correlation between body condition and intensity of infection, whereas a weak positive correlation was observed in adults. Neither body temperature, nor haematocrit, two proxies used for pathology, could be linked to intensities of infection.ConclusionIf both weaker condition and younger age are associated with higher infection intensity, then the highest selection pressure exerted by P. murinus should be at the juvenile stage. Confusion over the identities and nomenclature of malarial-like parasites requires that molecular barcodes are coupled to accurate morphological descriptions.

Response of bat species to sylvo-pastoral abandonment

We assessed the effect of abandonment of sylvo-pastoral practices in chestnut orchards (Castanea sativa) on bats in southern Switzerland to determine practical recommendations for bat conservation. We compared bat species richness and foraging activities between traditionally managed and unmanaged chestnut orchards, testing the hypothesis that managed orchards provide better foraging opportunities and harbour more bat species. Echolocation calls of foraging bats were sampled simultaneously at paired sites of managed and unmanaged orchards using custom made recorders. Vegetation structure and aerial insect availability were sampled at the recording sites and used as explanatory variables in the model. In a paired sampling design, we found twice the number of bat species (12) and five times higher total foraging activity in the managed chestnut orchards compared to the unmanaged ones. Bat species with low flight manoeuvrability were 14 times more common in managed orchards, whereas bats with medium to high manoeuvrability were only 5 times more common than in abandoned orchards. The vegetation structure was less dense in managed orchards. However, management did not affect relative insect abundance. Bats primarily visited the most open orchards, free of undergrowth. As a result of restricted access into the overgrown forests, the abandonment of chestnut orchards leads to a decline in bat species richness and foraging activities. Continued management of chestnut orchards to maintain an open structure is important for the conservation of endangered bat species in the southern Swiss Alps.

Identification of a novel determinant for membrane association in hepatitis C virus nonstructural protein 4B.

Nonstructural protein 4B (NS4B) plays an essential role in the formation of the hepatitis C virus (HCV) replication complex. It is a relatively poorly characterized integral membrane protein predicted to comprise four transmembrane segments in its central portion. Here, we describe a novel determinant for membrane association represented by amino acids (aa) 40 to 69 in the N-terminal portion of NS4B. This segment was sufficient to target and tightly anchor the green fluorescent protein to cellular membranes, as assessed by fluorescence microscopy as well as membrane extraction and flotation analyses. Circular dichroism and nuclear magnetic resonance structural analyses showed that this segment comprises an amphipathic alpha-helix extending from aa 42 to 66. Attenuated total reflection infrared spectroscopy and glycosylation acceptor site tagging revealed that this amphipathic alpha-helix has the potential to traverse the phospholipid bilayer as a transmembrane segment, likely upon oligomerization. Alanine substitution of the fully conserved aromatic residues on the hydrophobic helix side abrogated membrane association of the segment comprising aa 40 to 69 and disrupted the formation of a functional replication complex. These results provide the first atomic resolution structure of an essential membrane-associated determinant of HCV NS4B.

Preactivation of B lymphocytes does not enhance mouse mammary tumor virus infection.

We investigated whether mouse mammary tumor virus (MMTV) favors preactivated or naive B cells as targets for efficient infection. We have demonstrated previously that MMTV activates B cells upon infection. Here, we show that polyclonal activation of B cells leads instead to lower infection levels and attenuated superantigen-specific T-cell responses in vivo. This indicates that naive small resting B cells are the major targets of MMTV infection and that the activation induced by MMTV is sufficient to allow efficient infection.

Association of the Epstein-Barr virus latent membrane protein 1 with lipid rafts is mediated through its N-terminal region.

The latent membrane protein 1 (LMP1) encoded by the Epstein-Barr virus acts like a constitutively activated receptor of the tumor necrosis factor receptor (TNFR) family and is enriched in lipid rafts. We showed that LMP1 is targeted to lipid rafts in transfected HEK 293 cells, and that the endogenous TNFR-associated factor 3 binds LMP1 and is recruited to lipid rafts upon LMP1 expression. An LMP1 mutant lacking the C-terminal 55 amino acids (Cdelta55) behaves like the wild-type (WT) LMP1 with respect to membrane localization. In contrast, a mutant with a deletion of the 25 N-terminal residues (Ndelta25) does not concentrate in lipid rafts but still binds TRAF3, demonstrating that cell localization of LMP1 was not crucial for TRAF3 localization. Moreover, Ndelta25 inhibited WT LMP1-mediated induction of the transcription factors NF-kappaB and AP-1. Morphological data indicate that Ndelta25 hampers WT LMP1 plasma membrane localization, thus blocking LMP1 function.

