bluuebunny/crossref_metadata_2025 huggingface dataset:
Infection of a child in Hong Kong by an influenza A H3N2 virus closely related to viruses circulating in European pigs
Authors: Gregory V. , Lim W. , Cameron K. , Bennett M. , Marozin S. , Klimov A. , Hall H. , Cox N. , Hay A. , Lin Y. P.
Published: August 2015

Influenza virus A/Hong Kong/1774/99, isolated from a young child with mild influenza, was shown to be similar in its antigenic and genetic characteristics to H3N2 viruses circulating in pigs in Europe during the 1990s and in particular to be closely related to viruses isolated from two children in the Netherlands in 1993. Similar viruses had previously not been identified outside Europe. Although there is little evidence as to how the child contracted the infection, it appears likely that pigs in southern China were the source of infection. Characteristics shared with the European swine viruses include resistance to the anti-influenza drugs amantadine and rimantadine. Thus not only does this incident once again highlight the potential of pigs as a source of novel human influenza viruses, but also indicates the potential for emergence of amantadine-resistant human viruses.
Inhibitors of human cytomegalovirus replication drastically reduce the activity of the viral protein kinase pUL97
Authors: Marschall Manfred , Stein-Gerlach Matthias , Freitag Martina , Kupfer Regina , van den Bogaard Miriam , Stamminger Thomas
Published: August 2015

The UL97-encoded protein kinase (pUL97) of human cytomegalovirus (HCMV) plays a critical role in the control of virus replication. Deletion of the UL97 gene results in a drastic reduction in the replication efficiency. Although the exact function of pUL97 remains unclear and its sensitivity to specific inhibitors is speculative, protein kinase inhibitors of the indolocarbazole class are effective inhibitors of cytomegalovirus. Based on the phosphorylation of ganciclovir (GCV), a novel quantification system for pUL97 kinase activity was established: the phosphorylated form of GCV exerts an easily quantifiable cytotoxic effect in transfected cells. Importantly, the addition of indolocarbazole compounds, Gö6976 and NGIC-I, which were highly effective at nanomolar concentrations while other protein kinase inhibitors were not, led to a significant reduction of pUL97 kinase activity. It was also demonstrated that a catalytically inactive mutant of pUL97, K355M, and a GCV-resistant mutant, M460I, were both negative for GCV phosphorylation, although protein phosphorylation remained detectable for the latter mutant.In vitrokinase assays were used to confirm the levels of pUL97-mediated phosphorylation recorded. To generate a tool for screening large numbers of putative inhibitors that preferentially interfere with GCV as well as protein phosphorylation, pUL97-expressing cell clones with stable pUL97 kinase activity were selected. This study demonstrates that certain indolocarbazole compounds are potent pUL97 inhibitors and, therefore, represent novel candidates for antiviral drugs that target viral protein kinase functions.
Defining CAR as a cellular receptor for the avian adenovirus CELO using a genetic analysis of the two viral fibre proteins
Authors: Tan Poi Kiang , Michou Anne-Isabelle , Bergelson Jeffrey M. , Cotten Matt
Published: August 2015

The coxsackievirus and adenovirus receptor (CAR) is a high affinity receptor used by adenoviruses, including adenovirus type 5 (Ad5). The adenovirus fibre molecule bears the high affinity cell binding domain of Ad5, allowing virions to attach to CAR. The avian adenovirus CELO displays two fibre molecules on its capsid and it was logical to expect that the cell binding functions of CELO might also reside in one or both of these fibres. We had previously shown that the cell binding properties of CELO resemble Ad5, suggesting that the two viruses use similar receptors. Experiments with CAR-deficient CHO cells and CHO cells modified to express CAR demonstrated that CELO has CAR-dependent transduction behaviour like Ad5. Mutations were introduced into the CELO genome to disrupt either the long fibre 1 or the short fibre 2. A CELO genome with fibre 2 disrupted did not generate virus, demonstrating that fibre 2 is essential for some stage in virus growth, assembly or spread. However, a CELO genome with disrupted fibre 1 gene produced virus (CELOdF1) that was capable of entering chicken cells, but had lost both the ability to efficiently transduce human cells and the CAR-specific transduction displayed by wild-type CELO. The ability of CELOdF1 to transduce chicken cells suggests that CELOdF1 may still bind, probably via fibre 2, to a receptor expressed on avian but not mammalian cells. CELOdF1 replication was dramatically impaired in chicken embryos, demonstrating that fibre 1 is important for the in vivo biology of CELO.
Use of a vector based on Potato virus X in a whole plant assay to demonstrate nuclear targeting of Potato spindle tuber viroid
Authors: Zhao Yan , Owens Robert A. , Hammond Rosemarie W.
Published: August 2015

Potato spindle tuber viroid (PSTVd) is a covalently closed circular RNA molecule of 359 nucleotides that replicates within the nucleus of host cells. To determine how this small, highly structured RNA enters the nucleus, we have developed a virus-based, whole plant in vivo assay that uses green fluorescent protein (GFP) as the reporter molecule. The coding region of GFP was interrupted by insertion of an intron derived from the intervening sequence 2 of the potato ST-LS1 gene. A cDNA copy of the complete PSTVd genome was, in turn, embedded within the intron, and this construct was delivered into Nicotiana benthamiana plants via a vector based on Potato virus X. The intron-containing GFP subgenomic RNA synthesized during virus infection cannot produce a functional GFP unless the RNA is imported into the nucleus, where the intron can be removed and the spliced RNA returned to the cytoplasm. The appearance of green fluorescence in leaf tissues inoculated with constructs containing a full-length PSTVd molecule embedded in the intron indicates that nuclear import and RNA splicing events did occur.
A comparison of the solution structures of tobacco rattle and tobacco mosaic viruses from Raman optical activity
Authors: Blanch Ewan W. , Robinson David J. , Hecht Lutz , Barron Laurence D.
Published: August 2015

