bluuebunny/crossref_metadata_2025 huggingface dataset:
The sites for fatty acylation, phosphorylation and intermolecular disulphide bond formation of influenza C virus CM2 protein
Authors: Li Zhu-Nan , Hongo Seiji , Sugawara Kanetsu , Sugahara Kazuhiko , Tsuchiya Emi , Matsuzaki Yoko , Nakamura Kiyoto
Published: August 2015

The sites for fatty acylation, disulphide bond formation and phosphorylation of influenza C virus CM2 were investigated by site-specific mutagenesis. Cysteine 65 in the cytoplasmic tail was identified as the site for palmitoylation. Removal of one or more of three cysteine residues in the ectodomain showed that all of cysteines 1, 6 and 20 can participate in the formation of disulphide-linked dimers and/or tetramers, although cysteine 20 may play the most important role in tetramer formation. Furthermore, it was found that serine 78, located within the recognition motifs for mammary gland casein kinase and casein kinase I, is the predominant site for phosphorylation, although serine 103 is phosphorylated to a minor extent by proline-dependent protein kinase. The effects of acylation and phosphorylation on the formation of disulphide-linked oligomers were also studied. The results showed that, while palmitoylation has no role in oligomer formation, phosphorylation accelerates tetramer formation without influencing dimer formation. CM2 mutants defective in acylation, phosphorylation or disulphide bond formation were all transported to the cell surface, suggesting that none of these modifications is required for proper oligomerization. When proteins solubilized in detergent were analysed on sucrose gradients, however, the mutant lacking cysteines 1, 6 and 20 sedimented as monomers, raising the possibility that disulphide bond formation, although not essential for proper oligomerization, may stabilize the CM2 multimer. This was supported by the results of chemical cross-linking analysis, which showed that the triple-cysteine mutant can form multimers.
Apoptosis is induced by infectious bursal disease virus replication in productively infected cells as well as in antigen-negative cells in their vicinity
Authors: Jungmann Annett , Nieper Hermann , Müller Hermann
Published: August 2015

The kinetics of infectious bursal disease virus (IBDV) replication and induction of apoptosis were investigated in vitro and in vivo. After infection of chicken embryo (CE) cells with IBDV strain Cu-1, the proportion of apoptotic cells increased from 5·8% at 4 h post-infection (p.i.) to 64·5% at 48 h p.i. The proportion of apoptotic cells correlated with IBDV replication. UV-inactivated IBDV particles did not induce apoptosis. Double labelling revealed that, early after infection, the majority of antigen-expressing cells were not apoptotic; double-labelled cells appeared more frequently at later times. Remarkably, apoptotic cells were frequently located in the vicinity of antigen-expressing cells. This indicated that an apoptosis-inducing factor(s) might be released by cells that replicate IBDV. Since interferon (IFN) production has been demonstrated after IBDV infection, IFN was considered to be one of several factors. However, supernatants of infected CE cells in which virus infectivity had been neutralized were not sufficient to induce apoptosis.Similar results were observed in the infected bursae of Fabricius: early after infection, most of the cells either showed virus antigens or were apoptotic. Again, double-labelled cells appeared more frequently late after infection. This suggests that indirect mechanisms might also be involved in the induction of apoptosis in vivo, contributing to the rapid depletion of cells in the IBDV-infected bursa.
The effect of latency-associated transcript on the herpes simplex virus type 1 latency-reactivation phenotype is mouse strain-dependent
Authors: Perng Guey-Chuen , Slanina Susan M. , Ghiasi Homayon , Nesburn Anthony B. , Wechsler Steven L.
Published: August 2015

Herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) null mutants reactivate poorly in the rabbit ocular model. The situation in mice is less clear. Reports concluding that LAT null mutants reactivate poorly in the mouse explant-induced reactivation (EIR) model are contradicted by a similar number of reports of normal EIR of LAT− mutants in mice. To determine if the EIR phenotype might be mouse strain-dependent we infected BALB/c and Swiss Webster mice with LAT− or LAT+ virus and assessed EIR in individual trigeminal ganglia. Compared to LAT+ virus, LAT− virus reactivated poorly in Swiss Webster mice (P<0·05). In contrast, the EIR phenotype of these viruses was similar in BALB/c mice (P>0·1). Thus, LAT appeared to have a much greater impact on the EIR phenotype in Swiss Webster mice than in BALB/c mice. The mouse strain therefore appeared consequential in the HSV-1 EIR phenotype in mice.
Late temporal gene expression from the human cytomegalovirus pp28US (UL99) promoter when integrated into the host cell chromosome
Authors: Wu Jun , O’Neill Joseph , Barbosa Miguel S.
Published: August 2015

Toward understanding the temporal regulation of human cytomegalovirus (HCMV) late genes, we studied the regulation of the late gene promoter (pp28US, UL99) when outside the context of the viral genome and its response to the immediate early (IE) proteins. Expression of the luciferase reporter gene, regulated by the pp28US promoter, was synchronous with that of the endogenous viral pp28 gene, independently of whether the reporter was episomal or integrated into the glioblastoma cell line U373MG. Cotransfection of the reporter with expression vectors for each of the three major IE genes, IE72, IE86 and IE55, indicated that only IE86 transactivated the pp28US promoter. However, the magnitude of the promoter activation upon HCMV infection suggested that additional factors are also required for higher promoter activity. The promoter activation was specific to HCMV, as herpes simplex virus type 1 infection did not induce luciferase expression.
Epstein–Barr virus gene polymorphisms in Chinese Hodgkin’s disease cases and healthy donors: identification of three distinct virus variants
Authors: Zhou X.-G. , Sandvej K. , Li P.-J. , Ji X.-L. , Yan Q.-H. , Zhang X.-P. , Da J.-P. , Hamilton-Dutoit S. J.
Published: August 2015

