bluuebunny/crossref_metadata_2025 huggingface dataset:
Hepatitis delta virus genotypes I and II cocirculate in an endemic area of Yakutia, Russia
Authors: Ivaniushina Valeria , Radjef Nadjia , Alexeeva Marfa , Gault Elyanne , Semenov Sergei , Salhi Mohammed , Kiselev Oleg , Dény Paul
Published: August 2015

Currently, three genotypes of hepatitis delta virus (HDV) are described. The most common, genotype I, has a worldwide distribution; in contrast, genotype II has been found previously only in Japan and Taiwan, while genotype III is found exclusively in South America. Considering the high prevalence of HDV in Northern Siberia (Russia), restriction fragment length polymorphism (RFLP) was used to analyse HDV genotypes from 29 infected patients living in Yakutia. Of these isolates, 11 were characterized by partial nucleotide sequencing and two isolates were completely sequenced. Phylogenetic inference methods included maximum parsimony, maximum likelihood and distance analyses. A restriction pattern consistent with HDV genotype I was found in 14 samples, while the remaining 15 showed a different restriction pattern, inconsistent with any known genotype. Five Yakutian HDV isolates with the type I restriction pattern were sequenced and confirmed to be affiliated with genotype I, although the phylogenetic results indicate that they were heterogeneous and did not cluster together. Sequencing of eight isolates with the new RFLP pattern revealed that these isolates were most closely related to HDV genotype II. In contrast to HDV Yakutian genotype I sequences, all of these type II sequences formed a well-defined clade on phylogenetic trees. Comparison of clinical presentations during hospitalization between patients infected with HDV type I (n=14) and type II (n=15) did not reveal any differences in the severity of infection. These data indicate that the distribution of genotype II is not restricted to Taiwan or Japan, but spreads over Northern Asia, appearing in the native population of Yakutia. Type II Yakutian strains appeared to form a well-defined subclade and could be associated with severe chronic hepatitis in this area.
gpUL73 (gN) genomic variants of human cytomegalovirus isolates are clustered into four distinct genotypes
Authors: Pignatelli S. , Dal Monte P. , Landini M. P.
Published: August 2015

Clinical isolates of human cytomegalovirus (HCMV) show differences in tissue tropism, severity of clinical manifestations and ability to establish persistent or latent infections, characteristics that are thought to be related to genomic variation among strains. This work analysed the genomic variants of a new HCMV polymorphic locus, open reading frame (ORF) UL73. This ORF encodes the envelope glycoprotein gpUL73 (gN), which associates in a high molecular mass complex with its counterpart, gM, and induces a neutralizing antibody response in the host. Detailed sequence analysis of ORF UL73 and its gene product from clinical isolates and laboratory-adapted strains shows that this glycoprotein is highly polymorphic, in the N-terminal region in particular. gpUL73 hypervariability is not randomly distributed, but the identified genomic variants are clearly clustered into four distinct genotypes (gN-1, gN-2, gN-3 and gN-4), which are not associated with the gB subtype.
Ovine herpesvirus-2 glycoprotein B sequences from tissues of ruminant malignant catarrhal fever cases and healthy sheep are highly conserved
Authors: Dunowska Magdalena , Letchworth Geoffrey J. , Collins James K. , DeMartini James C.
Published: August 2015

Ovine herpesvirus-2 (OHV-2) infection has been associated with malignant catarrhal fever (MCF) in susceptible ruminants. In order to further investigate whether OHV-2 is an aetiological agent for sheep-associated (SA) MCF in cattle and bison, the entire sequences of OHV-2 glycoprotein B (gB) from different sources of viral DNA were compared. Target DNA was derived from tissues of bovine and bison cases of SA-MCF, from a lymphoblastoid cell line established from another bovine case of SA-MCF, and from a healthy sheep. The divergence between deduced amino acid sequences of OHV-2 gB ranged from 0·5 to 1·2%. The high degree of similarity between gB sequences from a healthy sheep and clinical cases of SA-MCF in cattle and bison suggests that OHV-2 is an ovine virus that is occasionally transmitted to other ruminant species, in which it can cause severe disease.
Immunity against both polyomavirus VP1 and a transgene product induced following intranasal delivery of VP1 pseudocapsid–DNA complexes
Authors: Clark B. , Caparrós-Wanderley W. , Musselwhite G. , Kotecha M. , Griffin B. E.
Published: August 2015

Murine polyomavirus VP1 virus-like particles (VLPs) can bind plasmid DNA and transport it into cells both in vitro and in vivo. Long-term expression of the transgene can be observed, suggesting that VP1 VLPs may be used as DNA delivery vehicles for gene therapy. In this study we have analysed the in vitro efficiency of transfection using different DNA/VLP molar ratios and the immune response induced following intranasal administration of these complexes to mice. Our results indicate that in short-term in vitro culture VP1 VLP–DNA complexes appear to be as efficient as DNA alone at transfecting cell monolayers. They also show that VP1 VLPs are very immunogenic, inducing high proliferative cell responses and both serum and mucosal antibodies. Moreover, VP1 VLP–DNA complexes appear to be capable of inducing a stronger immune response to the transgene product (β-galactosidase) than immunization with DNA only. The results suggest that polyomavirus VP1 VLPs derived from the wild-type sequence may be too immunogenic for repeated use as gene delivery vehicles in gene therapy. However, due to their high immunogenicity and apparent adjuvant properties, they could be modified and used as vaccines either on their own or complexed with DNA.
Antigenic variation among isolates of infectious salmon anaemia virus correlates with genetic variation of the viral haemagglutinin gene
Authors: Kibenge Frederick S. B. , Kibenge Molly J. T. , McKenna Patricia K. , Stothard Paul , Marshall Rebecca , Cusack R. Roland , McGeachy Sandi
Published: August 2015