Vibrational Raman optical activity (ROA) spectra of tobacco rattle virus (TRV) and tobacco mosaic virus (TMV) were measured and compared with a view to obtaining new information about the coat protein subunit structure of TRV. A sharp strong positive band observed at ∼1344 cm−1 in the ROA spectra of the two viruses is evidence that both contain a significant amount of a hydrated form of α-helix, but more in TRV than in TMV. Although the ROA spectrum of TMV shows significant positive intensity in the range ∼1297–1312 cm−1 characteristic of α-helix in a hydrophobic environment, as expected from the helix interface residues in the four-helix bundles that constitute the basic motif of the TMV coat protein fold, that of TRV shows little positive ROA intensity here. Instead TRV shows a strong positive ROA band at ∼1315 cm−1, of much greater intensity than bands shown here by TMV, that is characteristic of polyproline II (PPII) helix. This suggests that the additional long central and C-terminal sequences of the TRV coat proteins contain a significant amount of PPII structure, plus perhaps some β-strand judging by a prominent sharp negative ROA band shown by TRV at ∼1236 cm−1, but little α-helix. The open flexible hydrated nature of PPII helical structure is consistent with the earlier suggestions that the additional sequences are exposed and, together with a larger amount of hydrated α-helix, could serve to fill the extra volume required by the larger diameter of the cylindrical TRV particles relative to those of TMV.
Epstein–Barr virus nuclear antigen 5 interacts with HAX-1, a possible component of the B-cell receptor signalling pathway
Authors: Dufva Martin , Olsson Maria , Rymo Lars
Published: August 2015

Using a yeast two-hybrid screen of a B-cell cDNA library with an Epstein–Barr nuclear antigen 5 (EBNA5) molecule containing seven repeats of the W1W2domain as bait, we have isolated the EBNA5-interacting protein HAX-1. HAX-1 has previously been shown to associate with HS1, a protein specifically expressed in cells of the haematopoietic lineage, and is thought to be involved in signal transduction in B-cells. Immunofluorescence experiments showed that HAX-1 co-localized with the hsp60 protein that is associated with the mitochondria in the cell cytoplasm. Pull down experiments with a fusion protein between glutathioneS-transferase and the seven copy repeat EBNA5 synthesized in bacteria and in yeast cells confirmed that HAX-1 can interact with EBNA5in vitro. Conventionally, EBNA5 is regarded as a nuclear protein. However, we show here that the smallest EBNA5 species, composed of the unique Y domain and only one copy of the W1W2repeat domain, like HAX-1, co-localizes with the mitochondrial hsp60 protein in the B-cell cytoplasm. Furthermore, immunoprecipitation experiments demonstrate that the single repeat EBNA5 associates with HAX-1 in transfected B-lymphoblastoid cells.
Feline leukaemia provirus load during the course of experimental infection and in naturally infected cats
Authors: Hofmann-Lehmann Regina , Huder Jon B. , Gruber Sabine , Boretti Felicitas , Sigrist Brigitte , Lutz Hans
Published: August 2015

Feline leukaemia virus (FeLV) infection in domestic cats can vary in its outcome (persistent, transient, no infection) for reasons that are not entirely known. It was hypothesized that the initial virus and provirus load could significantly influence the course of retrovirus infection. To determine the role of provirus loads, two methods of PCR, a nested PCR and a fluorogenic probe-based (TaqMan) real-time quantitative PCR, which were specific to the U3 region of FeLV-A were established. FeLV provirus in naturally and experimentally infected cats was then measured. Only 3 weeks after experimental FeLV-A infection, persistently infected cats demonstrated higher provirus loads and lower humoral immune responses than cats that had overcome antigenaemia. Lower initial provirus loads were associated with successful humoral immune responses. Unexpectedly, provirus in the buffy-coat cells of two cats that tested negative for the p27 antigen (a marker for viraemia) was also detected. In 597 Swiss cats, comparison of p27 antigen levels with PCR results revealed broad agreement. However, similar to the experimental situation, a significant number of animals (10%) was negative for the p27 antigen and FeLV-positive by PCR. These cats had a mean provirus load 300-fold lower than that of animals testing positive for the p27 antigen. In conclusion, an association between the provirus load and the outcome of FeLV infection was found. Detection of provirus carriers should contribute to further the control of FeLV. In addition, quantification of provirus loads will lead to a better understanding of FeLV pathogenesis and anti-retrovirus protective mechanisms.
The envelope of Mason–Pfizer monkey virus has immunosuppressive properties
Authors: Blaise Sandra , Mangeney Marianne , Heidmann Thierry
Published: August 2015

We have demonstrated previously that the envelope protein of a murine retrovirus, Moloney murine leukaemia virus, has immunosuppressive propertiesin vivo. This property was manifested by the ability of the protein, when expressed by tumour cells normally rejected by engrafted mice, to allow theenv-expressing cells to escape immune rejection and to proliferate. Here, it is shown that this property is not restricted to the envelope of a murine retrovirus, but is also shared by the envelope encoded by a primate retrovirus, Mason–Pfizer monkey virus.
Phylogenetic reconstruction of intrapatient evolution of human immunodeficiency virus type 1: predominance of drift and purifying selection
Authors: Kils-Hütten Laurens , Cheynier Rémi , Wain-Hobson Simon , Meyerhans Andreas
Published: August 2015

