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Funding Information: Outside the submitted work, the authors disclose the following. Bausell H received personal fees from BioMarin, Ultragenyx, Horizon and Vitaflo. Bélanger-Quintana A reports personal fees from BioMarin, Nutricia, Vitaflo, Orphan Europe, Takeda and Genzyme. Rocha JC received research grants from BioMarin, Glutamine and Cambrooke, as well as personal fees from BioMarin, Applied Pharma Research, Nutricia, Merck Serono, Vitaflo, Cambrooke, PIAM and Lifediet. MacDonald A reports research funding from BioMarin, Nutricia, Applied Pharma Research, Vitaflo, Galen, Metax, Mevalia and Arla, as well as lecture fees from BioMarin, Applied Pharma Research, Nutricia and Vitaflo, and consultancy fees from BioMarin, Applied Pharma Research, Arla, Nutricia and Vitaflo. Met Ed reports grant funding from BioMarin, Nutricia, Vitaflo and Horizon Pharmaceuticals. Bernstein L and Rohr F report lecture fees from Vitaflo. Publisher Copyright: © 2021 The Authors Copyright: Copyright 2021 Elsevier B.V., All rights reserved. Background: The metabolic dietitian/nutritionist (hereafter ‘dietitian’) plays an essential role in the nutritional management of patients with phenylketonuria (PKU), including those on pegvaliase. Currently, more educational support and clinical experience is needed to ensure that dietitians are prepared to provide optimal nutritional management and counselling of pegvaliase-treated patients. Methods: Via a face-to-face data-review meeting, followed by a virtual consolidation meeting, a group of expert dietitians and one paediatrician discussed and developed a series of recommendations on the nutritional evaluation and management of patients receiving pegvaliase. The consensus group consisted of 10 PKU experts: six dietitians and one paediatrician from Europe and three dietitians from the US. One European and three US dietitians had experience with pegvaliase-treated patients. Results: The consensus group recommended that a physician, dietitian and nurse are part of the pegvaliase treatment team. Additionally, a psychologist/counsellor should be included if available. Practical proposals for the nutritional evaluation of pegvaliase-treated patients at baseline, during the induction and titration phases and for long-term maintenance were developed. The consensus group suggested assessment of blood Phe at least monthly or every 2 weeks in the event of low blood Phe (i.e., blood Phe

An external quality assessment of yellow fever virus (YFV) molecular detection in European laboratories was organised in rapid response to an increase in human cases in Brazil in 2018 with risk of import to Europe. Detection of YFV was assessed among 32 laboratories in 23/31 European Union (EU) and European Economic Area (EEA) countries and two laboratories in one non-EU/EEA country. Adequate capabilities were lacking in 10/23 countries; five did not participate as they lacked implemented assays.

Background: Tyrosinaemia type 1 is a rare inherited metabolic disease caused by an enzyme defect in the tyrosine degradation pathway. It is treated using nitisinone and a low-protein diet. In a workshop in 2013, a group of nutritional specialists from Germany, Switzerland and Austria agreed to advocate a simplified low-protein diet and to allow more natural protein intake in patients with tyrosinaemia type 1. This retrospective study evaluates the recommendations made at different treatment centers and their impact on clinical symptoms and metabolic control. Methods: For this multicenter study, questionnaires were sent to nine participating treatment centers to collect data on the general therapeutic approach and data of 47 individual patients treated by those centers. Results: Dietary simplification allocating food to 3 categories led to increased tyrosine and phenylalanine blood concentrations without weighing food. Phenylalanine levels were significantly higher in comparison to a strict dietary regimen whereas tyrosine levels in plasma did not change. Non-inferiority was shown for the simplification and liberalization of the diet. Compliance with dietary recommendations was higher using the simplified diet in comparison to the stricter approach. Age correlates negatively with compliance. Conclusions: Simplification of the diet with increased natural protein intake based on three categories of food may be implemented in the diet of patients with tyrosinaemia type 1 without significantly altering metabolic control. Patient compliance is strongly influencing tyrosine blood concentrations. A subsequent prospective study with a larger sample size is necessary to get a better insight into the effect of dietary recommendations on metabolic control.

Dietary management of 418 adult patients with galactosaemia (from 39 centres/12 countries) was compared. All centres advised lactose restriction, 6 restricted galactose from galactosides +/- fruits and vegetables and 12 offal. 38% (n = 15) relaxed diet by: 1) allowing traces of lactose in manufactured foods (n = 13) or 2) giving fruits, vegetables and galactosides (n = 2). Only 15% (n = 6) calculated dietary galactose. 32% of patients were lost to dietetic follow-up. In adult galactosaemia, there is limited diet relaxation. (C) 2015 Elsevier Inc. All rights reserved.

Experimental intranasal infection of marmosets (Callithrix jacchus) with calpox virus results in fatal disease. Route and dose used for viral inoculation of the test animals mimics the natural transmission of smallpox, thus representing a suitable model to study pathogenesis and to evaluate new vaccines against orthopoxvirus infection. However, the pathogenic mechanisms leading to death are still unclear. Therefore, our study aimed at investigating the kinetics of pathological alterations to clarify the pathogenesis in calpox virus infection. Following intranasal inoculation with two different viral doses, common marmosets were sacrificed on days 3, 5, 7, 10 and 12 post inoculation. Collected tissue was screened using histopathology, immunohistochemistry, transmission electron microscopy, and virological assays. Our data suggest that primary replication took place in nasal and bronchial epithelia followed by secondary replication in submandibular lymph nodes and spleen. Parallel to viremia at day 7, virus was detectable in many organs, mainly located in epithelial cells and macrophages, as well as in endothelial cells. Based on the onset of clinical signs, the histological and ultrastructural lesions and the immunohistochemical distribution pattern of the virus, the incubation period was defined to last 11 days, which resembles human smallpox. In conclusion, the data indicate that the calpox model is highly suitable for studying orthopoxvirus-induced disease.