Epstein–Barr virus (EBV) is associated with several malignancies. Specific EBV gene variants, e.g. the BamHI f configuration, a C-terminal region 30 bp deletion in the latent membrane protein-1 (LMP1) gene (del-LMP) and the loss of an XhoI site in LMP1 (XhoI-loss), are found in Chinese cases of nasopharyngeal carcinoma (NPC), suggesting that EBV sequence variation may be involved in oncogenesis. In order to understand better the epidemiology of these EBV variants, they were studied in virus isolates from EBV-positive Chinese cases of Hodgkin’s disease (HD; n=71) and donor throat washings from healthy Chinese. Sequencing was performed of 15 representative EBV isolates, including the first analysis of the LMP1 promoter in Asian wild-type EBV isolates. The following observations were made. (i) Three EBV LMP1 variants were identified, designated Chinese groups (CG) 1–3. In both EBV-associated HD and in healthy Chinese, CG1-like viruses showing del-LMP1 and XhoI-loss were predominant. (ii) CG1viruses were distinct from European and African variants, suggesting that this profile is useful for epidemiological studies. (iii) Specific patterns of mutations were present in the LMP1 promoter in both CG1 and CG2. (iv) The BamHI f variant was not found in Chinese HD, in contrast to Chinese NPC and European HD. This study confirms that EBV isolates in Chinese HD and other tumours differ from those reported in Western cases. However, this reflects the predominant virus strain present in the healthy Chinese population, suggesting that these are geographically restricted polymorphisms rather than tumour-specific strains.
Demonstration by single-cell PCR that Reed–Sternberg cells and bystander B lymphocytes are infected by different Epstein–Barr virus strains in Hodgkin’s disease
Authors: Faumont Nathalie , Al Saati Talal , Brousset Pierre , Offer Claudie , Delsol Georges , Meggetto Fabienne
Published: August 2015

Epstein–Barr virus (EBV) is associated with Hodgkin’s disease (HD). However, EBV-positive Reed–Sternberg (RS) cells and EBV-positive B lymphocytes co-exist in the same EBV-positive lymph node affected by HD. In a previous report, using total lymph node DNA, the presence of two distinct EBV strains was demonstrated, but their cellular localization (i.e. RS cells vs B lymphocytes) could not be determined. To address this question, three patients with EBV-associated HD were selected in the present study and single-cell PCR of the latent membrane protein-1 (LMP-1) gene from isolated RS cells was performed. In one case, it was clear that RS cells and B lymphocytes were infected by different EBV strains. In the two remaining cases, only one band was detected from total lymph node DNA. However, single-cell PCR showed that RS cells in each sample were infected by single EBV strains, which were different from those detected in lymphoblastoid cell lines derived from EBV-positive B lymphocytes of lymph node cell suspensions from these two patients.
Major histocompatibility complex class I molecules are down-regulated at the cell surface by the K5 protein encoded by Kaposi’s sarcoma-associated herpesvirus/human herpesvirus-8
Authors: Haque Muzammel , Ueda Keiji , Nakano Kazushi , Hirata Yuko , Parravicini Carlo , Corbellino Mario , Yamanishi Koichi
Published: August 2015

The expression of major histocompatibility complex class I (MHC-I) molecules at the cell surface was down-regulated in BC-3 cells infected with Kaposi’s sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 at early times after treatment with 12-O-tetradecanoylphorbol acetate (TPA), and in HeLa cells transfected with the K5 gene of KSHV. However, an immunoprecipitation study on these cells with anti-MHC-I monoclonal antibody revealed that there was no significant reduction in the synthesis of MHC-I molecules. A pulse–chase analysis followed by endoglycosidase H digestion also demonstrated the stability and transport of MHC-I molecules from the endoplasmic reticulum to at least the medial-Golgi. K5 antigen was clearly detected by immunohistological examination of samples from Kaposi’s sarcoma, primary effusion lymphoma and Castleman’s disease. These results suggest that the down-regulation of MHC-I molecules by K5 gene expression during reactivation may be important for evading immunological surveillance in the host.
Characterization of the murine gammaherpesvirus 68 ORF74 product: a novel oncogenic G protein-coupled receptor
Authors: Wakeling Madeleine N. , Roy Douglas J. , Nash Anthony A. , Stewart James P.
Published: August 2015