Infectious salmon anaemia virus (ISAV), an orthomyxovirus-like virus, is an important fish pathogen in marine aquaculture. Virus neutralization of 24 ISAV isolates in the TO cell line using rabbit antisera to the whole virus and comparative sequence analysis of their haemagglutinin (HA) genes have allowed elaboration on the variation of ISAV isolates. The 24 viruses were neutralized to varying degrees, revealing two major antigenic groups, one American and one European. Sequence analysis of the HA gene also revealed two groups of viruses (genotypes) that correlated with the antigenic groupings. The two HA subtypes had nucleotide sequence identity of only ⩽79·4% and amino acid sequence identity of ⩽84·5% whereas, within each subtype, the sequence identities were 90·7% or higher. This grouping was also evident upon phylogenetic analysis, which revealed two distinct phylogenetic families. Between the two groups, the amino acid sequence was most variable in the C-terminal region and included deletions of 4–16 amino acids in all isolates relative to ISAV isolate RPC/NB-980 280-2. In order to view the relationships among these sequences and the HA sequences of the established orthomyxoviruses, a second phylogenetic tree was constructed which showed the ISAV sequences to be more closely related to sequences from Influenzavirus A and Influenzavirus B than to sequences from Influenzavirus C and Thogotovirus. The extensive deletions in the gene of European ISAV isolates lead us to speculate that the archetypal ISAV was probably of Canadian origin.
Vaccine-enhanced respiratory syncytial virus disease in cotton rats following immunization with Lot 100 or a newly prepared reference vaccine
Authors: Prince Gregory A. , Curtis Spencer J. , Yim Kevin C. , Porter David D.
Published: August 2015

A formalin-inactivated respiratory syncytial virus vaccine was used to immunize infants in the mid-1960s; when these children later were naturally infected by the virus they developed markedly accentuated disease, and two died. For the present work, a new batch of vaccine was prepared using the original formula. Administration of either the old or new vaccines resulted in enhanced lesions in immunized cotton rats subsequently challenged with live virus, although administration of the vaccine reduced virus replication by 90%. Animals primed with formalin-inactivated virus and challenged developed markedly accentuated lesions of the same type as in animals undergoing primary or secondary infection. In addition, the animals with the vaccine-enhanced disease developed alveolitis and interstitial pneumonitis, which appear to be specific markers for the vaccine enhancement. The newly prepared vaccine appears suitable as a reference standard for studying the mechanism of vaccine-enhanced disease caused by this virus. Additionally, we reviewed the lesions in the lungs of the two humans who died with the vaccine-enhanced disease in 1967, and found that they were similar to, but more severe than those seen in the cotton rats.
Induction of the measles virus receptor SLAM (CD150) on monocytes
Authors: Minagawa Hiroko , Tanaka Kotaro , Ono Nobuyuki , Tatsuo Hironobu , Yanagi Yusuke
Published: August 2015

Wild-type strains of measles virus (MV) isolated in B95a cells use the signalling lymphocyte activation molecule (SLAM; also known as CD150) as a cellular receptor, whereas the Edmonston strain and its derivative vaccine strains can use both SLAM and the ubiquitously expressed CD46 as receptors. Among the major target cells for MV, lymphocytes and dendritic cells are known to express SLAM after activation, but monocytes have been reported to be SLAM-negative. In this study, SLAM expression on monocytes was examined under different conditions. When freshly isolated from the peripheral blood, monocytes did not express SLAM on the cell surface. However, monocytes became SLAM-positive after incubation with phytohaemagglutinin, bacterial lipopolysaccharide or MV. Anti-SLAM monoclonal antibodies efficiently blocked infection of activated monocytes with a wild-type strain of MV. These results indicate that SLAM is readily induced and acts as a monocyte receptor for MV.
The NS5A protein of bovine viral diarrhoea virus interacts with the α subunit of translation elongation factor-1
Authors: Johnson Craig M. , Perez Daniel R. , French Roy , Merrick William C. , Donis Ruben O.
Published: August 2015

A cellular protein that interacts with the NS5A polypeptide of bovine viral diarrhoea virus (BVDV) was identified in a yeast two-hybrid screen. The NS5A interactor was identified as the α subunit of bovine translation elongation factor 1A (eEF1A). Cell-free binding studies were performed with chimeric NS5A fused to glutathioneS-transferase (GST–NS5A) expressed in bacteria. GST–NS5A bound specifically to bothin vitro-translated and mammalian cell-expressed eEF1A. Moreover, purified eEF1A bound specifically to GST–NS5A attached to a solid phase. Conservation of this interaction was then analysed using a set of NS5A proteins derived from divergent BVDV strains encompassing known biotypes and genotypes. NS5A from all BVDV strains tested so far interacted with eEF1A. The conserved association of eEF1A with virus molecules involved in genome replication and the postulated role of pestivirus and hepacivirus NS5A in replication indicate that this interaction may play a role in the replication of BVDV.
MC159L protein from the poxvirus molluscum contagiosum virus inhibits NF-κB activation and apoptosis induced by PKR
Authors: Gil Jesús , Rullas Joaquín , Alcamí José , Esteban Mariano
Published: August 2015