The Global Virus Network (GVN) was established in 2011 in order to strengthen research and responses to current viral causes of human disease and to prepare against new viral pandemic threats. There are now 38 GVN Centers of Excellence and 6 Affiliate laboratories in 24 countries. GVN scientists meet annually to learn about each other's current research, address collaborative priorities and plan future programs. The 2016 meeting was held from October 23–25 in Hokkaido, Japan, in partnership with the Japanese Society for Virology, the National Institute of Infectious Diseases of Japan and the Research Center for Zoonosis Control of Hokkaido University. This report highlights the accomplishments of GVN researchers in many priority areas of medical virology, including the current Zika epidemic, infections by human papillomavirus, influenza, Ebola, Lassa, dengue, HIV, hepatitis C, and chikungunya viruses, and the development of improved diagnostics and new vaccines., Highlights • The GVN is an international research network comprised of 38 Centers of Excellence and 6 Affiliates in 24 countries. • The 2016 Global Virus Network (GVN) Meeting was held in Sapporo, Japan from October 23–25. • New data were presented on all various aspects of medical virology, therapy, and vaccines were presented. • International collaboration is needed to develop effective viral vaccines and therapeutics. • The 2017 international GVN meeting will be held on September 25–28 in Melbourne, Australia.

Background: There is no published data comparing dietary management of urea cycle disorders (UCD) in different countries.Methods: Cross-sectional data from 41 European Inherited Metabolic Disorder (IMD) centres (17 UK, 6 France, 5 Germany, 4 Belgium, 4 Portugal, 2 Netherlands, 1 Denmark, 1 Italy, 1 Sweden) was collected by questionnaire describing management of patients with UCD on prescribed protein restricted diets.Results: Data for 464 patients: N-acetylglutamate synthase (NAGS) deficiency, n = 10; carbamoyl phosphate synthetase (CPS1) deficiency, n = 29; ornithine transcarbamoylase (OTC) deficiency, n = 214; citrullinaemia, n = 108; argininosuccinic aciduria (ASA), n = 80; arginase deficiency, n = 23 was reported. The majority of patients (70%; n = 327) were aged 0-16 y and 30% (n = 137) >16 y. Prescribed median protein intake/kg body weight decreased with age with little variation between disorders. The UK tended to give more total protein than other European countries particularly in infancy. Supplements of essential amino acids (EAA) were prescribed for 38% [n = 174] of the patients overall, but were given more commonly in arginase deficiency (74%), CPS (48%) and citrullinaemia (46%). Patients in Germany (64%), Portugal (67%) and Sweden (100%) were the most frequent users of EAA. Only 18% [n = 84] of patients were prescribed tube feeds, most commonly for CPS (41%); and 21% [n = 97] were prescribed oral energy supplements.Conclusions: Dietary treatment for UCD varies significantly between different conditions, and between and within European IMD centres. Further studies examining the outcome of treatment compared with the type of dietary therapy and nutritional support received are required. (C) 2013 Elsevier Inc. All rights reserved.

Data are missing on the diversity of Plasmodium spp. infecting apes that live in their natural habitat, with limited possibility of human-mosquito-ape exchange. We surveyed Plasmodium spp. diversity in wild chimpanzees living in an undisturbed tropical rainforest habitat and found 5 species: P. malariae, P. vivax, P. ovale, P. reichenowi, and P. gaboni.

The Hepatitis C virus (HCV) infection exhibits a high global prevalence frequently associated with hepatocellular carcinoma, taking years to develop. Despite the standardization of highly sensitive HCV quantitative RT-PCR (qRT-PCR) detection methods, false-negative diagnoses may be generated with current methods, mainly due to the presence of PCR inhibitors and/or low viral loads in the patient's sample. These false-negative diagnoses impact both public health systems, in developing countries, and an in lesser extent, in developed countries, including both the risk of virus transmission during organ transplantation and/or blood transfusion and the quality of the antiviral treatment monitoring. To adopt an appropriate therapeutic strategy to improve the patient's prognosis, it is urgent to increase the HCV detection sensitivity. Based upon previous studies on HBV, we worked on the capacity of the scavenger acute phase protein, Apolipoprotein H (ApoH) to interact with HCV. Using different approaches, including immunoassays, antibody-inhibition, oxidation, ultracentrifugation, electron microscopy and RT-PCR analyses, we demonstrated specific interactions between HCV particles and ApoH. Moreover, when using a two-step HCV detection process, including capture of HCV by ApoH-coated nanomagnetic beads and a home-made real-time HCV-RT-PCR, we confirmed the presence of HCV for all samples from a clinical collection of HCV-seropositive patients exhibiting an RT-PCR COBAS (R) Taq-Man (R) HCV Test, v2.0 (COBAS)-positive result. In contrast, for HCV-seropositive patients with either low HCV-load as determined with COBAS or exhibiting HCV-negative COBAS results, the addition of the two-step ApoH-HCV-capture and HCV-detection process was able to increase the sensitivity of HCV detection or more interestingly, detect in a genotype sequence-independent manner, a high-proportion (44%) of HCV/RNA-positive among the COBAS HCV-negative patients. Thus, the immune interaction between ApoH and HCV could be used as a sample preparation tool to enrich and/or cleanse HCV patient's samples to enhance the detection sensitivity of HCV and therefore significantly reduce the numbers of false-negative HCV diagnosis results.