The intra-host evolution of 73 human immunodeficiency virus type 1 quasispecies was analysed by split decomposition analysis. Non-synonymous and synonymous nucleotide substitutions were counted along the shortest path connecting all sequences and compared with the numbers expected under the assumption of a random model of mutation. For the majority of substitutions, drift and negative selection seemed to prevail.
Mutagenesis of the dengue virus type 2 NS3 proteinase and the production of growth-restricted virus
Authors: Matusan Anita E. , Kelley Peter G. , Pryor Melinda J. , Whisstock James C. , Davidson Andrew D. , Wright Peter J.
Published: August 2015

The N-terminal one-third of the NS3 protein of Dengue virus type 2 (DEN-2) complexes with co-factor NS2B to form an active serine proteinase which cleaves the viral polyprotein. To identify sites within NS3 that may interact with NS2B, seven regions within the NS3 proteinase outside the conserved flavivirus enzyme motifs were mutated by alanine replacement. Five sites contained clusters of charged residues and were hydrophilic. Two sites were hydrophobic and highly conserved among flaviviruses. The effects of five mutations on NS2B/3 processing were examined using a COS cell expression system. Four retained significant proteinase activity. Three of these mutations and two more were introduced into genomic-length cDNA and tested for their effects on virus replication. The five mutant viruses showed reduced plaque size and two of the five showed significantly reduced titres. All seven mutations were mapped on the X-ray crystal structure of the DEN-2 NS3 proteinase: three were located at the N terminus and two at the C terminus of the NS2B-binding cleft. Two mutations were at the C terminus of the proteinase domain and one was solvent-exposed. The study demonstrated that charged-to-alanine mutagenesis in the viral proteinase can be used to produce growth-restricted flaviviruses that may be useful in the production of attenuated vaccine strains.
Induction of a protective response in swine vaccinated with DNA encoding foot-and-mouth disease virus empty capsid proteins and the 3D RNA polymerase
Authors: Cedillo-Barrón Leticia , Foster-Cuevas Mildred , Belsham Graham J. , Lefèvre François , Parkhouse R. Michael E.
Published: August 2015

This work focuses on the development of a potential recombinant DNA vaccine against foot-and-mouth disease virus (FMDV). Such a vaccine would have significant advantages over the conventional inactivated virus vaccine, in particular having none of the risks associated with the high security requirements for working with live virus. The principal aim of this strategy was to stimulate an antibody response to native, neutralizing epitopes of empty FMDV capsids generated in vivo. Thus, a plasmid (pcDNA3.1/P1–2A3C3D) was constructed containing FMDV cDNA sequences encoding the viral structural protein precursor P1–2A and the non-structural proteins 3C and 3D. The 3C protein was included to ensure cleavage of the P1–2A precursor to VP0, VP1 and VP3, the components of self-assembling empty capsids. The non-structural protein 3D was also included in the construct in order to provide additional stimulation of CD4+ T cells. When swine were immunized with this plasmid, antibodies to FMDV and the 3D polymerase were synthesized. Furthermore, neutralizing antibodies were detected and, after three sequential vaccinations with DNA, some of the animals were protected against challenge with live virus. Additional experiments suggested that the antibody response to FMDV proteins was improved by the co-administration of a plasmid encoding porcine granulocyte–macrophage colony-stimulating factor. Although still not as effective as the conventional virus vaccine, the results encourage further work towards the development of a DNA vaccine against FMDV.
Characterization of a new H-2Dk-restricted epitope prominent in primary influenza A virus infection
Authors: Tourdot Sophie , Herath Shan , Gould Keith G.
Published: August 2015

Influenza A virus infection of mice has been used extensively as a model to investigate the mechanisms of antigen presentation to cytotoxic T lymphocytes (CTL) and the phenomenon of immunodominance in antiviral CTL responses. The different virus-encoded epitopes that are recognized in H-2band H-2dmice have been characterized and their relative immunodominance has been well-studied. In H-2kmice, four different Kk-restricted influenza virus epitopes have been described, but the dominance hierarchy of these epitopes is unknown and there is also an uncharacterized Dk-restricted response against the virus. In this study, a Dk-restricted epitope derived from the influenza virus A/PR/8/34 polymerase protein PB1, corresponding to amino acid residues 349–357 (ARLGKGYMF), was identified. This peptide is the major epitope within the PB1 polymerase and is at least as dominant as any of the four Kk-restricted epitopes that are recognized in CBA mice following primary influenza virus infection. The PB1 epitope is only the fourth Dk-presented peptide to be reported and the sequence of this epitope confirms a Dk-restricted peptide motif, consisting of arginine at position two, arginine or lysine at position five and a hydrophobic residue at the carboxy terminus.
Expression, purification and characterization of the Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) DNA polymerase and interaction with the SpliNPV non-hr origin of DNA replication
Authors: Huang Jianhe , Levin David B.
Published: August 2015