Molluscum contagiosum virus (MCV) is a human poxvirus that causes abnormal proliferation of epithelial cells. MCV encodes specific molecules to control host defences, such as MC159L, which as previously shown prevents apoptosis induced by death receptors. However, unlike most poxviruses, MCV lacks a homologue to the E3L and K3L proteins of vaccinia virus, which are involved in the control of the key antiviral and pro-apoptotic dsRNA-dependent protein kinase, PKR. In this study, we analysed the relationship of MC159L to PKR. We found that MC159L is not a direct inhibitor of PKR since it does not associate with PKR and cannot block PKR-induced phosphorylation of eIF-2α. However, expression of MC159L inhibits apoptosis triggered by PKR through death receptor-mediated pathways. In addition, MC159L inhibits NF-κB activation induced in response to PKR. Expression of MC159L cannot counteract the PKR-mediated antiviral action in the context of a poxvirus infection, despite its ability to affect these signalling events. These findings show that MC159L is able to interfere with downstream events triggered by PKR in the absence of a direct physical interaction, and assign a role to MC159L in the control of some PKR-mediated biological effects.
Elevated expression of c-myc in lymphoblastoid cells does not support an Epstein–Barr virus latency III-to-I switch
Authors: Pajic Alexander , Polack Axel , Staege Martin S. , Spitkovsky Dimitry , Baier Barbara , Bornkamm Georg W. , Laux Gerhard
Published: August 2015

Epstein–Barr virus (EBV) transforms primary B cells in vitro. Established cell lines adopt a lymphoblastoid phenotype (LCL). In contrast, EBV-positive Burkitt’s lymphoma (BL) cells, in which the proto-oncogene c-myc is constitutively activated, do not express a lymphoblastoid phenotype in vivo. The two different phenotypes are paralleled by two distinct programmes of EBV latent gene expression termed latency type I in BL cells and type III in LCL. Human B cell lines were established from a conditional LCL (EREB2-5) by overexpression of c-myc and inactivation of EBV nuclear protein 2 (EBNA2). These cells (A1 and P493-6) adopted a BL phenotype in the absence of EBNA2. However, the EBV latency I promoter Qp was not activated. Instead, the latency III promoter Cp remained active. These data suggest that the induction of a BL phenotype by overexpression of c-myc in an LCL is not necessarily paralleled by an EBV latency III-to-I switch.
Varicella-zoster virus gH:gL contains a structure reactive with the anti-human gamma chain of IgG near the glycosylation site
Authors: Yokoyama Tomonori , Ayabe Satoko , Miyagi Huminori , Sugano Toru , Otsu Akira , Sato Hitoshi , Kageyama Seiji , Fujii Takao , Shiraki Kimiyasu
Published: August 2015

Varicella-zoster virus (VZV) glycoproteins were purified from infected cells using monoclonal antibodies and gH:gL was found to react with antibodies to the γ chain of human IgG (h-IgG), whereas gE:gI and gB did not. When gH:gL was captured by concanavalin A, it lost reactivity with the anti-h-IgG γ chain (anti-h-γ-IgG). gH:gL reacted with anti-h-γ-IgG in an ELISA assay and gave a K
d value of 2·16×10−7 M in a BIAcore assay. The K
d value of the human monoclonal antibody to gH (TI-57) used for the purification of gH:gL was 4·45×10−10 M. Virus pretreated with anti-h-IgG was five times more resistant to neutralization with TI-57. Although the nature of the binding was not clear, gH:gL bound to anti-h-γ-IgG. If this interaction results from immunological similarity between gH:gL and h-IgG, it may cause immune evasion in the pathogenesis of VZV infection.
Herpesvirus saimiri protein StpB associates with cellular Src
Authors: Hör Simon , Ensser Armin , Reiss Christine , Ballmer-Hofer Kurt , Biesinger Brigitte
Published: August 2015

Subgroup B isolates of Herpesvirus saimiri are less efficient in T lymphocyte transformation when compared with subgroups A or C. Here it is shown that subgroup B strain SMHI encodes a protein, StpB, at a position equivalent to those of the ORFs for the saimiri transforming proteins (Stp) of subgroups A and C. StpB shares little similarity with StpA or StpC, but interacts with the SH2 domain of cellular Src, as does StpA. Thus, factors other than c-Src binding determine the efficiency of primary T cell transformation by Herpesvirus saimiri.
The C-terminal residues of poliovirus proteinase 2Apro are critical for viral RNA replication but not for cis- or trans-proteolytic cleavage
Authors: Li Xiaoyu , Lu Hui-Hua , Mueller Steffen , Wimmer Eckard
Published: August 2015

Poliovirus proteinase 2Aprois an essential enzyme involved in cleavages of viral and cellular proteins during the infectious cycle. Evidence has been obtained that 2Aprois also involved in genome replication. All enteroviruses have a negatively charged cluster of amino acids at their C terminus (EE/DE/DAMEQ–NH2), a common motif suggesting function. When aligned with enterovirus sequences, the 2Aproproteinase of human rhinovirus type 2 (HRV2) has a shorter C terminus (EE…Q–NH2) and, indeed, the HRV2 2Aprocannot substitute for poliovirus 2Aproto yield a viable chimeric virus. Here evidence is provided that the C-terminal cluster of amino acids plays an unknown role in poliovirus genome replication. Deletion of the EEAME sequence from poliovirus 2Aprois lethal without significantly influencing proteinase function. On the other hand, addition of EAME to HRV2 2Apro, yielding a C terminus of this enzyme ofEEEAMEQ, stimulated RNA replication of a poliovirus/HRV2 chimera 100-fold. The novel role of the C-terminal sequence motif is manifested at the level of protein function, since silent mutations in its coding region had no effect on virus proliferation. Poliovirus type 1 Mahoney 2Aprocould be providedin transto rescue the lethal deletion EEAME in the poliovirus variant. Encapsidation studies left open the question of whether the C terminus of poliovirus 2Aprois involved in particle formation. It is concluded that the C terminus of poliovirus 2Aprois an essential domain for viral RNA replication but is not essential for proteolytic processing.
Measles virus and canine distemper virus target proteins into a TAP-independent MHC class I-restricted antigen-processing pathway
Authors: Neumeister Claudia , Nanan Ralph , Cornu Tatjana I. , Lüder Carsten G. K. , ter Meulen Volker , Naim Hussein , Niewiesk Stefan
Published: August 2015

