Pathogen-reduced Ebola virus convalescent plasma: first steps towards standardization of manufacturing and quality control including assessment of Ebola-specific neutralizing antibodies.

BACKGROUND: Ebola virus disease is a public health emergency of international concern, and enormous efforts are being made in the development of vaccines and therapies. Ebola virus convalescent plasma is a promising anti-infective treatment of Ebola virus disease. Therefore, we developed and implemented a pathogen-reduced Ebola virus convalescent plasma concept in accordance with national, European and global regulatory framework. MATERIALS AND METHODS: Ebola virus convalescent plasma manufacture and distribution was managed by a collection centre, two medical centres and an expert group from the European Blood Alliance. Ebola virus convalescent plasma was collected twice with an interval of 61 days from a donor recovering from Ebola virus disease in Germany. After pathogen reduction, the plasma was analysed for Ebola virus-specific immunoglobulin G (IgG) antibodies and its Ebola virus neutralizing activity. RESULTS: Convalescent plasma could be collected without adverse events. Anti-Ebola virus IgG titres and Ebola-specific neutralizing antibodies in convalescent plasma were only slightly reduced after pathogen reduction treatment with S59 amotosalen/UVA. A patient in Italy with Ebola virus disease was treated with convalescent plasma without apparent adverse effects. DISCUSSION: As proof of principle, we describe a concept and practical implementation of pathogen-reduced Ebola virus convalescent plasma manufacture, quality control and its clinical application to an Ebola virus disease patient.

Specific detection by real-time reverse-transcription PCR assays of a novel avian influenza A(H7N9) strain associated with human spillover infections in China.

In response to a recent outbreak in China, detection assays for a novel avian influenza A(H7N9) virus need to be implemented in a large number of public health laboratories. Here we present real-time reverse-transcription polymerase chain reaction (RT-PCR) assays for specific detection of this virus, along with clinical validation data and biologically-safe positive controls.

Fatal cowpox virus infection in captive banded mongooses (Mungos mungo).

Cowpox virus infections have been described in various domestic and exotic animal species. This report is the first on an outbreak of fatal generalized cowpox virus infection among captive banded mongooses (Mungos mungo, suborder Feliformia). All animals of a colony of 8 mongooses showed a fulminant course of disease. The whole population died (n=7) or was euthanized (n=1) within 11 days. Postmortem examinations were performed on 4 animals. All animals showed extensive necrotizing inflammation of retropharyngeal lymph nodes, typical poxviral skin lesions, and multiple necrotic foci in liver and spleen. Three animals exhibited an ulcerating stomatitis. Pulmonary lesions, a common feature of fatal cowpox virus infections in other feliform species, were not obvious. Histopathologically, characteristic cytoplasmic inclusion bodies were detected in all affected organs but the spleen. Based on transmission electron microscopy and cell culture, Orthopoxvirus was identified as the etiology. The virus was further characterized by polymerase chain reaction and sequence analysis, identifying it as cowpox virus. A survey in the habitat suggests wild brown rats (Rattus norvegicus) as the most likely source of infection.

Detection of influenza A(H1N1)v virus by real-time RT-PCR.

Influenza A(H1N1)v virus was first identified in April 2009. A novel real-time RT-PCR for influenza A(H1N1)v virus was set up ad hoc and validated following industry-standard criteria. The lower limit of detection of the assay was 384 copies of viral RNA per ml of viral transport medium (95% confidence interval: 273-876 RNA copies/ml). Specificity was 100% as assessed on a panel of reference samples including seasonal human influenza A virus H1N1 and H3N2, highly pathogenic avian influenza A virus H5N1 and porcine influenza A virus H1N1, H1N2 and H3N2 samples. The real-time RT-PCR assay for the influenza A matrix gene recommended in 2007 by the World Health Organization was modified to work under the same reaction conditions as the influenza A(H1N1)v virus-specific test. Both assays were equally sensitive. Clinical applicability of both assays was demonstrated by screening of almost 2,000 suspected influenza (H1N1)v specimens, which included samples from the first cases of pandemic H1N1 influenza imported to Germany. Measuring influenza A(H1N1)v virus concentrations in 144 laboratory-confirmed samples yielded a median of 4.6 log RNA copies/ml. The new methodology proved its principle and might assist public health laboratories in the upcoming influenza pandemic.

Site directed mutagenesis of the carboxyl terminus of human cytomegalovirus glycoprotein B leads to attenuation of viral growth in cell culture.

A viable human cytomegalovirus (HCMV) mutant was generated harbouring a glycoprotein B (gB) in which the carboxyl-terminal amino acids DRLRHR (aa 885-900) were changed to AALREE. Characterization of the phenotype of the recombinant virus revealed significant reduction of infectious progeny release and only moderate reduction of viral DNA replication indicating its diminished specific infectivity. This observation was in line with immunogold labeling of extracellular virions demonstrating that the amount of gB protein was markedly reduced in the envelope of the mutant virus. Our results suggest that the conserved carboxyl-terminus of the gB molecule is critical for HCMV maturation.

Open reading frame III of borna disease virus encodes a nonglycosylated matrix protein.