The DNA polymerase from Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) was expressed in, and purified from, prokaryotic and eukaryotic expression systems. While less protein was obtained from the E. coli expression system, SpliNPV DNAPOL purified from E. coli displayed similar biochemical characteristics to DNAPOL expressed in, and subsequently purified from, insect cells (Sf9) using a baculovirus expression system. Biochemical analyses suggested that the DNA polymerase and the 3′–5′ exonuclease activities are intrinsic to the protein. Deletion of the first 80 amino acid residues from the N terminus of the DNAPOL affected neither the DNA polymerase nor the exonuclease activities of the enzyme. Replication products from single-stranded M13 DNA demonstrated that the DNA synthesis activity of SpliNPV DNAPOL is highly processive. Transient expression assays with a set of deletion clones containing the putative SpliNPV non-hr origin of DNA replication permitted functional characterization of sequence elements within the origin fragment. Purified SpliNPV DNAPOL stimulated origin-dependent DNA replication in a cell-free replication assay.
Comparative pathogenesis of Helicoverpa zea S nucleopolyhedrovirus in noctuid larvae
Authors: Washburn Jan O. , Wong James F. , Volkman Loy E.
Published: August 2015

We used a recombinant of Helicoverpa zea S nucleopolyhedrovirus containing the hsp70/lacZ reporter cassette (HzSNPV-hsp70/lacZ) to quantify mortality relationships and to elucidate early pathogenesis in two permissive hosts, Heliothis virescens and Helicoverpa zea, and one semi-permissive host, Trichoplusia ni. Fourth instar T. ni were highly resistant to fatal infection both by oral injection of occlusions and by intrahaemocoelic injection of budded virus, indicating the presence of both midgut and systemic mechanisms of resistance. In bioassays, newly moulted (40) H. zea were significantly more susceptible than 40
H. virescens to fatal infection, but mortality levels were the same for larval cohorts inoculated 16 h after the moult (416). Developmental resistance was stronger in H. zea and in both hosts, partially reversed by administration of the optical brightener M2R. In both species, developmental resistance was correlated with a reduced ability of HzSNPV to establish and/or maintain primary midgut infections. In time-course experiments using a dosage of 15 occlusions (∼LD90), lacZ expression marking the onset of primary and secondary infection was first observed in midgut columnar and tracheal cells at 4 and 12 h, respectively. Inoculation of 40 larvae resulted in approximately twofold more foci in H. zea larvae than in H. virescens, but H. zea larvae sloughed infected midgut cells at a faster rate. For both heliothines, interaction of occlusion-derived virus with primary cellular targets within the midgut epithelium was critical to the outcome of infection and a key process underlying acquisition of developmental resistance.
Genomic organization of RNA2 of Tomato ringspot virus: processing at a third cleavage site in the N-terminal region of the polyprotein in vitro
Authors: Carrier Karma , Xiang Yu , Sanfaçon Hélène
Published: August 2015

The proteinase ofTomato ringspot virus(genusNepovirus) is responsible for proteolytic cleavage of the RNA2-encoded polyprotein (P2) at two cleavage sites, allowing definition of the domains for the movement protein (MP) and coat protein. In this study, we have characterized a third cleavage site in the N-terminal region of P2 using anin vitroprocessing assay and partial cDNA clones. Results from site-directed mutagenesis of putative cleavage sites suggest that cleavage occurs at dipeptide Q301/G. Cleavage at this site is predicted to result in the release of two proteins from the N-terminal region of P2: a 34 kDa protein located at the N terminus of P2 (assuming translation initiation at the first AUG codon) and a 71 kDa protein located immediately upstream of the MP domain. In contrast, only one protein domain is present in the equivalent region of the P2 polyprotein of other characterized nepoviruses.
Quasispecies nature of the genome of Grapevine fanleaf virus
Authors: Naraghi-Arani Pejman , Daubert Steve , Rowhani Adib
Published: August 2015

Genetic diversity was characterized in 14 isolates ofGrapevine fanleaf virus(GFLV) recovered from grapevine (Vitis vinifera). Virions were collected by immunocapture, and a 1557 bp fragment containing part of the viral coat protein gene and part of the untranslated region to its 3′ side was amplified by RT–PCR. Sequence variation among isolates was characterized by restriction fragment length polymorphism (RFLP) analysis and by sequencing. TheAvaII-generated RFLP patterns from the various isolates were highly variable. The isolates were passaged inChenopodium quinoa. The RFLP patterns altered with passage through the alternate host, but the variation stabilized after a number of passages. Individual genomes were recovered by cloning. The subcloned sequences were found to vary from each other by as much as 13%, and the encoded amino acid sequences by as much as 9%. The data suggest that the GFLV genome consists of quasispecies populations.
Identification of a novel type C porcine endogenous retrovirus: evidence that copy number of endogenous retroviruses increases during host inbreeding
Authors: Mang Rui , Maas Jolanda , Chen Xianghong , Goudsmit Jaap , van der Kuyl Antoinette C.
Published: August 2015

Different classes of porcine endogenous retroviruses (PERVs), which have the potential to infect humans during xenotransplantation, have been isolated from the pig genome. Because vertebrate genomes may contain numerous endogenous retrovirus sequences, the pig genome was examined for additional endogenous retroviruses, resulting in the isolation of a novel, complete endogenous retrovirus genome, designated PERV-E. The gag, pol and env genes of PERV-E are closely related to those of human endogenous retrovirus (HERV) 4-1, which belongs to the HERV-E family. Results of studies to determine the presence and copy number of PERVs demonstrated that PERV-E and PERV-A/B-like proviruses were present in all genomes tested, but that PERV-C was not found in two of the species examined, including wild boar. Multiple copies of PERVs could be found in each pig genome. Among all of the pig genomes tested, the wild boar genome had the lowest copy number of all PERVs, suggesting that the number of integrations of complete endogenous retroviruses is increased by inbreeding.
The haemagglutinin protein is an important determinant of measles virus tropism for dendritic cells in vitro
Authors: Ohgimoto Shinji , Ohgimoto Kaori , Niewiesk Stefan , Klagge Ingo M. , Pfeuffer Joanna , Johnston Ian C. D. , Schneider-Schaulies Jürgen , Weidmann Armin , ter Meulen Volker , Schneider-Schaulies Sibylle
Published: August 2015