Background: There appears little consensus concerning protein requirements in phenylketonuria (PKU). Methods: A questionnaire completed by 63 European and Turkish IMD centres from 18 countries collected data on prescribed total protein intake (natural/intact protein and phenylalanine-free protein substitute [PS]) by age, administration frequency and method, monitoring, and type of protein substitute. Data were analysed by European region using descriptive statistics. Results: The amount of total protein (from PS and natural/intact protein) varied according to the European region. Higher median amounts of total protein were prescribed in infants and children in Northern Europe (n = 24 centres) (infants 2-3 g/kg/day; 1-3 years of age, >2-3 g/kg/day; 4-10 years of age, >1.5-2.5 g/kg/day) and Southern Europe (n = 10 centres) (infants 2-2.5 g/kg/day; 4-10 years of age, >1.5-2 g/kg/day) and with Western Europe (n = 25 centres) giving the least (infants 2-2.5 g/kg/day, 1-3 years of age, 1.5-2 g/kg/day; 4-10 years of age, 1-1.5 g/kg/day). Total protein prescription was similar in patients aged >10 years (1-1.5 g/kg/day) and maternal patients (1-1.5 g/kg/day). Conclusions: The amounts of total protein prescribed varied between European countries and appeared to be influenced by geographical region. In PKU, all gave higher than the recommended 2007 WHO/FAO/UNU safe levels of protein intake for the general population. (C) 2015 Elsevier Inc. All rights reserved.

An epizootic infection was observed in a colony of 80 New World monkeys consisting of various species including a group of marmosets and Saguinus species. During the summer and autumn of 2002, 30 animals died of unknown diseases. Six animals were sent to the German Primate Center for investigation of the cause of death. A complete pathologic and histologic investigation was carried out. The animals exhibited erosive-ulcerative lesions of the oral mucous membranes. Advanced stages of the disease were characterised by hemorrhagic lesions on the skin distributed randomly over the body, but principally on the face, scrotal region, soles, and palms. Electron microscopy revealed virus particles with orthopox-like morphology within intracytoplasmic inclusions in epithelial cells. The DNA samples from various tissues were analyzed by use of a set of orthopox virus-specific, real-time polymerase chain reaction assays. Amplification products were sequenced to define the virus more precisely. Sequencing confirmed the presence of an orthopox virus. Sequence data indicated that all six animals were infected with the same virus. Propagation of the virus on Vero cells resulted in a rapidly progressive cytopathogenic effect. Preliminary phylogenetic analyses of two genes revealed closest homology to cowpox viruses. The origin of this poxvirus outbreak remains unexplained, and the strain and genus of the virus need to be determined in detail.

HISTORY AND CLINICAL FINDINGS A 53-year-old West African man presented two years after a travel to Guinea because of severe headache, neck stiffnes, fever and pruritus. The patient had been in orthopedical treatment for the last five months. INVESTIGATIONS Stool microscopy revealed a high number of Strongyloides stercoralis larvae. Hematology, biochemistry and all other parasitology results were normal. HIV-1/2 testing was negative and CD4+-lymphocyte count was normal. Concomitant infection by Human T Cell lymphotropic virus type 1 (HTLV-1) was confirmed by serology and PCR. The phylogenetic analysis confirmed African origin of the virus. TREATMENT The infection responded to a five-day course of albendazol at 400 mg/d but during the following five years repeat recrudescences were observed inspite of high-dosage and prolonged antiparasitic treatments. Eventually, eradication of the infection was achieved by a four day course of ivermectin 0.2 mg/kg/d. CONCLUSIONS Although both strongyloidiasis and HTLV-1 infections occur most frequently in tropical areas, these may also be observed in temperate regions. Suppression of the immune system by HTLV-1 differs from that by HIV. CD4+-lymphocytes were rarely decreased. Prolonged treatment with ivermectin in a dosage exceeding the current recommendations may be required in HTLV-1 infected patients and was well tolerated. The unusual presentation of the infection with muscular symptoms contributed to the delay of the diagnosis. HTLV-1 positive patients must be monitored for years. They and their partners must be instructed how to prevent transmission of the virus.

Objective: Mycosis fungoides (MF) is the most frequent form of cutaneous T cell lymphoma (CTCL). Human T cell leukemia virus type 1 (HTLV-1) involvement in MF progression is a matter of debate. The goal of the investigation was to search for HTLV-1 markers in a group of MF patients from a nonendemic area to HTLV-1. Materials and Methods: Fifty MF patients and 60 healthy donors from Moscow and the Moscow region were examined for HTLV-1 markers by Western blot, PCR, nested PCR, PCR/Southern hybridization, TaqMan real-time PCR and sequencing. Results: Plasma samples from MF patients were repeatedly negative for antibodies to HTLV-1 structural proteins. HTLV-1 tax-related sequences (corresponding to the second exon) were found in blood from 20 of 50 MF patients and in 3 of 5 saliva specimens. Three of 8 sequenced tax-like amplimers were identical and 5 of 8 contained 1–2 substitutions. tax transcripts and antibodies to p40tax were detected in some ‘PCR-tax’-positive MF patients. Defective HTLV-1 genomes were demonstrated in 2 of 50 MF patients. Phylogenetic analysis of the defective genome 5′-LTR sequence revealed a relationship with HTLV-1a sequences from the transcontinental subgroup of HTLV-1. Conclusions: HTLV-1 tax-like sequences were revealed in blood and for the first time in saliva from MF patients living in an HTLV-1 nonendemic region. Expression of tax-like sequences was confirmed by both reverse transcription PCR and Western blot.