After infection of CEM174.T2 cells [deficient for the transporter of antigen presentation (TAP)] with measles virus (MV) the nucleocapsid protein is recognized by Ld-restricted cytotoxic T cells in a TAP-independent, chloroquine-sensitive fashion. Presentation via the TAP-independent pathway requires virus replication. During MV infection of the cell the nucleocapsid as well as the matrix protein enter the endolysosomal compartment as indicated by colocalization with the lysosomal-associated membrane protein 1 (LAMP-1). Similarly, the nucleocapsid protein of canine distemper virus (CDV) is recognized in a TAP-independent fashion. In addition, a recombinant MV expressing bacterial β-galactosidase protein is able to introduce the recombinant antigen into the TAP-independent pathway whereas a vaccinia virus expressing β-galactosidase is not. These data and a report about TAP-independent recognition of parainfluenza virus type 1 suggest that members of theParamyxoviridaefamily regularly introduce viral proteins into the TAP-independent antigen-processing pathway.
Cell-to-cell movement of potato virus X involves distinct functions of the coat protein
Authors: Fedorkin O. N. , Solovyev A. G. , Yelina N. E. , Zamyatnin A. A. , Zinovkin R. A. , Mäkinen K. , Schiemann J. , Yu. Morozov S.
Published: August 2015

Complementation of movement-deficient potato virus X (PVX) coat protein (CP) mutants, namely PVX.CP-Xho lacking the 18 C-terminal amino acid residues and PVX.ΔCP lacking the entire CP gene, was studied by transient co-expression with heterologous proteins. These data demonstrated that the potyvirus CPs and both the major and minor CPs of beet yellows closterovirus could complement cell-to-cell movement of PVX.CP-Xho but not PVX.ΔCP. These data also indicated that the C-terminally truncated PVX CP lacked a movement function which could be provided in trans by the CPs of other filamentous viruses, whereas another movement determinant specified by some region outside the most C-terminal part of the PVX CP could not be complemented either by potyvirus or closterovirus CPs. Surprisingly, the CP of spherical cocksfoot mottle sobemovirus rescued all of the PVX CP movement functions, complementing the spread of PVX.CP-Xho and, to a lesser extent, PVX.ΔCP. Both these mutants were also rescued by the tobacco mosaic virus (TMV) movement protein (MP). To shed light on the movement function of PVX CP, attempts were made to complement PVX.CP-Xho by a series of TMV MP mutants. An internal deletion abolished complementation, suggesting that the internal region of TMV MP, which includes a number of overlapping functional domains important for cell-to-cell transport, provides an activity complementing movement determinant(s) specified by the C-terminal region of PVX CP.
Re-analysis of human immunodeficiency virus type 1 isolates from Cyprus and Greece, initially designated ‘subtype I’, reveals a unique complex A/G/H/K/? mosaic pattern
Authors: Paraskevis D. , Magiorkinis M. , Vandamme A. M. , Kostrikis L. G. , Hatzakis A.
Published: August 2015

Human immunodeficiency virus type 1 (HIV-1) has been classified into three main groups and 11 distinct subtypes. Moreover, several circulating recombinant forms (CRFs) of HIV-1 have been recently documented to have spread widely causing extensive HIV-1 epidemics. A subtype, initially designated I (CRF04_cpx), was documented in Cyprus and Greece and was found to comprise regions of sequence derived from subtypes A and G as well as regions of unclassified sequence. Re-analysis of the three full-length CRF04_cpx sequences that were available revealed a mosaic genomic organization of unique complexity comprising regions of sequence from at least five distinct subtypes, A, G, H, K and unclassified regions. These strains account for approximately 2% of the total HIV-1-infected population in Greece, thus providing evidence of the great capability of HIV-1 to recombine and produce highly divergent strains which can be spread successfully through different infection routes.
Extended nucleocapsid protein is cleaved from the Gag–Pol precursor of human immunodeficiency virus type 1
Authors: Chen Nissim , Morag Abraham , Almog Nava , Blumenzweig Immanuel , Dreazin Orna , Kotler Moshe
Published: August 2015

Human immunodeficiency virus type 1 Gag and Gag–Pol precursors are translated from an mRNA which is indistinguishable from the full-length genomic RNA. The ratio of Gag to Gag–Pol polyproteins is approximately 20:1 and is controlled by a frameshift of the reading frame, which takes place downstream of the p7 nucleocapsid (NC) in the N terminus of the p1 peptide. The viral precursors Gag and Gag–Pol are cleaved by the virus-encoded protease (PR) into the structural proteins, and into p6Pol, PR, reverse transcriptase and integrase. Due to the frameshift event, the cleavage site at the C terminus of NC coded in the Gag frame (ERQAN-FLGKI) changes either to ERQANFLRED or ERQANFFRED. The results presented in this report demonstrate that the NC released from the Gag–Pol precursor is 8 amino acid residues longer than the NC cleaved from the Gag polyprotein. Our results also show that truncated Gag–Pol precursors bearing cleavage site mutation at the NC/p6Pol, and/or p6Pol/PR junctions, undergo autoprocessing in bacterial and eukaryotic cells, indicating that PR is active when part of the precursor.
Identification and characterization of a new intron in Borna disease virus
Authors: Cubitt Beatrice , Ly Calvin , de la Torre Juan Carlos
Published: August 2015