Recombinant measles viruses (MV) in which the authentic glycoprotein genes encoding the fusion and the haemagglutinin (H) proteins of the Edmonston (ED) vaccine strains were swapped singly or doubly for the corresponding genes of a lymphotropic MV wild-type virus (strain WTF) were used previously to investigate MV tropism in cell lines in tissue culture. When these recombinants and their parental strains, the molecular ED-based clone (ED-tag) and WTF, were used to infect cotton rats, only viruses expressing the MV WTF H protein replicated in secondary lymphatic tissues and caused significant immunosuppression.In vitro, viruses containing the ED H protein revealed a tropism for human peripheral blood lymphocytes as documented by enhanced binding and virus production, whereas those containing the WTF H protein replicated well in monocyte-derived dendritic cells (Mo-DC). This did not correlate with more efficient binding of these viruses to DC, but with an enhancement of uptake, virus spread, accumulation of viral antigens and virus production. Thus, replacement of the ED H protein with WTF H protein was sufficient to confer the DC tropism of WTF to ED-tagin vitro. This study suggests that the MV H protein plays an important role in determining cell tropism to immune cells and this may play an important role in the induction of immunosuppressionin vivo.
Comparisons among the larger genome segments of six nodaviruses and their encoded RNA replicases
Authors: Johnson Karyn N. , Johnson Kyle L. , Dasgupta Ranjit , Gratsch Theresa , Ball L. Andrew
Published: August 2015

The Nodaviridae are a family of isometric RNA viruses that infect insects and fish. Their genomes, which are among the smallest known for animal viruses, consist of two co-encapsidated positive-sense RNA segments: RNA1 encodes the viral contribution to the RNA-dependent RNA polymerase (RdRp) which replicates the viral genome, whereas RNA2 encodes the capsid protein precursor. In this study, the RNA1 sequences of two insect nodaviruses – Nodamura virus (the prototype of the genus) and Boolarra virus – are reported as well as detailed comparisons of their encoded RdRps with those of three other nodaviruses of insects and one of fish. Although the 5′ and 3′ untranslated regions did not reveal common features of RNA sequence or secondary structure, these divergent viruses showed similar genome organizations and encoded RdRps that had from 26 to 99% amino acid sequence identity. All six RdRp amino acid sequences contained canonical RNA polymerase motifs in their C-terminal halves and conserved elements of predicted secondary structure throughout. A search for structural homologues in the protein structure database identified the poliovirus RdRp, 3Dpol, as the best template for homology modelling of the RNA polymerase domain of Pariacoto virus and allowed the construction of a congruent three-dimensional model. These results extend our understanding of the relationships among the RNA1 segments of nodaviruses and the predicted structures of their encoded RdRps.
Mapping of a dengue virus neutralizing epitope critical for the infectivity of all serotypes: insight into the neutralization mechanism
Authors: Thullier Philippe , Demangel Caroline , Bedouelle Hugues , Mégret Françoise , Jouan Alain , Deubel Vincent , Mazié Jean-Claude , Lafaye Pierre
Published: August 2015

Dengue virus infections are a growing public health concern and strategies to control the spread of the virus are urgently needed. The murine monoclonal antibody 4E11 might be of interest, since it neutralizes dengue viruses of all serotypes by binding to the 296–400 segment of the major dengue virus envelope glycoprotein (DE). When phage-displayed peptide libraries were screened by affinity for 4E11, phage clone C1 was selected with a 50% frequency. C1 shared three of nine residues with DE306–314and showed significant reactivity to 4E11 in ELISA. C1-induced antibodies cross-reacted with DE296–400in mice, suggesting that it was a structural equivalent of the native epitope of 4E11 on DE. Accordingly, 4E11 bound to the DE306–314synthetic peptide and this reaction was inhibited by DE296–400. Moreover, DE306–314could block dengue virus infection of target cells in anin vitroassay. A three-dimensional model of DE revealed that the three amino acids shared by DE296–400and C1 were exposed to the solvent and suggested that most of the amino acids comprising the 4E11 epitope were located in the DE306–314region. Since 4E11 blocked the binding of DE296–400to heparin, which is a highly sulfated heparan sulfate (HSHS) molecule, 4E11 may act by neutralizing the interaction of DE306–314with target cell-displayed HSHS. Our data suggest that the DE306–314segment is critical for the infectivity of all dengue virus serotypes and that molecules that block the binding of DE306–314to HSHS may be antiviral reagents of therapeutic interest.
Complete nucleotide sequence and genome organization of Grapevine fleck virus
Authors: Sabanadzovic Sead , Ghanem-Sabanadzovic Nina Abou , Saldarelli Pasquale , Martelli Giovanni P.
Published: August 2015