To study the genetics of atopy systematically we established a mouse model that provides the general phenotype of atopy: the early response characteristic of IgE-dependent eczema or atopic dermatitis, and the diagnostic test of atopy, the skin-prick test. Using an immediate cutaneous hypersensitivity test (ICHS) against birch pollen extract we could classify A/J and C57BL/6 (B6) inbred mouse strains respectively as high responder and low responders. The F1 hybrids were found to be high responders with incomplete penetrance. Backcrossing F1 mice to the low responder B6 strain yielded three classes of responders, high, intermediate, and low. A genome-wide microsatellite screen of the backcross progeny disclosed suggestive linkage to a microsatellite marker on chromosome 6 close to the locus of the IL-5 receptor alpha chain. Its allelic variation in A/J and B6 strains was investigated and two major differences were detected. Firstly, a nucleotide exchange in the 5' untranslated region of B6 mRNA resulted in increased transcription/translation of a reporter construct. Higher expression of the receptor on the cell surface would be expected to favor an allergic immune response. Secondly, the two alleles are differentially spliced so as to yield two soluble isoforms in A/J mice versus one in B6 mice. Higher expression of soluble IL-5R would be expected to reduce the level of allergy through capture of IL-5. Thus both findings conform to the expectation based on susceptibility to atopy and thus identify the IL-5R alpha chain as a likely contributor to the genetics of atopy.

Chimeric bcr/abl fusion proteins are thought to be the molecular 'pathogen' of chronic myelogenous leukaemia (CML). Expression levels of the respective fusion RNAs reflect disease progression as well as remission upon therapeutic intervention in CML patients. However, there is no quick and reliable method that would allow the quantitative routine monitoring of bcr/abl hybrid transcripts. A fluorescent probe-based PCR assay (TaqMan) has been described to quantitfy the exact amount of target sequences. We have established TaqMan real-time RT-PCRs for M-bcr/abl (b2a2, b2a3, b3a2, b3a3) and m-bcr/abl (e1a2) fusion transcripts as well as for β-actin. All PCRs quantified as little as 10 copies/100 ng total cDNA. In order to investigate whether this procedure is appropriate for routine diagnostic monitoring, we performed retrospective measurements on 9 documented CML disease courses. Our data show that ongoing or relapsing CML is paralleled by increasing peripheral levels of bcr/abl fusion RNAs. Furthermore, sucessful anti-leukemic treatment is reflected by decreasing absolute bcr/abl transcript numbers. In contrast with conventional bcr/abl PCR techniques we could distinguish single positive values and gradually increasing copy numbers. Int. J. Cancer 86:741–746, 2000. © 2000 Wiley-Liss, Inc.

Background: Within Europe, the management of pyridoxine (B-6) non-responsive homocystinuria (HCU) may vary but there is limited knowledge about treatment practice.Aim: A comparison of dietetic management practices of patients with B-6 non-responsive HCU in European centres.Methods: A cross-sectional audit by questionnaire was completed by 29 inherited metabolic disorder (IMD) centres: (14 UK, 5 Germany, 3 Netherlands, 2 Switzerland, 2 Portugal, 1 France, 1 Norway, 1 Belgium).Results: 181 patients (73% >16 years of age) with HCU were identified. The majority (66%; n= 119) were on dietary treatment (1-10 years, 90%; 11-16 years, 82%; and >16 years, 58%) with or without betaine and 34% (n = 62) were on betaine alone. The median natural protein intake (g/day) on diet only was, by age: 1-10 years, 12 g; 11-16 years, 11 g; and > 16 years, 45g. With diet and betaine, median natural protein intake (g/day) by age was: 1-10 years, 13 g; 11-16 years, 20g; and >16 years, 38g. Fifty-two percent (n = 15) of centres allocated natural protein by calculating methionine rather than a protein exchange system. A methionine-free L-amino acid supplement was prescribed for 86% of diet treated patients. Fifty-two percent of centres recommended cystine supplements for low plasma concentrations. Target treatment concentrations for homocystine/homocysteine (free/total) and frequency of biochemical monitoring varied.Conclusion: In B-6 non-responsive HCU the prescription of dietary restriction by IMD centres declined with age, potentially associated with poor adherence in older patients. Inconsistencies in biochemical monitoring and treatment indicate the need for international consensus guidelines. (C) 2013 Elsevier Inc. All rights reserved.

Approximately one third of all local recurrences of breast cancer are incurable at the time of diagnosis. Locoregional intraarterial chemotherapy is one of the new therapy modalities besides laser therapy and combined radiotherapy/hyperthermia. The results of a phase I-II study, in which 15 patients with advanced, partly pretreated local recurrences as well as 2 patients with T4N2/N3 tumours were included, are reported as follows. All in all, 39 superselective intraarterial chemotherapy courses were carried out. Mitomycin (10 mg) and Mitoxantrone (25 mg) were infused over 90 min. The side effects due to the catheter system were two haematomas and one thrombosis attributed to an insufficient heparin dose. Locally, the chemotherapy was well tolerated. One severe systemic side effect, a leucopenia WHO 4 degrees was observed. Nausea, thrombocytopenia and alopecia rates were low (7 x nausea 1 degree, 5 x thrombocytopenia 1 degree, 2 x thrombocytopenia 2 degrees, 2 x alopecia 1 degree). 6 Complete remissions (3 x pCR, 3 x cCR) as well as 6 partial remissions and 5 no changes were found. We believe that this method, because of the low side-effect profile and the temporary good results, represents a good alternative in otherwise incurable locoregional recurrence of breast carcinoma and in specified cases of locally advanced disease. 1 degree and 2 x thrombocytopenia 2 degrees, 2 x alopecia 1 degree). Altogether, 6 complete remissions were found (3 x cCR, 3 x pCR), 6 partial remissions with 4 x no change occurred.(ABSTRACT TRUNCATED AT 250 WORDS)

Increasing infections with Monkeypox and Cowpox viruses pose a continuous and growing threat to human health. The standard method for detecting poxvirus neutralizing antibodies is the plaque-reduction neutralization test that is specific but also time-consuming and laborious. Therefore, a rapid and reliable method was developed to determine neutralizing antibody titers within twelve hours. The new assay measures viral mRNA transcription as a marker for actively replicating virus after incomplete neutralization using real-time PCR.