Murine gammaherpesvirus (MHV-68) is well established as a small animal model for the study of gammaherpesviruses. The MHV-68 genome contains an open reading frame (ORF74) that has significant sequence homology with mammalian G-protein coupled receptors (GPCRs) and the GPCR from the related Kaposi’s sarcoma-associated herpesvirus (KSHV). Here we show that the MHV-68 ORF74 is predicted to encode a GPCR since it has seven potential transmembrane helices and that it has other sequence motifs in common with GPCRs. Of interest is the observation that the sequence around a conserved arginine at the start of the second intracellular loop suggests that the ORF74 product may signal constitutively (agonist independent). Given that the ORF74 product is predicted to encode a GPCR we named it MHV-GPCR. In studies on the transcription of the MHV-GPCR, we determined that it was encoded on multiple early transcripts of 3·4, 4·4, 6·6 and 8·7 kb in size. At least one of these transcripts was bicistronic, containing the ORF encoding the Bcl-2 homologue also.In vivo, we found that MHV GPCR was expressed during acute infection but also during persistence, particularly in the lungs of infected mice. Immunofluorescence studies indicated that the MHV GPCR protein was expressed on the surface of cells in patches. Finally, like the KSHV GPCR, expression of the MHV GPCR resulted in transformation of NIH 3T3 cells. We surmise, therefore, that the MHV GPCR may act in concert with genes with which it is expressed such as vBcl-2 to enhance the growth and survival of MHV-68-infected cells.
Genotypes of JC virus in East, Central and Southwest Europe
Authors: Agostini Hansjürgen T. , Deckhut Alison , Jobes David V. , Girones Rosina , Schlunck Günther , Prost Marcin G. , Frias Carolina , Pérez-Trallero E. , Ryschkewitsch Caroline F. , Stoner Gerald L.
Published: August 2015

Distinctive genotypes of JC virus have been described for the major continental landmasses. Studies on European-Americans and small cohorts in Europe showed predominantly Type 1. Types 2 and 7 are found in Asia, and Types 3 and 6 in Africa. These genotypes differ in sequence by about 1–3%. Each genotype may have several subtypes which differ from each other by about 0·5–1%. The genotypes can be defined by a distinctive pattern of nucleotides in a typing region of the VP1 gene. This genotyping approach has been confirmed by phylogenetic reconstruction using the entire genome exclusive of the rearranging regulatory region. In this first large European study, we report on the urinary excretion of JCV DNA of 350 individuals from Poland, Hungary, Germany and Spain. We included Gypsy cohorts in Hungary (Roma), Germany (Sinti), and Spain (Gitano), as well as Basques in Spain. We show that while Type 1 predominates in Europe, the proportions of Type 1A and 1B may differ from East to Southwest Europe. Type 4, closely related to the Type 1 sequence (only ∼1% difference) was a minor genotype in Germany, Poland and Spain, but represented the majority in Basques. The Gitanos in Spain showed a variant Type 4 sequence termed ‘Rom-1’. Interestingly, neither the Gitanos in Spain, nor Sinti or Roma in Germany or Hungary showed the Type 2 or Type 7 genotype that might be expected if their origins were in an Asian population.
Identification of a genetic determinant of pathogenicity in chicken anaemia virus
Authors: Yamaguchi S. , Imada T. , Kaji N. , Mase M. , Tsukamoto K. , Tanimura N. , Yuasa N.
Published: August 2015

The molecular basis of pathogenicity of the chicken anaemia virus (CAV) needs to be clarified in order to develop a safe, live virus vaccine. In this study, several high- and low-pathogenic infectious DNA clones were obtained from field virus samples after 12 or 38 passages in MDCC-MSB1 cells. The high-pathogenic clones induced a low haematocrit, low weight gain and high mortality. Nucleotide sequence analyses identified one amino acid, at residue 394 of the VP1 capsid protein, as a major determinant of pathogenicity. To determine the role of this amino acid in pathogenicity, chimeric infectious DNA clones and point-mutated clones were used for chicken pathogenicity tests. These analyses clearly demonstrated that residue 394 of VP1 was crucial for the pathogenicity of CAV; all of the cloned viruses with glutamine at this position were highly pathogenic, whereas those with histidine had low pathogenicity. Low-pathogenic CAV, based on an infectious DNA clone, is a candidate for a genetically homogeneous and stable CAV live vaccine.
An Arabidopsis thaliana protein interacts with a movement protein of Turnip crinkle virus in yeast cells and in vitro
Authors: Lin Bing , Heaton Louis A.
Published: August 2015

Plant virus movement proteins bind host components to promote virus movement from initially infected cells to neighbouring cells. In this study, cDNA clones encoding p8 and p9, two small proteins required for the movement ofTurnip crinkle virus, were used as ‘bait’ in a yeast two-hybrid system to screen anArabidopsis thalianacDNA library for interactive proteins. OneA.thalianaclone was identified that encodes a protein, designated Atp8, which interacted with p8 in yeast cells andin vitro. The apparent full-length of Atp8 mRNA was sequenced and shown to encode a protein with two possible transmembrane helices, several potential phosphorylation sites and two ‘RGD’ sequences.
Humoral and CD4+ T helper (Th) cell responses to the hepatitis C virus non-structural 3 (NS3) protein: NS3 primes Th1-like responses more effectively as a DNA-based immunogen than as a recombinant protein
Authors: Lazdina Una , Hultgren Catharina , Frelin Lars , Chen Margaret , Lodin Karin , Weiland Ola , Leroux-Roels Geert , Quiroga Juan A. , Peterson Darrell L. , Milich David R. , Sällberg Matti
Published: August 2015