Borna disease virus (BDV) has a non-segmented, negative-strand (NNS) RNA genome. In contrast to all other known NNS RNA animal viruses, BDV replication and transcription occur in the nucleus of infected cells. Moreover, BDV uses RNA splicing for the regulation of its genome expression. Two introns (I and II), both present in two viral primary transcripts of 2·5 and 7·2 kb, have been reported in BDV. Here, evidence is provided of a new BDV intron, intron III, generated by alternative 3′ splice-site choice. Intron III-spliced mRNAs were detected at early times post-infection and found to be present in cells from different types and species. Intron III-spliced mRNAs have coding capability for two new viral proteins with predicted molecular masses of 8·4 and 165 (p165) kDa. p165 is a deleted form of the BDV L polymerase, containing three RGD motifs and a signal peptide signal that could target it into the secretory pathway. These findings underscore the proteomic complexity exhibited by BDV.
2000 Fleming Lecture. The origin and evolution of hepatitis viruses in humans
Authors: Simmonds Peter
Published: August 2015

The spread and origins of hepatitis C virus (HCV) in human populations have been the subject of extensive investigations, not least because of the importance this information would provide in predicting clinical outcomes and controlling spread of HCV in the future. However, in the absence of historical and archaeological records of infection, the evolution of HCV and other human hepatitis viruses can only be inferred indirectly from their epidemiology and by genetic analysis of contemporary virus populations. Some information on the history of the latter may be obtained by dating the time of divergence of various genotypes of HCV, hepatitis B virus (HBV) and the non-pathogenic hepatitis G virus (HGV)/GB virus-C (GBV-C). However, the relatively recent times predicted for the origin of these viruses fit poorly with their epidemiological distributions and the recent evidence for species-associated variants of HBV and HGV/GBV-C in a wide range of non-human primates. The apparent conservatism of viruses over long periods implied by these latter observations may be the result of constraints on sequence change peculiar to viruses with single-stranded genomes, or with overlapping reading frames. Large population sizes and intense selection pressures that optimize fitness may be the factors that set virus evolution apart from that of their hosts.
A small region of the dengue virus-encoded RNA-dependent RNA polymerase, NS5, confers interaction with both the nuclear transport receptor importin-β and the viral helicase, NS3
Authors: Johansson Magnus , Brooks Andrew J. , Jans David A. , Vasudevan Subhash G.
Published: August 2015

The dengue virus RNA-dependent RNA polymerase, NS5, and the protease/helicase, NS3, are multidomain proteins that have been shown to interact bothin vivoandin vitro. A hyperphosphorylated form of NS5 that does not interact with NS3 has been detected in the nuclei of virus-infected cells, presumably as the result of the action of a functional nuclear localization sequence within the interdomain region of NS5 (residues 369–405). In this study, it is shown by using the yeast two-hybrid system that the C-terminal region of NS3 (residues 303–618) interacts with the N-terminal region of NS5 (residues 320–368). Further, it is shown that this same region of NS5 is also recognized by the cellular nuclear import receptor importin-β. The interaction between NS5 and importin-β and competition by NS3 with the latter for the same binding site on NS5 were confirmed by pull-down assays. The direct interaction of importin-β with NS5 has implications for the mechanism by which this normally cytoplasmic protein may be targetted to the nucleus.
The early pathogenesis of foot-and-mouth disease in pigs infected by contact: a quantitative time-course study using TaqMan RT–PCR
Authors: Alexandersen Soren , Oleksiewicz Martin B. , Donaldson Alex I.
Published: August 2015

Foot-and-mouth disease (FMD) is a highly contagious, economically important virus disease of cloven-hoofed animals. The objective of the present study was to examine the early pathogenesis of FMD in pigs by a quantitative time-course study. Under experimental conditions, recipient pigs were infected by contact with donor pigs affected by FMD. Every 24 h from day 1 to day 4 after exposure, two recipient pigs were selected randomly, killed and necropsied. A range of tissues were analysed by a quantitative TaqMan RT–PCR method and by titration of FMD virus on primary bovine thyroid cells. The titres of virus determined by assay in cell culture and calculated from the quantitative TaqMan data correlated strongly (r>0·9), thereby establishing the validity of the TaqMan calculations. The data indicated that the replication of virus in the lungs contributes only in small part to airborne virus excretion. Sites in the pharynx, trachea and nasal mucosa are probably more important in that regard. The sites of earliest virus infection and possibly replication in recipient pigs appeared to be in the pharynx (soft palate, tonsil and floor of pharynx). The data indicated that FMD virus replication in pigs is rapid and that the majority of virus amplification occurs in the skin. A model for the progression of infection is proposed, indicating initial spread from the pharyngeal region, through regional lymph nodes and via the blood to epithelial cells, resulting in several cycles of virus amplification and spread.
Mapping the domains on the phosphoprotein of bovine respiratory syncytial virus required for N–P and P–L interactions using a minigenome system
Authors: Khattar Sunil K. , Yunus Abdul S. , Samal Siba K.
Published: August 2015