BackgroundDespite ongoing - and in some regions escalating -morbidity and mortality associated with malaria para-sites, evolutionary epidemiology of Plasmodium is notwell characterized. Recent studies using molecularapproaches demonstrated that wild and captive gorillasand captive bonobos and chimpanzees are infected withP. falciparum and that these apes harbor parasitesbroadly related to P. falciparum[1]. Captive chimpanzeesand bonobos had malaria parasites related to humanP. ovale and P. malariae and various monkeys and onesemi-wild chimpanzee hadP. vivax[2]. It is not clearwhether these apes harbor naturally these parasites orwhether they are transmitted from humans. Most of theexamined animals had close contact with humans, com-parable studies in wild living apes are missing. We pro-vide the first survey ofPlasmodium diversity in wildchimpanzees living in an undisturbed tropical rainforestin Africa.MethodsWe examined tissue from 16 wild West African chim-panzees that lived in the Tai National Park, Ivory Coast,where human contact with animals is limited toresearchers who access the territory only during the day.Samples were collected from animals that died primarilyfrom anthrax or respiratory disease. Generic real timePCR assay was used to detect all knownPlasmodiumspecies.Results11/16 (68%) animals tested positive. Sequence analysesof cytB and 18S rRNA genes identifiedP. malariae,P. ovale, P. vivax, P. gaboni, P. reichenowi, P. billcollinsiand P. billbraii.DiscussionPrevious examination of the role of our closest phyloge-netic relatives, the great apes, in the evolution andpersistence of human malarias has been limited by a lackof data from wild ape populations. Interpretation ofpatterns of malaria infection in captive ape populationsmust consider ample opportunities for human to apetransmission, negating the opportunity to investigate theevolutionary origins and public health-related risks ofthese parasites. Our examination of malaria parasites inwild chimpanzees demonstrates that these apes are mostlikely naturally infected with Plasmodium species homolo-gous toP. malariae, P. vivaxand P. ovale as well asP. falciparum. Whether wild great apes are the origin ofthese malaria types requires further investigation but theymay act as reservoir of infection. These results haveimportant implications for global efforts underway to era-dicate malaria in humans including vaccine developmentbased on animal variants of human parasites.

Little is known about Hepatitis B Virus (HBV) infections in chimpanzees. Therefore, we investigated the prevalence of chimpanzee HBV (chHBV) infections in captive, wild born chimpanzees in the sanctuary on Ngamba Island, Uganda and one sample from a wild free ranging chimpanzee. In one third of the plasma samples (32.4%; 12/37) we detected antibodies to Hepatitis B (core) antigen. Amongst those individuals HBV DNA was detected in one captive wild born and the wild chimpanzee. In contrast to the only available earlier described HBV sequence from the subspecies Pan troglodytes schweinfurthii, there was no evidence of recombination with human HBV. Our sequences therefore are likely to present the "original" chHBV from P. t. schweinfurthii.

Human endogenous retroviruses (HERVs) are discussed as causative agents of various diseases including cancers. Expression of endogenous retroviral sequences can be induced by ultraviolet (UV) light, which is also considered as a cofactor in the development of cutaneous melanoma. Therefore, we investigated whether ultraviolet C (UVC) induces HERV-K rec and np9 expression in normal human epidermal melanocytes (NHEM) and in melanoma cell lines. NHEM and four different melanoma cell lines were irradiated with 10 and 30 mJ/cm(2) UVC, respectively. Expression of the HERV-K transcripts rec and np9 was measured 0, 6, 12, and 24 h after UV exposure by quantitative real-time PCR. In NHEM, HERV-K rec expression was significantly induced 24 h after UV exposure with 10 mJ/cm(2) UVC, whereas np9 expression transiently increased 6 and 12 h after irradiation with both UV doses. In contrast, in melanoma cell lines MEWO, G-361 and GR-M rec, and np9 expression was downregulated or remained unchanged after UV treatment. UVC irradiation induced HERV-K rec and np9 expression in NHEM, which might be an indicator of a functional role of Rec and/or Np9 in melanoma formation.

The intentional re-introduction of Variola virus (VARV), the agent of smallpox, into the human population is of great concern due its bio-terroristic potential. Moreover, zoonotic infections with Cowpox (CPXV) and Monkeypox virus (MPXV) cause severe diseases in humans. Smallpox vaccines presently available can have severe adverse effects that are no longer acceptable. The efficacy and safety of new vaccines and antiviral drugs for use in humans can only be demonstrated in animal models. The existing nonhuman primate models, using VARV and MPXV, need very high viral doses that have to be applied intravenously or intratracheally to induce a lethal infection in macaques. To overcome these drawbacks, the infectivity and pathogenicity of a particular CPXV was evaluated in the common marmoset (Callithrix jacchus). A CPXV named calpox virus was isolated from a lethal orthopox virus (OPV) outbreak in New World monkeys. We demonstrated that marmosets infected with calpox virus, not only via the intravenous but also the intranasal route, reproducibly develop symptoms resembling smallpox in humans. Infected animals died within 1–3 days after onset of symptoms, even when very low infectious viral doses of 56102 pfu were applied intranasally. Infectious virus was demonstrated in blood, saliva and all organs analyzed. We present the first characterization of a new OPV infection model inducing a disease in common marmosets comparable to smallpox in humans. Intranasal virus inoculation mimicking the natural route of smallpox infection led to reproducible infection. In vivo titration resulted in an MID50 (minimal monkey infectious dose 50%) of 8.36102 pfu of calpox virus which is approximately 10,000-fold lower than MPXV and VARV doses applied in the macaque models. Therefore, the calpox virus/marmoset model is a suitable nonhuman primate model for the validation of vaccines and antiviral drugs. Furthermore, this model can help study mechanisms of OPV pathogenesis. peerReviewed