The non-structural 3 (NS3) protein is one of the most conserved proteins of hepatitis C virus, and T helper 1 (Th1)-like responses to NS3 in humans correlate with clearance of infection. Several studies have proposed that DNA-based immunizations are highly immunogenic and prime Th1-like responses, although few head-to-head comparisons with exogenous protein immunizations have been described. A full-length NS3/NS4A gene was cloned in eukaryotic vectors with expression directed to different subcellular compartments. Inbred mice were immunized twice in regenerating tibialis anterior (TA) muscles with either plasmid DNA or recombinant NS3 (rNS3). After two 100 μg DNA immunizations, specific antibody titres of up to 12960 were detected at week 5, dominated by IgG2a and IgG2b. NS3-specific CD4+T cell responses in DNA-immunized mice peaked at day 13, as measured by proliferation and IL-2 and IFN-γ production. Mice immunized with 1–10 μg rNS3 without adjuvant developed antibody titres comparable to those of the DNA-immunized mice, but dominated instead by IgG1. CD4+T cell responses in these mice showed peaks of IL-2 response at day 3 and IL-6 and IFN-γ responses at day 6. With adjuvant, rNS3 was around 10-fold more immunogenic with respect to speed and magnitude of the immune responses. Thus, immunization with rNS3 in adjuvant is superior to DNA immunization with respect to kinetics and quantity in priming specific antibodies and CD4+T cells. However, as a DNA immunogen, NS3 elicits stronger Th1-like immune responses, whereas rNS3 primes a mixed Th1/Th2-like response regardless of the route, dose or adjuvant.
Jaagsiekte sheep retrovirus can be detected in the peripheral blood during the pre-clinical period of sheep pulmonary adenomatosis
Authors: González L. , García-Goti M. , Cousens C. , Dewar P. , Cortabarría N. , Extramiana A. B. , Ortín A. , De las Heras M. , Sharp J. M.
Published: August 2015

Peripheral blood leukocytes (PBLs) and tissue samples from 36 sheep were examined for jaagsiekte sheep retrovirus (JSRV) by hemi-nested PCR. Animals were classified according to the status of sheep pulmonary adenomatosis (SPA), which was confirmed by pathological examination, as follows: (i) sheep with classical SPA (cSPA, n=10), (ii) sheep with atypical SPA (aSPA, n=6), (iii) non-affected sheep from SPA-affected flocks (in-contact, n=10) and (iv) non-affected sheep from SPA-free flocks (control, n=10). JSRV proviral DNA was detected in the PBLs of 10/10 cSPA, 5/6 aSPA, 4/10 in-contact and 0/10 control sheep. Lung tumours and lymphoid organs were also found to be JSRV-positive. The number of positive PCR results was greater for sheep in the cSPA group than for those in the aSPA and in-contact groups. For the first time, it is concluded that JSRV can be detected in naturally infected sheep before the onset of clinical disease and even before the development of discernible tumours.
Furin cleavage of the respiratory syncytial virus fusion protein is not a requirement for its transport to the surface of virus-infected cells
Authors: Sugrue Richard J. , Brown Craig , Brown Gaie , Aitken James , McL. Rixon Helen W.
Published: August 2015

The intracellular cleavage of respiratory syncytial virus (RSV) fusion (F) protein by furin was examined. In RSV-infected LoVo cells, which express an inactive form of furin, and in RSV-infected Vero cells treated with the furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (dec-RVKR-cmk), the F protein was expressed as a non-cleaved 73 kDa species. In both cases the F protein was initially expressed as an endoglycosidase H (Endo H)-sensitive precursor (F0EHs) which was modified approximately 40 min post-synthesis by the addition of complex carbohydrates to produce the Endo H-resistant form (F0EHr). The size and glycosylation state of F0EHr were identical to a transient intermediate form of non-cleaved F protein which was detected in RSV-infected Vero cells in the absence of inhibitor. Cell surface biotinylation and surface immunofluorescence staining showed that F0EHr was present on the surface of RSV-infected cells. RSV filaments have been shown to be the predominant form of the budding virus that is detected during virus replication. Analysis of the RSV-infected cells using scanning electron microscopy (SEM) showed that, in the presence of dec-RVKR-cmk, virus budding was impaired, producing fewer and much smaller viral filaments than in untreated cells. A comparison of immunofluorescence and SEM data showed that F0EHr was routed to the surface of virus-infected cells but not located in these smaller structures. Our findings suggest that activation of the F protein is required for the efficient formation of RSV filaments.
Plasma membrane requirements for cell fusion induced by herpes simplex virus type 1 glycoproteins gB, gD, gH and gL
Authors: Browne Helena , Bruun Birgitte , Minson Tony
Published: August 2015

Herpes simplex virus type 1 (HSV-1) glycoproteins gB, gD and gHL are capable of inducing cell fusion when expressed from plasmid vectors in the absence of any other virus components. Fusion requires the expression of all four glycoproteins on the same membrane, since they are unable to cooperate in trans to induce syncytium formation. In addition, the fusion event is dependent on the expression of a gD receptor on target cell membranes and does not require the presence of cell-surface glycosaminoglycans.
In vitro selection of human cytomegalovirus variants unable to transfer virus and virus products from infected cells to polymorphonuclear leukocytes and to grow in endothelial cells
Authors: Grazia Revello M. , Baldanti Fausto , Percivalle Elena , Sarasini Antonella , De-Giuli Luciana , Genini Emilia , Lilleri Daniele , Labò Nazarena , Gerna Giuseppe
Published: August 2015