The interaction of bovine respiratory syncytial virus (BRSV) phosphoprotein (P) with nucleocapsid (N) and large polymerase (L) proteins was investigated using an intracellular BRSV–CAT minigenome replication system. Coimmunoprecipitation assays using P-specific antiserum revealed that the P protein can form complexes with N and L proteins. Deletion mutant analysis of the P protein was performed to identify the regions of P protein that interact with N and L proteins. The results indicate that two independent N-binding sites exist on the P protein: an internal region of 161–180 amino acids and a C-terminal region of 221–241 amino acids. The L-binding site was mapped to a region of P protein encompassing amino acids 121–160. The data suggest that N and L protein binding domains on the P protein do not overlap.
An immunodominant neutralization epitope on the ‘thumb’ subdomain of human immunodeficiency virus type 1 reverse transcriptase revealed by phage display antibodies
Authors: Ohba Hiroyoshi , Soga Takatoshi , Tomozawa Takanori , Nishikawa Yoshifumi , Yasuda Atsushi , Kojima Asato , Kurata Takeshi , Chiba Joe
Published: August 2015

An antibody phage display library was produced from the splenocytes of mice immunized with an infectious vaccinia virus recombinant (WRRT) expressing the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1). The library was panned against HIV-1 RT. Two clones, 5F and 5G, which produced Fab fragments specific for RT, were isolated. Surprisingly, both 5F and 5G Fab fragments were capable of strongly inhibiting the RNA-dependent DNA polymerase activity of HIV-1 RT. A hybridoma cell line that produces the monoclonal antibody 7C4, which strongly inhibits RT activity, was established previously using splenocytes from mice immunized with WRRT by the same immunization protocol. The epitope recognized by 7C4 exists in the region of the template primer-binding sites (or the ‘helix clump’) of RT. By epitope mapping and competitive ELISA analysis, it was shown that the 5F and 5G Fab fragments were directed against the same, or a very closely related, epitope that is recognized by 7C4. The neutralizing activities of the 5F, 5G and 7C4 Fab fragments correlated with their affinities for HIV-1 RT. DNA sequencing indicated that the immunoglobulin genes of the heavy chains of 5G and 7C4, as well as those of the light chains of 5F and 5G, had the same origin. These results suggest that the neutralizing epitope, which is recognized by these antibodies, becomes immunodominant after repeated immunization of mice with WRRT. This unique epitope, HIV-1 RT-specific and immunodominant neutralizing epitope (HRSINE), is a logical target for new types of HIV-1 RT inhibitors and gene therapy.
Intracellular localization of the hepatitis B virus HBx protein
Authors: Henkler Frank , Hoare Jonathan , Waseem Naushin , Goldin Robert D. , McGarvey Michael J. , Koshy Rajen , King Ian A.
Published: August 2015

The hepatitis B virus (HBV) X protein (HBx) was originally suggested to be a viral transcriptional activator, but its functional mechanisms are still unclear. In this study we have analysed the intracellular localization of HBx in transfected cells and demonstrate that its compartmentalization is dependent on overall expression levels. HBx was exclusively or predominantly localized in the nuclei in weakly expressing cells. However, elevated cellular levels correlated with its accumulation in the cytoplasm, suggesting that the capacity of HBx for nuclear compartmentalization might be limited. Cytoplasmic HBx was detected either as punctate granular staining or in dispersed, finely granular patterns. We have further analysed the detailed cytoplasmic compartmentalization, using confocal microscopy, and show no association with the endoplasmic reticulum, plasma membrane or lysosomes, but a substantial association of HBx with mitochondria. However, a major fraction of cytoplasmic HBx did not localize in mitochondria, indicating the presence of two distinctly compartmentalized cytoplasmic populations. Furthermore, high levels of HBx expression led to an abnormal mitochondrial distribution, involving clumping and organelle aggregation, which was not observed at lower expression levels. The data presented here provide novel insights into the compartmentalization of HBx and may prove important for future evaluations of its functions, both in the viral life-cycle and in the pathology of HBV-related liver disease.
Analysis of two genomic variants of orang-utan hepadnavirus and their relationship to other primate hepatitis B-like viruses
Authors: Verschoor Ernst J. , Warren Kristin S. , Langenhuijzen Susan , Heriyanto  , Swan Ralph A. , Heeney Jonathan L.
Published: August 2015

We recently described orang-utan hepadnavirus (OuHV) (Warren et al., Journal of Virology, 73, 7860–7865, 1999). Phylogenetic analyses indicated that the various isolates of OuHV can be divided into two genomic variants. Two representatives from each genomic cluster were analysed both molecularly and phylogenetically. Their genome organization was highly similar to other hepadnaviruses of apes and humans. The complete genome sequences of the two OuHV types had an overall 5% sequence difference. Research on 25 seropositive Bornean orang-utans showed that, of the 19 animals infected with one variant, 12 originated from East Kalimantan. Phylogenetic analysis was performed using the full-length genomes of various primate hepadnaviruses. The tree topology revealed one cluster of Old World hepadnaviruses that is divided into two subclusters, one consisting of the ape viruses, and the other comprising the human genotypes A–E. These data suggest that the great apes and gibbons have been infected with a common ancestor hepadnavirus.
Geographically distant isolates of the crinivirus Cucurbit yellow stunting disorder virus show very low genetic diversity in the coat protein gene
Authors: Rubio Luis , Abou-Jawdah Yusuf , Lin Han-Xin , Falk Bryce W.
Published: August 2015

The population structure and genetic variation ofCucurbit yellow stunting disorder virus(CYSDV) isolates were estimated by single-strand conformation polymorphism and nucleotide sequence analyses of the CYSDV coat protein gene. Analysis of 71 isolates collected from Spain, Jordan, Turkey, Lebanon, Saudi Arabia and North America showed that, from a genetic viewpoint, these isolates could be divided into two diverged subpopulations: an Eastern subpopulation composed of Saudi Arabian isolates and a Western subpopulation containing the rest of the CYSDV isolates. The genetic variation within the Western subpopulation was very small (nucleotide identity >99%) in spite of the extensive and discontinuous geographical distribution and different years of collection. We also estimated the within-isolate genetic structure and variation of three CYSDV isolates by analysing 30 clones per isolate. Our results showed that these CYSDV isolates had a quasispecies structure.
Recognition of cis-acting sequences in RNA 3 of Prunus necrotic ringspot virus by the replicase of Alfalfa mosaic virus
Authors: Aparicio F. , Sánchez-Navarro J. A. , Olsthoorn R. C. L. , Pallás V. , Bol J. F.
Published: August 2015