Swine Hepatitis E virus (HEV) can be transmitted from pigs to humans causing hepatitis. A high prevalence of HEV in wild boar populations is reported for several European countries, but actual data for Germany are missing. During the hunting season from October to December 2007 liver, bile and blood samples were collected from wild boars in four different German regions. The samples were tested for HEV RNA by quantitative PCR (qPCR) and anti-HEV IgG antibodies by two different ELISAs and a Line immunoassay. A seroprevalence of 29.9% using ELISA and 26.2% in the Line immunoassay was determined. The seroprevalence rate varied greatly within the analyzed regions. However, qPCR analysis revealed a higher prevalence of 68.2% positive animals with regional differences. Surprisingly, also adult wild sows and wild boars were highly HEV positive by qPCR. Compared to liver and serum samples, bile samples showed a higher rate of positive qPCR results. Sequencing and phylogenetic analysis of a 969 nt fragment within ORF 2 revealed that all isolates clustered within genotype 3 but differed in the subtype depending on the hunting spot. Isolates clustered within genotypes 3i, 3h, 3f and 3e. Within one population HEV isolates were closely related, but social groups of animals in close proximity might be infected with different subtypes. Two full-length genomes of subtypes 3i and 3e from two different geographic regions were generated. The wild boar is discussed as one of the main sources of human autochthonous infections in Germany.

Mosquito-borne infections cause some of the most debilitating human diseases, including yellow fever and malaria, yet we lack an understanding of how disease risk scales with human-driven habitat changes. We present an approach to study variation in mosquito distribution and concomitant viral infections on the landscape level. In a pilot study we analyzed mosquito distribution along a 10-km transect of a West African rainforest area, which included primary forest, secondary forest, plantations, and human settlements. Variation was observed in the abundance of Anopheles, Aedes, Culex, and Uranotaenia mosquitoes between the different habitat types. Screening of trapped mosquitoes from the different habitats led to the isolation of five uncharacterized viruses of the families Bunyaviridae, Coronaviridae, Flaviviridae, and Rhabdoviridae, as well as an unclassified virus. Polymerase chain reaction screening for these five viruses in individual mosquitoes indicated a trend toward infection with specific viruses in specific mosquito genera that differed by habitat. Based on these initial analyses, we believe that further work is indicated to investigate the impact of anthropogenic landscape changes on mosquito distribution and accompanying arbovirus infection.

Pregnancy management in phenylketonuric women includes continuous dietary control starting before conception, aiming to maintain blood phenylalanine concentrations in a desirable range, irrespective of the fetal genetic PKU status. While the maternal phenylalanine hydroxylase (PAH) genotype will influence metabolic control, an effect of the fetal genetic PKU status on maternal metabolic control during pregnancy has not been described. We monitored three pregnancies of women with classical PKU by dietary protocols of daily phenylalanine intake, phenylalanine blood concentrations, and obstetric care. Patients 1 and 2 carried a heterozygous (not PKU-affected) fetus, while patient 3 was pregnant with a PKU-affected fetus (PAH p.R408W and p.R408W). The expected increase in phenylalanine tolerance during the course of pregnancy was observed in patients 1 and 2 in whom phenylalanine intake could be steadily increased from 400 to 1700 mg/day while phenylalanine blood concentrations remained in the desired range. Gain of body weight was 13.0 and 17.7 kg, respectively. In patient 3, the phenylalanine tolerance did not rise above 600 mg/day, and phenylalanine blood concentrations were above the desired range on several occasions. Caloric intake was therefore encouraged, which led to a weight gain of 20.0 kg. The course of pregnancy was otherwise normal in all three cases, and infants with normal birth weight and head circumference were born. The different phenylalanine tolerance in pregnancies with PKU-affected and non-affected fetuses suggests that PAH genotype and metabolic situation of the fetus influence maternal metabolic control. A phenylalanine tolerance remaining low in the third trimester of pregnancy may indicate fetal PKU.

Friend replication-competent murine leukemia virus (F-MuLV), clone 57, induces a severe early hemolytic anemia and a later erythroleukemia after inoculation of newborn IRW or ICFW mice, whereas Moloney MuLV (M-MuLV) induces only lymphoid leukemia. We have shown previously that the attenuated hemolytic and erythroleukemogenic abilities of an F-MuLV variant, clone B3, were due mostly to changes in the env gene and long terminal repeat, respectively. For the present study, we derived two constructs exchanging env fragments of F-MuLV 57 and M-MuLV and compared them with two constructs described by Chatis et al. (J. Virol. 52:248-254, 1984) exchanging the U3 region of the long terminal repeat of the same parental viruses. When comparing the hemolytic effect of these constructs with those of the parent, we found that the U5-gag-pol region of F-MuLV was required for development of severe early hemolytic anemia and that, unlike the env of F-MuLV B3, the env of M-MuLV was fully competent in inducing severe early hemolytic anemia when associated with the F-MuLV U5-gag-pol and U3 regions. As expected, induction of erythroleukemia depended on the presence of the F-MuLV U3 region; however, the presence of both the U3 and U5-gag-pol regions of F-MuLV appeared to be synergistic and was associated with a more rapid appearance of erythroleukemia.