Determinants of hepatitis A vaccine immunity in a cohort of human immunodeficiency virus-infected children living in Switzerland.

Vaccination in HIV-infected children is often less effective than in healthy children. The goal of this study was to assess vaccine responses to hepatitis A virus (HAV) in HIV-infected children. Children of the Swiss Mother and Child HIV Cohort Study (MoCHiV) were enrolled prospectively. Recommendations for initial, catch-up, and additional HAV immunizations were based upon baseline antibody concentrations and vaccine history. HAV IgG was assessed by enzyme-linked immunosorbent assay (ELISA) with a protective cutoff value defined as ≥10 mIU/ml. Eighty-seven patients were included (median age, 11 years; range, 3.4 to 21.2 years). Forty-two patients were seropositive (48.3%) for HAV. Among 45 (51.7%) seronegative patients, 36 had not received any HAV vaccine dose and were considered naïve. Vaccine responses were assessed after the first dose in 29/35 naïve patients and after the second dose in 33/39 children (25 initially naïve patients, 4 seronegative patients, and 4 seropositive patients that had already received 1 dose of vaccine). Seroconversion was 86% after 1 dose and 97% after 2 doses, with a geometric mean concentration of 962 mIU/ml after the second dose. A baseline CD4(+) T cell count below 750 cells/μl significantly reduced the post-2nd-dose response (P = 0.005). Despite a high rate of seroconversion, patients with CD4(+) T cell counts of <750/μl had lower anti-HAV antibody concentrations. This may translate into a shorter protection time. Hence, monitoring humoral immunity may be necessary to provide supplementary doses as needed.

Kaposi sarcoma herpes virus antibody response and viremia following highly active antiretroviral therapy in the Swiss HIV Cohort study.

OBJECTIVE: To describe the effect of HAART on Kaposi sarcoma herpes virus (KSHV) antibody response and viremia among HIV-positive MSM. DESIGN: A follow-up study of 272 HIV-positive MSM (including 22 with Kaposi sarcoma) who first initiated HAART between January 1996 and July 2004 in the Swiss HIV Cohort Study. METHODS: For each individual, two serum samples, one at HAART initiation and another 24 months later, were tested for latent and lytic KSHV antibodies using immunofluorescence assays, and for KSHV viremia using PCR. Factors associated with changes in KSHV antibody titers and viremia were evaluated. RESULTS: At HAART initiation, 69.1 and 75.0% of patients were seropositive to latent and lytic KSHV antibodies, respectively. Seropositivity was associated with the presence of Kaposi sarcoma, older age, lower CD8 cell count and higher CD4/CD8 ratio. Prevalence of KSHV viremia at HAART initiation was 6.4%, being significantly higher among patients with Kaposi sarcoma (35.0%), and those with HIV viral loads 100 000 copies/ml (11.7%) or higher. At 24-month follow-up, geometric mean titers (GMTs) among KSHV seropositive patients increased and antibody seroprevalence was higher. Having Kaposi sarcoma and/or CD4 cell counts less than 50 cells/microl at HAART initiation was associated both with higher probability for antibody titers to increase (including seroconversion) and larger increases in GMTs. Only one of 17 viremic patients at HAART initiation had viremia at 24-month follow-up. CONCLUSION: HAART increases KSHV-specific humoral immune response and clearance of viremia among HIV-infected MSM, consistent with the dramatic protection offered by HAART against Kaposi sarcoma.

Recognition of RNA virus by RIG-I results in activation of CARD9 and inflammasome signaling for interleukin 1 beta production.

Interleukin 1 beta (IL-1 beta) is a potent proinflammatory factor during viral infection. Its production is tightly controlled by transcription of Il1b dependent on the transcription factor NF-kappaB and subsequent processing of pro-IL-1 beta by an inflammasome. However, the sensors and mechanisms that facilitate RNA virus-induced production of IL-1 beta are not well defined. Here we report a dual role for the RNA helicase RIG-I in RNA virus-induced proinflammatory responses. Whereas RIG-I-mediated activation of NF-kappaB required the signaling adaptor MAVS and a complex of the adaptors CARD9 and Bcl-10, RIG-I also bound to the adaptor ASC to trigger caspase-1-dependent inflammasome activation by a mechanism independent of MAVS, CARD9 and the Nod-like receptor protein NLRP3. Our results identify the CARD9-Bcl-10 module as an essential component of the RIG-I-dependent proinflammatory response and establish RIG-I as a sensor able to activate the inflammasome in response to certain RNA viruses.