Four human cytomegalovirus (HCMV) isolates from different clinical sources were extensively propagated in human embryonic lung fibroblasts (HELF). Plaque isolates from each of the four virus strains were evaluated for their ability to be transferred to polymorphonuclear leukocytes (PMNL) and to grow in endothelial cells (EC). While all four of the clinical strains were found to be both PMNL- and EC-tropic, variants were identified from each of the four strains that lacked both biological properties, while three of the four parental strains lost their transfer capacity before passage 50 in HELF. It was demonstrated that one of the four field isolates (VR6110) and its transfer-deficient variant were genetically related, but showed different curves of virus yield in HELF. In addition, neither the immediate-early (IE) mRNA nor the IE protein p72 were found to be transferred to PMNL before 72 h post-infection (late in infection) or in the presence of viral DNA replication inhibitors. These findings link EC and PMNL tropism and suggest that PMNL tropism is a late HCMV function.
Epstein–Barr virus-encoded protein kinase BGLF4 mediates hyperphosphorylation of cellular elongation factor 1δ (EF-1δ): EF-1δ is universally modified by conserved protein kinases of herpesviruses in mammalian cells
Authors: Kato Kentaro , Kawaguchi Yasushi , Tanaka Michiko , Igarashi Mie , Yokoyama Akihiko , Matsuda Go , Kanamori Mikiko , Nakajima Kaori , Nishimura Yorihiro , Shimojima Masayuki , Phung Hang T. T. , Takahashi Eiji , Hirai Kanji
Published: August 2015

Translation elongation factor 1δ (EF-1δ) is hyperphosphorylated in various mammalian cells infected with alpha-, beta- and gammaherpesviruses and EF-1δ modification is mediated by viral protein kinases, including UL13 of herpes simplex virus type 1 and UL97 of human cytomegalovirus. In this study, the following is reported. (i) BGLF4 encoded by the prototype gammaherpesvirus Epstein–Barr virus was purified as a fusion protein that was labelled with [γ-32P]ATP and labelling was eliminated by phosphatase. (ii) The ratio of the hyperphosphorylated form of human EF-1δ was increased both in Sf9 cells after infection with baculoviruses expressing GST–BGLF4 fusion proteins and in COS-7 cells after transfection with a BGLF4 expression plasmid. These results indicate that purified BGLF4 possesses protein kinase activity and mediates EF-1δ hyperphosphorylation. These data also support the hypothesis that the protein kinases that are conserved by herpesviruses universally mediate EF-1δ modification in mammalian cells.
Cellular transcription factors that interact with p6 promoter elements of parvovirus B19
Authors: Raab Ulla , Bauer Birgit , Gigler Andreas , Beckenlehner Karin , Wolf Hans , Modrow Susanne
Published: August 2015

All transcripts of the human parvovirus B19 identified so far are regulated by a single promoter at map unit 6 of the viral genome, the so-called p6 promoter. This promoter is active in a wide variety of different cells. In order to identify cellular transcription factors involved in regulating promoter activity, we performed gel-retardation and supershift assays using the parts of the p6 promoter sequence shown previously to be protected in footprint experiments. Thereby, binding was demonstrated of the Oct-1 protein to an octamer motif within the p6 promoter and of the transcription factor Sp1 to three GC boxes. A specific preferential interaction of the factor Sp3 with one of these boxes was observed, indicating that the ratio Sp1:Sp3 may be involved in the regulation of promoter activity. Consensus sites for the regulatory protein YY1 are located close to the GC boxes and the octamer motif, to which this factor binds efficiently.
Genes Ia, II, III, IV and V of Soybean chlorotic mottle virus are essential but the gene Ib product is non-essential for systemic infection
Authors: Takemoto Yutaka , Hibi Tadaaki
Published: August 2015

Soybean chlorotic mottle virus(SbCMV) is the type species of the genus ‘Soybean chlorotic mottle-like viruses’, within the familyCaulimoviridae. The double-stranded DNA genome of SbCMV (8178 bp) contains eight major open reading frames (ORFs). Viral genes essential and non-essential for the replication and movement of SbCMV were investigated by mutational analysis of an infectious 1·3-mer DNA clone. The results indicated that ORFs Ia, II, III, IV and V were essential for systemic infection. The product of ORF Ib was non-essential, although the putative tRNAMetprimer-binding site in ORF Ib was proved to be essential. Immunoselection PCR revealed that an ORF Ia deletion mutant was encapsidated in primarily infected cells, indicating that ORF Ia is required for virus movement but not for replication. ORF IV was confirmed to encode a capsid protein by peptide sequencing of the capsid. Analysis of the viral transcripts showed that the SbCMV DNA genome gives rise to a pregenomic RNA and an ORF VI mRNA, as shown in the case ofCauliflower mosaic virus.
Nucleotide sequence of the coat protein gene of Lettuce big-vein virus
Authors: Sasaya Takahide , Ishikawa Koichi , Koganezawa Hiroki
Published: August 2015