The complete nucleotide sequence of Grapevine fleck virus (GFkV) genomic RNA was determined. The genome is 7564 nt in size, excluding the 3′-terminal poly(A) tail, is characterized by an extremely high cytosine content (ca. 50%), and contains four putative open reading frames and untranslated regions of 291 and 35 nt at the 5′ and 3′ ends, respectively. ORF 1 potentially encodes a 215·4 kDa polypeptide (p215), which has the conserved motifs of replication-associated proteins of positive-strand RNA viruses. ORF 2 encodes a 24·3 kDa polypeptide (p24) identified as the coat protein. ORFs 3 and 4 are located at the extreme 3′ end of the viral genome and encode proline-rich proteins of 31·4 kDa (p31) and 15·9 kDa (p16), respectively, of unknown function. Phylogenetic analysis of the viral replicase and coat protein genes showed that GFkV is related to members of the Tymovirus and Marafivirus genera. Two subgenomic RNAs were present in the GFkV preparations as ascertained by molecular hybridization. The genome organization of GFkV resembles to some extent that of tymoviruses and marafiviruses. However, differences in the biological and epidemiological behaviour, cytopathology and molecular properties (i.e. size of genomic RNA and coat protein, and number of ORFs) support the notion that GFkV is a separate virus belonging in a new genus.
Vaccinia virus semaphorin A39R is a 50–55 kDa secreted glycoprotein that affects the outcome of infection in a murine intradermal model
Authors: Gardner Jeremy D. , Tscharke David C. , Reading Patrick C. , Smith Geoffrey L.
Published: August 2015

Vaccinia virus (VV) protein A39R has amino acid similarity to the extracellular domain of a glycosylphosphatidylinositol-linked cell surface semaphorin (SEMA7A/CDw108) that has an immunological expression profile and binding properties, thereby implicating A39R as an immunomodulator. Previously, a closely related A39R protein expressed by ectromelia virus was shown to induce cytokine production and up-regulate ICAM-1 expression in mouse monocytesin vitro. In this study, we show that theA39Rgene of VV strain Copenhagen (COP) encodes a 50–55 kDa secreted glycoprotein and is expressed late during infection. The A39R protein was secreted by eight of 15 strains of VV, but not by strain Western Reserve (WR). To analyse the VV A39R function, several recombinant viruses were made, including anA39Rdeletion mutant of VV COP and a WR mutant containing theA39Rsequence from COP. Loss of the gene from COP did not affect virus growthin vitro, or VV virulence in a mouse intranasal model, and had only a slight effect on lesion size in an intradermal model. In contrast, expression of COPA39Rby VV WR was associated with an increase in the severity and persistence of skin lesions after intradermal infection of mice. Finally, a histological examination of mouse skin infected with recombinant viruses suggested that A39R has direct or indirect pro-inflammatory properties.
Differential permissivity to measles virus infection of human and CD46-transgenic murine lymphocytes
Authors: Evlashev Alexey , Valentin Hélène , Rivailler Pierre , Azocar Olga , Rabourdin-Combe Chantal , Horvat Branka
Published: August 2015

Analysis of measles virus (MV) pathogenesis requires the development of an adequate small animal model of MV infection. In this study, permissivity to MV infection was compared in human and transgenic murine T lymphocytes, expressing different levels of the human MV receptor, CD46. Whereas MV binding and entry correlated with CD46 expression, higher levels of MV replication were always observed in human T lymphocytes. This suggests the existence of intracellular factors, acting posterior to virus entry, that could limit MV replication in murine lymphocytes and should be considered when creating new animal models of MV infection.
Complete nucleotide sequences of Nipah virus isolates from Malaysia
Authors: Chan Y. P. , Chua K. B. , Koh C. L. , Lim M. E. , Lam S. K.
Published: August 2015

We have completely sequenced the genomes of two Nipah virus (NiV) isolates, one from the throat secretion and the other from the cerebrospinal fluid (CSF) of the sole surviving encephalitic patient with positive CSF virus isolation in Malaysia. The two genomes have 18246 nucleotides each and differ by only 4 nucleotides. The NiV genome is 12 nucleotides longer than the Hendra virus (HeV) genome and both genomes have identical leader and trailer sequence lengths and hexamer-phasing positions for all their genes. Both NiV and HeV are also very closely related with respect to their genomic end sequences, gene start and stop signals, P gene-editing signals and deduced amino acid sequences of nucleocapsid protein, phosphoprotein, matrix protein, fusion protein, glycoprotein and RNA polymerase. The existing evidence demonstrates a clear need for the creation of a new genus within the subfamily Paramyxovirinae to accommodate the close similarities between NiV and HeV and their significant differences from other members of the subfamily.
Antigenic variants with amino acid deletions clarify a neutralizing epitope specific for influenza B virus Victoria group strains
Authors: Nakagawa Naoko , Kubota Ritsuko , Nakagawa Toshimasa , Okuno Yoshinobu
Published: August 2015

To study the neutralizing epitopes of influenza B virus Victoria group strains, two monoclonal antibodies (MAbs) were used to select antigenic variants of the virus. MAbs 10B8 and 8E6 were found to react with B/Victoria group strains in three tests, peroxidase–antiperoxidase staining, haemagglutination inhibition and neutralization tests; no reactivity with B/Yamagata group strains was observed. Analysis of the deduced amino acid sequences of 10B8-induced variants identified a single amino acid deletion at residue 165 or 170, as well as a single amino acid substitution at residues 164 (Asp→Tyr), 165 (Asn→Ser or Thr) or 203 (Lys→Thr or Asn). A single amino acid substitution at residue 241 (Pro→Ser) was observed in 8E6-induced variants. Three-dimensional analysis showed that the epitopes for both MAbs were situated in close proximity to each other. Since B/Yamagata group strains are characterized by amino acid deletions at residues 164–166, the epitope for MAb 10B8 is strictly specific for B/Victoria group strains.
Evidence of Borna disease virus genome detection in French domestic animals and in foxes (Vulpes vulpes)
Authors: Dauphin G. , Legay V. , Sailleau C. , Smondack S. , Hammoumi S. , Zientara S.
Published: August 2015