Alfalfa mosaic virus (AMV) and Prunus necrotic ringspot virus (PNRSV) belong to the genera Alfamovirus and Ilarvirus, respectively, of the family Bromoviridae. Initiation of infection by AMV and PNRSV requires binding of a few molecules of coat protein (CP) to the 3′ termini of the inoculum RNAs and the CPs of the two viruses are interchangeable in this early step of the replication cycle. Cis-acting sequences in PNRSV RNA 3 that are recognized by the AMV replicase were studied in in vitro replicase assays and by inoculation of AMV–PNRSV RNA 3 chimeras to tobacco plants and protoplasts transformed with the AMV replicase genes (P12 plants). The results showed that the AMV replicase recognized the promoter for minus-strand RNA synthesis in PNRSV RNA 3 but not the promoter for plus-strand RNA synthesis. A chimeric RNA with PNRSV movement protein and CP genes accumulated in tobacco, which is a non-host for PNRSV.
Analysis of the aphthovirus 2A/2B polyprotein ‘cleavage’ mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal ‘skip’
Authors: Donnelly Michelle L. L. , Luke Garry , Mehrotra Amit , Li Xuejun , Hughes Lorraine E. , Gani David , Ryan Martin D.
Published: August 2015

The 2A region of the aphthovirus foot-and-mouth disease virus (FMDV) polyprotein is only 18 aa long. A ‘primary’ intramolecular polyprotein processing event mediated by 2A occurs at its own C terminus. FMDV 2A activity was studied in artificial polyproteins in which sequences encoding reporter proteins flanked the 2A sequence such that a single, long, open reading frame was created. The self-processing properties of these artificial polyproteins were investigated and the co-translational ‘cleavage’ products quantified. The processing products from our artificial polyprotein systems showed a molar excess of ‘cleavage’ product N-terminal of 2A over the product C-terminal of 2A. A series of experiments was performed to characterize our in vitro translation systems. These experiments eliminated the translational or transcriptional properties of the in vitro systems as an explanation for this imbalance. In addition, the processing products derived from a control construct encoding the P1P2 region of the human rhinovirus polyprotein, known to be proteolytically processed, were quantified and found to be equimolar. Translation of a construct encoding green fluorescent protein (GFP), FMDV 2A and β-glucuronidase, also in a single open reading frame, in the presence of puromycin, showed this antibiotic to be preferentially incorporated into the [GFP2A] translation product. We conclude that the discrete translation products from our artificial polyproteins are not produced by proteolysis. We propose that the FMDV 2A sequence, rather than representing a proteolytic element, modifies the activity of the ribosome to promote hydrolysis of the peptidyl(2A)-tRNAGly ester linkage, thereby releasing the polypeptide from the translational complex, in a manner that allows the synthesis of a discrete downstream translation product to proceed. This process produces a ribosomal ‘skip’ from one codon to the next without the formation of a peptide bond.
The genome of herpesvirus of turkeys: comparative analysis with Marek’s disease viruses
Authors: Kingham Brewster F. , Zelnık Vladimır , Kopáček Juraj , Majerčiak Vladimır , Ney Erik , Schmidt Carl J.
Published: August 2015

The complete coding sequence of the herpesvirus of turkeys (HVT) unique long (UL) region along with the internal repeat regions has been determined. This allows completion of the HVT nucleotide sequence by linkage to the sequence of the unique short (US) region. The genome is approximately 160 kbp and shows extensive similarity in organization to the genomes of Marek’s disease virus serotypes 1 and 2 (MDV-1, MDV-2) and other alphaherpesviruses. The HVT genome contains 75 ORFs, with three ORFs present in two copies. Sixty-seven ORFs were identified readily as homologues of other alphaherpesvirus genes. Seven of the remaining eight ORFs are homologous to genes in MDV, but are absent from other herpesviruses. These include a gene with similarity to cellular lipases. The final, HVT-unique gene is a virus homologue of the cellular NR-13 gene, the product of which belongs to the Bcl family of proteins that regulate apoptosis. No other herpesvirus sequenced to date contains a homologue of this gene. Of potential significance is the absence of a complete block of genes within the HVT internal repeat that is present in MDV-1. These include the pp38 and meq genes, which have been implicated in MDV-1-induced T-cell lymphoma. By implication, other genes present in this region of MDV-1, but missing in HVT, may play important roles in the different biological properties of the viruses.
Increased proteolytic activity and matrix metalloprotease expression in lungs during infection by porcine reproductive and respiratory syndrome virus
Authors: Girard Manon , Cléroux Patrick , Tremblay Pierre , Dea Serge , St-Pierre Yves
Published: August 2015