A sequence of 1425 nt was established that included the complete coat protein (CP) gene of Lettuce big-vein virus (LBVV). The LBVV CP gene encodes a 397 amino acid protein with a predicted M
r of 44486. Antisera raised against synthetic peptides corresponding to N-terminal or C-terminal parts of the LBVV CP reacted in Western blot analysis with a protein with an M
r of about 48000. RNA extracted from purified particles of LBVV by using proteinase K, SDS and phenol migrated in gels as two single-stranded RNA species of approximately 7·3 kb (ss-1) and 6·6 kb (ss-2). After denaturation by heat and annealing at room temperature, the RNA migrated as four species, ss-1, ss-2 and two additional double-stranded RNAs (ds-1 and ds-2). The Northern blot hybridization analysis using riboprobes from a full-length clone of the LBVV CP gene indicated that ss-2 has a negative-sense nature and contains the LBVV CP gene. Moreover, ds-2 is a double-stranded form of ss-2. Database searches showed that the LBVV CP most resembled the nucleocapsid proteins of rhabdoviruses. These results indicate that it would be appropriate to classify LBVV as a negative-sense single-stranded RNA virus rather than as a double-stranded RNA virus.
Analysis of canine parvovirus sequences from wolves and dogs isolated in Italy
Authors: Battilani Mara , Scagliarini Alessandra , Tisato Ernesto , Turilli Carlo , Jacoboni Irene , Casadio Rita , Prosperi Santino
Published: August 2015

The VP2 genes of Italian canine parvovirus (CPV) type 2 strains isolated from dogs and wolves were sequenced and a three-dimensional model of the VP2 capsid protein was constructed. Two mutations were detected in the VP2 sequences of the Italian strains: one at residue 297 and one at residue 265. Variant 297 is the predominant CPV isolate in Europe, whereas variant 265 has never been detected before. The mutation at residue 265 causes a disruption in a G strand of the β-barrel in the VP2 protein. Data on strains isolated from wolves demonstrated that the same strain of CPV can circulate among domestic and wild canids; therefore, this result leads us to exclude the possibility that a separate parvovirus pool exists in wild populations.
Cross-protection against mucosal simian immunodeficiency virus (SIVsm) challenge in human immunodeficiency virus type 2-vaccinated cynomolgus monkeys
Authors: Walther-Jallow Lilian , Nilsson Charlotta , Söderlund Johan , ten Haaft Peter , Mäkitalo Barbro , Biberfeld Peter , Böttiger Per , Heeney Jonathan , Biberfeld Gunnel , Thorstensson Rigmor
Published: August 2015

In this study we compared the efficacy of live attenuated human immunodeficiency virus type 2 (HIV-2) vaccine alone versus boosting with live non-pathogenic HIV-2 following priming with ALVAC HIV-2 (recombinant canarypox virus expressing HIV-2 env, gag and pol). Six monkeys were first inoculated intravenously with live HIV-2SBL-6669 and 7 to 10 months later were challenged intrarectally with 10 MID50 of cell-free simian immunodeficiency virus (SIV) strain SIVsm. One monkey was completely protected against SIV infection and all five monkeys that became SIV-infected showed a lower virus replication and an initial lower virus load as compared with a parallel group of six control animals. In another experiment five monkeys were immunized either three times with ALVAC HIV-2 alone or twice with ALVAC HIV-2 and once with purified native HIV-2 gp125. The monkeys were then challenged with HIV-2 given intravenously and finally with pathogenic SIVsm given intrarectally. After challenge with SIVsm, three of five monkeys were completely protected against SIVsm infection whereas the remaining two macaques became SIV-infected but with limited virus replication. In conclusion, vaccination with an ALVAC HIV-2 vaccine followed by exposure to live HIV-2 could induce cross-protection against mucosal infection with SIVsm and seemed to be more efficient than immunization with a live HIV-2 vaccine only.
Phylogeny of North American Powassan virus
Authors: Ebel Gregory D. , Spielman Andrew , Telford Sam R.
Published: August 2015

To determine whether Powassan virus (POW) and deer tick virus (DTV) constitute distinct flaviviral populations transmitted by ixodid ticks in North America, we analysed diverse nucleotide sequences from 16 strains of these viruses. Two distinct genetic lineages are evident, which may be defined by geographical and host associations. The nucleotide and amino acid sequences of lineage one (comprising New York and Canadian POW isolates) are highly conserved across time and space, but those of lineage two (comprising isolates from deer ticks and a fox) are more variable. The divergence between lineages is much greater than the variation within either lineage, and lineage two appears to be more diverse genetically than is lineage one. Application of McDonald–Kreitman tests to the sequences of these strains indicates that adaptive evolution of the envelope protein separates lineage one from lineage two. The two POW lineages circulating in North America possess a pattern of genetic diversity suggesting that they comprise distinct subtypes that may perpetuate in separate enzootic cycles.
Foot-and-mouth disease virus can utilize the C-terminal extension of coxsackievirus A9 VP1 for cell infection
Authors: Leippert Martina , Pfaff Eberhard
Published: August 2015