Borna disease virus (BDV) is an enveloped, non-segmented negative-stranded RNA virus which belongs to the Bornaviridae family. BDV is an aetiological agent of encephalitis in horses, sheep and several other vertebrate species. In order to extend our knowledge about the presence of BDV in France, a study based on BDV RNA detection by RT–nested-PCR was done with 196 animal tissues: 171 brain samples collected from different animal species (75 horses, 59 foxes, 31 cattle, 4 dogs, 1 sheep, 1 roe deer) and 25 horse blood samples. An RNA internal standard molecule was constructed and was co-amplified with the test template. This study reports the first detection of BDV RNA in France in 10 brain samples collected from horses, foxes and cattle, and from 14 horse blood samples. Detection of the BDV genome in the brains of six red foxes is the first evidence of BDV infection in this species.
Induction of simian immunodeficiency virus (SIV)-specific CTL in rhesus macaques by vaccination with modified vaccinia virus Ankara expressing SIV transgenes: influence of pre-existing anti-vector immunity
Authors: Sharpe Sally , Polyanskaya Natasha , Dennis Mike , Sutter Gerd , Hanke Tomáš , Erfle Volker , Hirsch Vanessa , Cranage Martin
Published: August 2015

A major aim in AIDS vaccine development is the definition of strategies to stimulate strong and durable cytotoxic T lymphocyte (CTL) responses. Here we report that simian immunodeficiency virus (SIV)-specific CTL developed in 4/4 macaques following a single intramuscular injection of modified vaccinia virus Ankara (MVA) constructs expressing both structural and regulatory/accessory genes of SIV. In two animals Nef-specific responses persisted, but other responses diminished and new responses were not revealed, following further vaccination. Vaccination of another two macaques, expressing Mamu A*01 MHC class I, with MVA constructs containingnefandgag–polunder the control of the moderate strength natural vaccinia virus early/late promoter P7.5, again induced an early Nef-specific response, whereas responses to Gag remained undetectable. Anti-vector immunity induced by this immunization was shown to prevent the efficient stimulation of CTL directed to the cognate Gag epitope, p11C C-M, following vaccination with another MVA construct expressing SIV Gag–Pol under a strong synthetic vaccinia virus-specific promoter. In contrast, vaccination of a previously unexposed animal resulted in a SIV-specific CTL response widely disseminated in lymphoid tissues including lymph nodes associated with the rectal and genital routes of SIV entry. Thus, despite the highly attenuated nature of MVA, repeated immunization may elicit sufficient anti-vector immunity to limit the effectiveness of later vaccination.
The novel picornavirus Equine rhinitis B virus contains a strong type II internal ribosomal entry site which functions similarly to that of Encephalomyocarditis virus
Authors: Hinton Tracey M. , Crabb Brendan S.
Published: August 2015

Equine rhinitis B virus (ERBV) has recently been classified as an Erbovirus, a new genus in the Picornaviridae family. ERBV is distantly related to members of the Cardiovirus and Aphthovirus genera which utilize a type II internal ribosome entry sequence (IRES) to initiate translation. We show that ERBV also possesses the core stem–loop structures (H–L) of a type II IRES. The function of the ERBV IRES was characterized using bicistronic plasmids that were analysed both by transfection into BHK-21 cells and by in vitro transcription and translation in rabbit reticulocyte lysates. In both systems, a region encompassed by nucleotides (nt) 189–920 downstream of the poly(C) tract was required for maximal translation. This sequence includes stem–loops H–L as well as four additional upstream stem–loops. Nt 904 corresponds to the second of three in-frame AUG codons located immediately downstream of the polypyrimidine tract (nucleotides 869–880). Site-directed mutagenesis demonstrated that AUG2 is the major initiation codon despite the appropriate positioning of AUG1 16 nt downstream of the polypyrimidine tract. In direct IRES competition experiments, the ERBV IRES was able to compete strongly for translation factors with the IRES of Encephalomyocarditis virus (EMCV). This was true when the assays were performed in vitro (with the IRESs competing either in cis or trans) and in vivo (with the IRESs competing in cis). A comparative analysis of the strength of several IRESs revealed that the ERBV IRES, like that of the EMCV, is a powerful inducer of translation and may have similar potential for use in mammalian expression systems.
Distribution of rotavirus-specific memory B cells in gut-associated lymphoid tissue after primary immunization
Authors: Moser Charlotte A. , Offit Paul A.
Published: August 2015

We found previously that mice inoculated orally with simian rotavirus strain RRV developed virus-specific memory B cell responses 16 weeks after immunization that were greater than those found 6 weeks after immunization. Memory B cell responses were defined as the quantity of virus-specific IgA detected in small intestinal lamina propria (LP) fragment cultures of immunized mice at various intervals after challenge. Enhanced memory B cell responses correlated with enhanced protection against shedding. In order to understand better the delayed onset of rotavirus-specific memory B cell responses, a method was developed to determine the frequencies of rotavirus-specific memory B cells in gut-associated lymphoid tissues (GALT). We found that protection against rotavirus challenge was determined by the frequency of rotavirus-specific memory B cells in GALT LP.
Frequency-dependent selection in human immunodeficiency virus type 1
Authors: Yuste Eloisa , Moya Andrés , López-Galíndez Cecilio
Published: August 2015