In wild chimpanzees in the Taï National Park, Côte d'Ivoire, sudden deaths which were preceded by respiratory problems had been observed since 1999. Two new clones of Streptococcus pneumoniae were identified in deceased apes on the basis of multilocus sequence typing analysis and ply , lytA , and pbp2x sequences. The findings suggest that virulent S. pneumoniae occurs in populations of wild chimpanzees with the potential to cause infections similar to those observed in humans.

We have developed an advanced tissue processing technique on porcine pulmonary heart valves for pulmonary valve replacement and its initial clinical application during the autograft operation according to Ross. The novel concept consists of a cell-free matrix achieved by deoxycholic acid treatment that is repopulated by host cells in vivo. Molecular biology, radioligand binding, and electron microscopy consistently showed that these valves are almost free of cellular components. Animal experiments and clinical investigations revealed excellent hemodynamic properties of the valves, no need for antithrombotic therapy, and repopulation by host cells without any signs of calcification. In juvenile sheep the internal diameter of the implanted valves significantly increased in growing animals by approximately 10 mm. The repopulation of the decellularized heart valves was found not only in sheep but also in humans, which indicates that the underlying mechanisms, presumably repair mechanisms, might be common in mammals. If these findings can be confirmed by others, they will lead to new concepts in the field of cardiovascular tissue engineering that will eliminate the need for in vitro construction of autologous heart valves.

Background Coxiella burnetii, the bacterium causing Q fever, is an obligate intracellular biosafety level 3 agent. Detection and quantification of these bacteria with conventional methods is time consuming and dangerous. During the last years, several PCR based diagnostic assays were developed to detect C. burnetii DNA in cell cultures and clinical samples. We developed and evaluated TaqMan-based real-time PCR assays that targeted the singular icd (isocitrate dehydrogenase) gene and the transposase of the IS1111a element present in multiple copies in the C. burnetii genome. Results To evaluate the precision of the icd and IS1111 real-time PCR assays, we performed different PCR runs with independent DNA dilutions of the C. burnetii Nine Mile RSA493 strain. The results showed very low variability, indicating efficient reproducibility of both assays. Using probit analysis, we determined that the minimal number of genome equivalents per reaction that could be detected with a 95% probability was 10 for the icd marker and 6.5 for the IS marker. Plasmid standards with cloned icd and IS1111 fragments were used to establish standard curves which were linear over a range from 10 to 107 starting plasmid copy numbers. We were able to quantify cell numbers of a diluted, heat-inactivated Coxiella isolate with a detection limit of 17 C. burnetii particles per reaction. Real-time PCR targeting both markers was performed with DNA of 75 different C. burnetii isolates originating from all over the world. Using this approach, the number of IS1111 elements in the genome of the Nine Mile strain was determined to be 23, close to 20, the number revealed by genome sequencing. In other isolates, the number of IS1111 elements varied widely (between seven and 110) and seemed to be very high in some isolates. Conclusion We validated TaqMan-based real-time PCR assays targeting the icd and IS1111 markers of C. burnetii. The assays were shown to be specific, highly sensitive and efficiently reproducible. Cell numbers in dilutions of a C. burnetii isolate were reliably quantified. PCR quantification suggested a high variability of the number of IS1111 elements in different C. burnetii isolates, which may be useful for further phylogenetic studies.

After eradication of variola virus the worldwide vaccination program was stopped to avoid the severe complications observed in a small fraction of vaccinees. Hence, there is at least one non-vaccinated generation in the human population that is immunologically naive with respect to variola virus infections. The possibility of a deliberate release of variola virus by bioterrorist attacks has led to the resumption of vaccination of hospital employees and military personnel with vaccinia virus in certain parts of the world. However, the appearance of a single confirmed smallpox case worldwide would result in vaccination of possible contact persons with vaccinia virus. Therefore, reliable confirmation of vaccinia virus in patients presenting with smallpox-like syndromes is required. A vaccinia virus-specific single nucleotide polymorphism was identified in the gene B8R coding for a vaccinia virus IFNgamma receptor. Based on this polymorphism, the LightCycler real-time PCR assay detects vaccinia virus DNA in a linear range from 10(6) to 10 genome equivalents and discriminates vaccinia virus from other orthopoxviruses by fluorescence melting curve analysis (DeltaT = 9 degrees C). While the assay amplifies generically DNA of all orthopoxviruses tested, amplification curves are only displayed for vaccinia virus strains including strains formerly used for vaccination. In addition, an internal amplification control is described that allows reliable interpretation of results.

International audience; - The objective of this study was to evaluate the effect of a commercial vaccine adjuvant on the clinical and pathological outcome of PCV2 experimentally infected 8 to 9-week-old conventional pigs. Forty-four pigs were divided into four groups: non-infected control pigs, pigs that received a vaccine adjuvant, pigs inoculated with PCV2, and pigs inoculated with PCV2 together with the vaccine adjuvant. Infection was monitored until 69 days post-inoculation (PI). Some PCV2 inoculated pigs had hyperthermia, but no other clinical signs were recorded. No characteristic PMWS gross or microscopic lesions were observed in any of the pigs. PCV2 DNA was detected in lymphoid tissues by in situ hybridisation in 6 PCV2 inoculated pigs on day 69 PI. All PCV2 inoculated pigs seroconverted between days 21 and 49 PI, shortly after viremia detection. Moreover, viremia was detected between days 7 and 69 PI using PCR. A peak of the virus load was detected by real-time quantitative PCR between days 14 and 21 PI. There were no significant differences in the proportion of PCV2 positive serum and in the viral load between PCV2 and PCV2 + adjuvant inoculated pigs. Although PMWS was not reproduced in neither PCV2 nor PCV2 + adjuvant inoculated pigs, viremia detection and seroconversion indicated that all PCV2 inoculated pigs developed a chronic long-term asymptomatic infection. An increase of PCV2 replication was not observed in pigs inoculated with the adjuvant. These results indicate that the principle of immunostimulation may not be applicable under the experimental conditions used, suggesting that not all adjuvants used in commercial vaccines are capable of triggering mechanisms for PMWS development.