Foot-and-mouth disease virus (FMDV) is known to employ the conserved Arg–Gly–Asp (RGD) tripeptide located on the variable βG–βH loop of the VP1 capsid protein for binding to cells. Coxsackievirus A9 (CAV9) also carries an RGD sequence, but on a short C-terminal extension of its VP1 and in a different amino acid context. This apparent relationship raised the question of whether insertion of the heterologous CAV9 sequence into FMDV would influence infection by the genetically modified FMDV. Four VP1 mutants were generated by PCR mutagenesis of a full-length FMDV cDNA plasmid. After transfection of BHK-21 cells, viral protein synthesis and virus particle formation could be detected. Two of the four mutants, mV9b and mV9d, could be propagated in BHK-21 cells, but not in CV-1 cells. Both of these mutants contained 17 amino acids of the C terminus of CAV9 VP1. Infection of BHK cells could be specifically inhibited by rabbit immune serum raised against a synthetic peptide representing the amino acid sequence of the C-terminal extension of CAV9 VP1. This demonstrated the direct involvement of the inserted sequence in cell infection. In fact, genetically modified FMDV O1K was capable of employing the VP1 C-terminal RGD region of CAV9 for infection of BHK cells. In addition, these results show that, even in cell culture-adapted viruses, the RGD-containing βG–βH loop plays an important role in virus infectivity.
Equine rhinitis A virus: structural proteins and immune response
Authors: Hartley Carol A. , Ficorilli Nino , Dynon Kemperly , Drummer Heidi E. , Huang Jin-an , Studdert Michael J.
Published: August 2015

Equine rhinitis A virus (ERAV) is a picornavirus that has been reclassified as a member of the Aphthovirus genus because of its resemblance to foot-and-mouth disease virus at the level of nucleotide sequence and overall genomic structure. The N-terminal amino acid sequence of three of the four capsid proteins of ERAV was determined and showed that the proteolytic cleavage sites within the precursor P1 polypeptide occur exactly as those predicted for an aphthovirus-like 3C protease, which generates the capsid proteins VP1 and VP3. However, the autocatalytic cleavage site between VP4 and VP2, which is independent of 3C protease cleavage, was different from that predicted previously. ERAV.393/76 antisera from horses and rabbits showed different reactivity to the viral structural proteins in both serum neutralization assays and Western blots. High neutralizing antibody titres appeared to correlate with strong reactivity to VP1 in Western blots.
High-level expression of a foreign gene from the most 3′-proximal locus of a recombinant Newcastle disease virus
Authors: Huang Zhuhui , Krishnamurthy Sateesh , Panda Aruna , Samal Siba K.
Published: August 2015

A previous report showed that insertion of a foreign gene encoding chloramphenicol acetyltransferase (CAT) between the HN and L genes of the full-length cDNA of a virulent Newcastle disease virus (NDV) yielded virus with growth retardation and attenuation. The NDV vector used in that study was pathogenic to chickens; it is therefore not suitable for use as a vaccine vector. In the present study, an avirulent NDV vector was generated and its potential to express CAT protein was evaluated. The CAT gene was under the control of NDV transcriptional start and stop signals and was inserted immediately before the open reading frame of the viral 3′-proximal nucleocapsid protein gene. A recombinant NDV expressing CAT activity at a high level was recovered. The replication and pathogenesis of the CAT-expressing recombinant NDV were not modified significantly. These results indicate the potential utility of an avirulent NDV as a vaccine vector.
Cloning and identification of the infectious salmon anaemia virus haemagglutinin
Authors: Krossøy Bjørn , Devold Magnus , Sanders Lisette , Knappskog Per Morten , Aspehaug Vidar , Falk Knut , Nylund Are , Koumans Sjo , Endresen Curt , Biering Eirik
Published: August 2015

Infectious salmon anaemia virus (ISAV) is an orthomyxo-like virus that causes serious disease in Atlantic salmon (Salmo salar). Like the orthomyxoviruses, ISAV has been shown to possess haemagglutinin (HA) activity. This study presents the cloning, expression and identification of the ISAV HA gene, which was isolated from a cDNA library by immunoscreening. The HA gene contained an ISAV-specific conserved nucleotide motif in the 5′ region and a 1167 bp open reading frame encoding a protein with a predicted molecular mass of 42·4 kDa. The HA gene was expressed in a baculovirus system. A monoclonal antibody (MAb) shown previously to be directed against the ISAV HA reacted with insect cells infected with recombinant baculovirus. Salmon erythrocytes also adsorbed to these cells and adsorption was inhibited by the addition of either the ISAV-specific MAb or a polyclonal rabbit serum prepared against purified virus, confirming the virus specificity of the reaction. Immunoblot analyses indicated that ISAV HA, in contrast to influenza virus HA, is not posttranslationally cleaved. Sequence comparisons of the HA gene from five Norwegian, one Scottish and one Canadian isolate revealed a highly polymorphic region that may be useful in epidemiological studies.
Gender-related differences in susceptibility, early virus dissemination and immunosuppression in mice infected with Friend murine leukaemia virus variant FIS-2
Authors: Bruland Torunn , Dai Hong Yan , Lavik Liss Anne S. , Kristiansen Lena I. , Dalen Are
Published: August 2015