Genetic variation is the main evolutionary strategy adopted by RNA viruses and retroviruses. Evolution operates through competition between different individuals in the same environment, resulting in the imposition of the fittest variant. The process of competition could be affected by various factors, including the frequency of the different competing individuals. In order to investigate this aspect, individual virus populations derived from a human immunodeficiency virus type 1 isolate were studied at different competing proportions. The dynamics of variant imposition in each competition experiment permitted the detection of frequency-dependent selection (FDS); i.e. the imposition of variants is related to their biological fitness, which is also affected by the proportions at which they compete. The existence of FDS in different viruses with RNA genomes would indicate a general mechanism favouring genetic heterogeneity.
Diversity of mosaic structures and common ancestry of human immunodeficiency virus type 1 BF intersubtype recombinant viruses from Argentina revealed by analysis of near full-length genome sequences
Authors: Thomson Michael M. , Delgado Elena , Herrero Isabel , Villahermosa María Luisa , Vázquez-de Parga Elena , Cuevas María Teresa , Carmona Rocío , Medrano Leandro , Pérez-Álvarez Lucía , Cuevas Laureano , Nájera Rafael
Published: August 2015

The findings that BF intersubtype recombinant human immunodeficiency type 1 viruses (HIV-1) with coincident breakpoints in pol are circulating widely in Argentina and that non-recombinant F subtype viruses have failed to be detected in this country were reported recently. To analyse the mosaic structures of these viruses and to determine their phylogenetic relationship, near full-length proviral genomes of eight of these recombinant viruses were amplified by PCR and sequenced. Intersubtype breakpoints were analysed by bootscanning and examining the signature nucleotides. Phylogenetic relationships were determined with neighbour-joining trees. Five viruses, each with predominantly subtype F genomes, exhibited mosaic structures that were highly similar. Two intersubtype breakpoints were shared by all viruses and seven by the majority. Of the consensus breakpoints, all nine were present in two viruses, which exhibited identical recombinant structures, and four to eight breakpoints were present in the remaining viruses. Phylogenetic analysis of partial sequences supported both a common ancestry, at least in part of their genomes, for all recombinant viruses and the phylogenetic relationship of F subtype segments with F subtype viruses from Brazil. A common ancestry of the recombinants was supported also by the presence of shared signature amino acids and nucleotides, either unreported or highly unusual in F and B subtype viruses. These results indicate that HIV-1 BF recombinant viruses with diverse mosaic structures, including a circulating recombinant form (which are widespread in Argentina) derive from a common recombinant ancestor and that F subtype segments of these recombinants are related phylogenetically to the F subtype viruses from Brazil.
Subunit composition and conformational stability of the oligomeric form of the avian reovirus cell-attachment protein σC
Authors: Grande Ana , Costas Celina , Benavente Javier
Published: August 2015

Previous work has shown that the avian reovirus cell-attachment sigma C (σC) protein is a multimer. In the first part of this study the oligomerization state of intracellularly synthesized σC was analysed by different approaches, including SDS–PAGE, chemical cross-linking, sedimentation and gel filtration analysis. All these approaches indicated that protein σC in its native state is a homotrimer. In the second part of the present work we investigated the effect of different factors and reagents on oligomer stability, in order to elucidate the nature of the forces that maintain the conformational stability of the homotrimer. Our results, based on the stabilizing effect conferred by reducing agents, demonstrate that the σC subunits are not covalently bound via disulfide linkages. They further suggest that the formation of an intrachain disulfide bond between the two cysteine residues of the σC polypeptide has a negative effect on oligomer stability. The susceptibility of the trimer to pH, temperature, ionic strength, chemical denaturants and detergents indicates that hydrophobic interactions contribute much more to oligomer stability than do ionic interactions and hydrogen bonding. Finally, our results also reveal that mammalian and avian reovirus cell attachment proteins follow different subunit dissociation pathways.
Pathological changes of renal epithelial cells in mice transgenic for the TT virus ORF1 gene
Authors: Yokoyama Hiroshi , Yasuda Jiro , Okamoto Hiroaki , Iwakura Yoichiro
Published: August 2015

TT virus (TTV) is a newly identified human DNA virus of the familyCircoviridae. Its genome consists of six putative open reading frames (ORFs). TTV was isolated originally from a patient with cryptogenic hepatitis and the association of TTV with hepatitis has been studied extensively, while its significance in other diseases is unknown. To examine the pathogenicity of TTV, mice transgenic for the ORF genes in various combinations were produced. A total of 11 independent founder mice was produced: two mice, which were found to carry the ORF1 gene, showed pathological changes in the kidney; other tissues were not affected. In these mice, the transgene was expressed most strongly in the kidney and the transcript was shown to be spliced to encode a protamine-like, highly basic protein. Mice from a line with high transgene expression developed renal failure with severe renal epithelial cell abnormalities resembling those seen in humans with nephrotic syndrome. The transgenic mice with severe ascites died before reaching the age of 5 weeks. Another founder mouse with low expression levels also showed similar, but milder, renal epithelial cell changes, indicating that these effects were not caused by the insertion of the transgene, but, rather, were caused by the ORF1 gene product. These observations suggest that TTV affects renal epithelial cells as part of the naturally occurring infection.