The local increase in the secretion of extracellular proteases, allowing cleavage of the extracellular matrix and thereby facilitating the infiltration of T cells, monocytes and neutrophils, is a hallmark of chronic inflammation and autoimmunity. In pulmonary genetic diseases, such as emphysema and cystic fibrosis, proteases can also favour the development of local immunodeficiency by degrading key regulators of the immune response, such as CD4, CD8, IgG, ICAM-1 and C3b receptors. Since several infectious agents can give rise to severe pulmonary disorders associated with opportunistic infections, we sought to determine whether an increase in proteolytic activity occurred during infection with porcine reproductive and respiratory syndrome virus (PRRSV), the causative agent of a new disease in swine characterized by severe respiratory problems in young pigs. Piglets were infected with the virus and bronchoalveolar lavages were collected at various times post-infection to measure the net proteolytic activity. It was shown that PRRSV infection leads to a significant increase in proteolytic activity in pulmonary fluids. Maximal activity was found at 7 and 14 days post-infection, with a return towards normal levels at day 42. Zymographic analyses showed a significant increase in the secretion of matrix metalloproteases (MMPs) 2 and 9, two enzymes involved in tissue remodelling. Histological analyses showed a correlation between the increase in proteolytic activity and the appearance of lesions that were characterized by massive lymphomononuclear cell infiltration. These results suggest that virus infection of the lungs can lead to a transient increase in proteolytic activity that could favour opportunistic infection.
Evidence for recombination in natural populations of dengue virus type 1 based on the analysis of complete genome sequences
Authors: Tolou H. J. G. , Couissinier-Paris P. , Durand J.-P. , Mercier V. , de Pina J.-J. , de Micco P. , Billoir F. , Charrel R. N. , de Lamballerie X.
Published: August 2015

Recombination events are known to occur in non-segmented RNA viruses like polioviruses or alphaviruses. Analysis of the subgenomic sequences of dengue virus type 1 (DENV-1) structural genes has recently allowed the identification of possible recombination breakpoints. Because DENV is a major human pathogen, this discovery might have important implications for virus pathogenicity, vaccine safety and efficiency, or diagnosis and, therefore, requires clear confirmation. We report the complete sequence determination of one Asian and two African strains of DENV-1 isolated from human patients. Rigorous sequence analysis provided strong evidence for the occurrence of intragenomic recombination events between DENV-1 strains belonging to different lineages. Singapore S275/90 strain appears to be the evolutionary product of a recombination event between viruses belonging to two distinct lineages: one lineage includes an African strain isolated in Abidjan (Ivory Coast) and the other includes isolates from Djibouti and Cambodia. The ‘Recombination Detection Program’, bootscanning and analysis of diversity plots provided congruent results concerning the existence of a two-switch recombination event and the localization of recombination breakpoints. Thus, the 5′ and 3′ genomic ends of the Singapore S275/90 strain were inherited from a Djibouti/Cambodia lineage ancestor and an internal fragment located in the envelope/NS1 region originated from an Abidjan lineage ancestor.
The role of IL-5, IL-6 and IL-10 in primary and vaccine-primed immune responses to infection with Friend retrovirus (Murine leukaemia virus)
Authors: Strestik Beatrice D. , Olbrich Anke R. M. , Hasenkrug Kim J. , Dittmer Ulf
Published: August 2015

The defence of a host against viral infections is strongly influenced by cytokines. We investigated the role of the B-cell stimulating cytokines IL-5 and IL-6, and the immuno-suppressive cytokine IL-10, during primary and secondary immune responses in mice against infection with Friend retrovirus (FV) (Murine leukaemia virus). IL-5−/−mice were comparable to C57BL/6 wild-type mice in their ability to control acute FV infection. In contrast, IL-6−/−and IL-10−/−mice showed significantly enhanced virus loads in spleen cells. However, this impaired control of acute FV replication did not alter the long-term control over persistent FV in IL-6−/−and IL-10−/−mice. Immunization with a live attenuated vaccine virus prior to challenge protected all three types of cytokine-deficient mice from high levels of spleen virus, despite the finding that the vaccinated IL-5- and IL-6-deficient mice had significantly reduced titres of virus-neutralizing IgG class antibodies. The results indicate that IL-6 and IL-10 contribute to primary immune responses against FV, but are dispensable during persistent infection and vaccine-primed secondary responses.
The role of the Type I interferon response in the resistance of mice to filovirus infection
Authors: Bray Mike
Published: August 2015

Adult immunocompetent mice inoculated with Ebola (EBO) or Marburg (MBG) virus do not become ill. A suckling-mouse-passaged variant of EBO Zaire ’76 (‘mouse-adapted EBO-Z’) causes rapidly lethal infection in adult mice after intraperitoneal (i.p.) inoculation, but does not cause apparent disease when inoculated subcutaneously (s.c.). A series of experiments showed that both forms of resistance to infection are mediated by the Type I interferon response. Mice lacking the cell-surface IFN-α/β receptor died within a week after inoculation of EBO-Z ’76, EBO Sudan, MBG Musoke or MBG Ravn, or after s.c. challenge with mouse-adapted EBO-Z. EBO Reston and EBO Ivory Coast did not cause illness, but immunized the mice against subsequent challenge with mouse-adapted EBO-Z. Normal adult mice treated with antibodies against murine IFN-α/β could also be lethally infected with i.p.-inoculated EBO-Z ’76 or EBO Sudan and with s.c.-inoculated mouse-adapted EBO-Z. Severe combined immunodeficient (SCID) mice became ill 3–4 weeks after inoculation with EBO-Z ’76, EBO Sudan or MBG Ravn, but not the other viruses. Treatment with anti-IFN-α/β antibodies markedly accelerated the course of EBO-Z ’76 infection. Antibody treatment blocked the effect of a potent antiviral drug, 3-deazaneplanocin A, indicating that successful filovirus therapy may require the active participation of the Type I IFN response. Mice lacking an IFN-α/β response resemble primates in their susceptibility to rapidly progressive, overwhelming filovirus infection. The outcome of filovirus transfer between animal species appears to be determined by interactions between the virus and the innate immune response.