Objective A new quantitative polymerase chain reaction (real-time PCR) was designed to detect Toxoplasma DNA in human body fluid samples. Methods Real-time fluorescence detection of amplification product formation on the basis of the TaqMan-System was established with Toxoplasma 18S rDNA as a target gene. Results The method provides a high sensitivity comparable to conventional nested PCR procedures and generates quantitative data when detecting toxoplasmic DNA in human blood, cerebrospinal or amniotic fluid. Moreover, data were obtained investigating blood samples from an immunocompromised patient with reactivated toxoplasmosis after allogeneic bone marrow transplantation, monitoring the therapeutic effect. Conclusions The potential application of this method to detect Toxoplasma DNA in body fluids and to follow the development of parasitemia under therapy could be demonstrated.

A real-time PCR assay was developed to quantify human cytomegalovirus (CMV) DNA. This assay was used to demonstrate a higher CMV DNA load in plasma of bone marrow transplant patients than in that of blood donors. The CMV load was higher in CMV antigen-positive patients than in antigen-negative patients.

A 17-year-old girl developed a cutaneous macular lesion in the area of the right sternoclavicular joint. In the 3-week period after initial symptoms were noted, the lesion passed through papular, vesicular, and pustular stages before forming a hard black eschar (diameter, 2 cm) with erythema and edema, as well as regional lymphangitis (figure 1). In the early stage of the disease, the patient experienced malaise and had subfebrile temperatures. After treatment with several different antibiotic regimens (clindamycin-clarithromycin, then fucidine-imipeFigure 2. Hard black eschar with erythema and edema in the area of the sternoclavicular joint.

Introduction: Increasing evidence has emerged that adenoviruses (AdV) can cause severe complications in immunocompromised hosts especially following hematopoietic stem cell transplantation (HSCT). So far protective immune responses in adenovirus infection have not been assessed in detail, but control of infection seems to be dependent on CD4+ T-cells. However, the main antigenic adenovirus targets for specific CD4+ T-cells are unknown. Methods: We here use a novel technique to directly assess the entire repertoire of CD4+ T-cells specific for a defined antigen according to antigen-induced CD154 expression after short-term ex vivo stimulation. AdV-lysate-specific and AdV-hexon-specific CD4+ T-cells were isolated, expanded in vitro and further assessed for their fine specificities using recombinant AdV-proteins and AdV-lysates from various AdV-serogroups. Additionally, we evaluated the cytokine profile of AdV-lysate- and AdV-hexon-specific CD4+ T-cells. Results: AdV-lysate-specific CD4+ T-cells reacted efficiently with AdV-hexon and penton proteins. Furthermore, CD4+ T-cells specific for hexon protein derived from AdV-serogroup B crossreact with recombinant hexon protein derived from various other AdV-serotypes and are characterized by a Th1-like cytokine profile. Conclusion: Our results imply that adenovirus hexon protein is a suitable candidate antigen for the ex vivo generation of adenovirus-specific, serogroup cross-reactive proinflammatory CD4+ T-cells for adoptive T-cell therapies.

Background. A unit of the European Mobile Laboratory (EMLab) consortium was deployed to the Ebola virus disease (EVD) treatment unit in Guéckédou, Guinea, from March 2014 through March 2015. Methods. The unit diagnosed EVD and malaria, using the RealStar Filovirus Screen reverse transcriptionpolymerase chain reaction (RT-PCR) kit and a malaria rapid diagnostic test, respectively. Results. The cleaned EMLab database comprised 4719 samples from 2741 cases of suspected EVD from Guinea. EVD was diagnosed in 1231 of 2178 hospitalized patients (57%) and in 281 of 563 who died in the community (50%). Children aged 74 years (90%) and low in patients aged 1019 years (40%). On admission, RT-PCR analysis of blood specimens from patients who died in the hospital yielded a lower median cycle threshold (Ct) than analysis of blood specimens from survivors (18.1 vs 23.2). Individuals who died in the community had a median Ct of 21.5 for throat swabs. Multivariate logistic regression on 1047 data sets revealed that low Ct values, ages of

West Nile virus (WNV) is a mosquito-borne Flavivirus with birds as its natural reservoir. Mosquitoes are able to transmit WNV to humans. In Europe, WNV is most probably imported by migrating birds from WNV-endemic regions. In this study, the risk of WNV infection during bird-ringing activities in Germany and Austria was investigated. Several serological test systems were used to study WNV antibody prevalence among 137 bird ringers. Neutralising antibodies were detected in three of the bird ringers. This seropositivity could be explained by alternative factors such as traveling to endemic areas. The application of different serological methods showed that WNV IgG ELISAs were less specific than WNV IgG immunofluorescence tests (IFT). The neutralisation test (NT) appears to be the gold standard for Flavivirus differentiation. At present, we assume that bird-ringing activity in Germany and Austria harbours a very low risk of WNV infection.