The open reading frame III of Borna disease virus (BDV) codes for a protein with a mass of 16 kDa, named p16 or BDV-M. p16 was described as an N-glycosylated protein in several previous publications and therefore was termed gp18, although the amino acid sequence of p16 does not contain any regular consensus sequence for N glycosylation. We examined glycosylation of p16 and studied its membrane topology using antisera raised against peptides, which comprise the N and the C termini. Neither an N- nor a C-terminal peptide is cleaved from p16 during maturation. Neither deglycosylation of p16 by endoglycosidases nor binding of lectin to p16 was detectable. Introduction of typical N-glycosylation sites at the proposed sites of p16 failed in carbohydrate attachment. Flotation experiments with membranes of BDV-infected cells on density gradients revealed that p16 is not an integral membrane protein, since it can be dissociated from membranes. Our experimental data strongly suggest that p16 is a typical nonglycosylated matrix protein associated at the inner surface of the viral membrane, as is true for homologous proteins of other members of the Mononegavirales order.

Multimerization potential of the cytoplasmic domain of the human cytomegalovirus glycoprotein B.

In the present study the coding sequence of the cytoplasmic tail of the human cytomegalovirus glycoprotein B (gB) was expressed. The secondary structure of the purified recombinant protein was analyzed by circular dichroism, and the quaternary structure was investigated by gel permeation chromatography, and electron microscopy. Our data indicate that the cytoplasmic gB domain contains alpha-helix structures and assembles into tetramers, suggesting that the authentic gB may represent a homotetramer.

A mono phenylalanine-based motif (F790) and a leucine-dependent motif (LI760) mediate internalization of furin.

The eukaryotic endoprotease furin, a member of the subtilisin-related family of prohormone convertases, is synthesized and transported within the constitutive secretory pathway to the plasma membrane, from where it recycles to the trans-Golgi network (TGN). Previous studies showed that TGN-residence and recycling are mediated by the cytoplasmic tail. Two targeting determinants have been described so far, the acidic signal CPSDSEEDEG783 containing two casein kinase II (CKII) phosphorylation sites and the internalization signal YKGL765. Refined analyses of the cytoplasmic domain of furin, which was mutagenized and tagged to the influenza hemagglutinin and to the membrane cofactor protein (CD46) as reporter molecules reveal two additional internalization determinants, a leucine-isoleucine signal, LI760, and a mono phenylalanine-based motif at F790, which functions without any specific neighboring amino acid sequence. Both signals are capable of independently mediating internalization, as has been shown previously for the tyrosine-based signal. Thus, furin internalization is mediated by at least three independent endocytosis signals.

Effect of cysteine substitutions on dimerization and interfragment linkage of human cytomegalovirus glycoprotein B (gp UL55).

Experiments were carried out to analyze the function of cysteine residues at amino acid positions 506 (cI), 550 (cII), 573 (cIII), and 610 (cIV), in dimerization and/or disulfide linkage of human cytomegalovirus (HCMV) glycoprotein B (gB). Single c-codons or pairs were substituted in the gB sequence of constructs which were used for transfection and selection of stable transfectants. Analysis of gB expression products revealed that single substitutions of cIII or cIV, but neither single nor double substitutions of cI or/and cII prevented gB dimerization. All substituted gB derivatives were, however, no longer processed by proteolytic cleavage. After deletion of the membrane anchor domain, correct proteolytic processing was again observed for anchorless gB forms. Substitutions of cI or cI/cII in secretory gB appeared to interfere with disulfide linkage between gB cleavage fragments. In the case of anchorless gB with substitutions of cII, cIII, or cIII/cIV, however, extracellular gB forms were not recovered. Using the Sindbis expression system recovery of all anchorless gB forms with cysteine substitutions was achieved. Analysis verified involvement of cI/II substitutions in intrachain disulfide linkage between cleavage fragments of HCMV gB.

Nuclear translocation of mutagenized forms of human cytomegalovirus glycoprotein B (gpUL55).

To define structural elements involved in translocation of human cytomegalovirus (HCMV) glycoprotein B (gB) to the inner nuclear membrane (INM) compartment, mutagenized gB derivatives with deletions of the potential membrane anchor domains or of portions of the cytoplasmic tail were stably expressed in human astrocytoma cells. Subcellular localization examined by immunofluorescence and cell fractionation suggested that all gB derivatives reached the INM; however, reduced amounts were found after deletion of the extreme carboxy terminus [amino acids 856-906; gB(Del3)]. Pulse-chase analysis revealed accumulation in nuclear fractions of all gB derivatives during the chase, except for gB(Del3), which exhibited impaired nuclear retention. A carboxy-terminal nucleoplasmin-like signal localized within the respective deletion may thus be involved in nuclear transport and retention of HCMV gB. Immunoprecipitation after 32P-radiolabelling of the gB transfectants verified that the gB molecule is phosphorylated at a carboxy-terminal consensus motif for casein kinase II.

Retrieval of human cytomegalovirus glycoprotein B from the infected cell surface for virus envelopment.

Surface biotinylation of human cytomegalovirus (HCMV)-infected fibroblasts under pulse-chase conditions was used to define the cellular route of the dominant viral envelope glycoprotein gB into the cytoplasmic compartment of viral maturational envelopment. The results showed that a major fraction of gB was re-internalized from the infected cell surface prior to incorporation into the viral envelope. Viral particles carrying biotinylated gB were subsequently released into the culture medium. Viral release appeared to be inhibited in the presence of gB-specific antibody or when infected cultures were incubated at room temperature, but was not reduced by inhibitors of cellular glycoprotein transport. To our knowledge this is the first report describing that HCMV gB is retrieved from the infected cell surface prior to viral envelopment.