An emerging amount of data indicates a correlation between gender-related factors and regulation of virus infection and supports what is known in clinical circles, that these topics are of great importance in many infectious diseases. In the present study we found that young adult NMRI male mice are more susceptible to infection by a variant of Friend murine leukaemia virus, FIS-2, than are female mice. We observed that the level of virus in serum, bone marrow and spleen was initially higher in male mice. Male mice were also more susceptible to FIS-2-induced immunosuppression. These results indicate a more efficient virus replication and dissemination in male mice. Studies with recombinant viruses between FIS-2 and the prototype Friend murine leukaemia virus revealed that FIS-2 LTR is one major factor contributing to the observed gender differences. A possible sex hormone influence on FIS-2 transcription due to the presence of a glucocorticoid response element in FIS-2 LTR is discussed.
Screening enteroviruses for β-cell tropism using foetal porcine β-cells
Authors: Roivainen Merja , Ylipaasto Petri , Ustinov Jarkko , Hovi Tapani , Otonkoski Timo
Published: August 2015

Primary adult human insulin-producing β-cells are susceptible to infection by prototype strains of coxsackieviruses (CV) and infection may result in impaired β-cell function and/or cell death, as shown for coxsackie B virus (CVB) types 4 and 5, or have no apparent immediate adverse effects, as shown for CVA-9. Because of the limited availability of human pancreatic β-cells, the aim of this study was to find out if foetal porcine pancreatic islets could be used as a substitute in enterovirus (EV) screening. These cells resemble human β-cells in several biological properties. CVB infection resulted in a rapid progressive decline of insulin content and reponsiveness to insulin release. The amount of virus inoculum sufficient for this destruction was small, corresponding to only 55 infectious units per pancreas. In contrast to CVBs, CVA-9 replicated poorly, and sometimes not at all, in foetal porcine β-cells. The first signs of functional impairment and cell destruction, if present at all, were seen only after 1–3 weeks of incubation. Furthermore, CVA-16, several strains of echoviruses and human parechovirus type 1 were unable to replicate in foetal porcine pancreatic β-cells. Based on these results, foetal porcine islets are somewhat more sensitive to CVB infection than adult human islets, whereas many other human EV strains do not infect porcine β-cells. Therefore, foetal porcine β-cells cannot be used for systematic screening of human EV strains and isolates for β-cell tropism, but they might provide a useful model for detailed studies on the interaction of CVBs with β-cells.
Mitogen stimulation favours replication of equine herpesvirus-1 in equine blood mononuclear cells by inducing cell proliferation and formation of close intercellular contacts
Authors: van der Meulen Karen M. , Nauwynck Hans J. , Pensaert Maurice B.
Published: August 2015

In the present study, equine herpesvirus-1 (EHV-1)-infected cells were identified in ionomycin/phorbol dibutyrate (IONO/PDB)-stimulated peripheral blood mononuclear cells (PBMC) and the mechanism by which stimulation increases the percentage of infected cells was examined. In the population of viral antigen-positive PBMC, 38·4±4·5% were CD5+T-lymphocytes (18·1±3·2% CD4+13·6±1·8% CD8+), 18·1±5·4% were B-lymphocytes, 8·5±3·9% were monocytes and 35% remained unidentified. The role of the cell cycle in the increased susceptibility to EHV-1 upon stimulation was examined by stimulating PBMC for 0, 12, 24 or 36 h prior to inoculation. A high correlation was found between the increase of cells in the S- (r=0·974) and G2/M-phase (r=0·927) at the moment of inoculation and the increase of infected cells at 12 h post-inoculation (p.i.). This suggests that a specific stage of the S-phase or S- and G2/M-phase facilitates virus replication. At 24 h p.i. lower correlations were found, suggesting that other effects are involved. From 12 h after addition of IONO/PDB, formation of clusters of PBMC became manifest. We examined whether close intercellular contacts in these clusters facilitated cell-to-cell transmission of EHV-1. Between 8 and 17 h p.i., the percentage of clusters containing adjacent infected cells increased from 1·6 to 13·4% and the maximal number of adjacent infected cells increased from two to four. Confocal microscopy visualized close intercellular contacts between adjacent infected cells. It can be concluded that mitogen stimulation favours EHV-1 infection of PBMC (i) by initiating specific cell cycle events and (ii) by inducing formation of clusters, thereby facilitating transmission of virus between cells.
Perforin and Fas in murine gammaherpesvirus-specific CD8+ T cell control and morbidity
Authors: Topham David J. , Cardin Rhonda C. , Christensen Jan P. , Brooks James W. , Belz Gabrielle T. , Doherty Peter C.
Published: August 2015

The immune system uses both virus-specific T cells and B cells to control the acute and latent phases of respiratory infection with the murine gammaherpesvirus 68 (γHV-68). We sought to further define the important effector mechanisms for CD8+T cells. First, depletion of the CD4+T cells resulted in a failure of most animals to drive the virus into latency, although lytic virus in the lung was reduced by approximately 1000-fold from its peak. Second, the absence of either perforin or Fas alone had no impact on the ability to reduce titres of lytic virus in the lung. Further neutralization of IFN-γ in CD4-depleted P+/+, P−/−or Fas−/−mice had no effect. To define the requirements for Fas or perforin more clearly, two sets of chimeric mice were constructed differing in perforin expression by the T cells, and Fas on infected epithelial cells or lymphocytes. Animals with P−/−T cells and a Fas−/−lung failed to limit the shedding of infectious virus, regardless of whether CD4 T cells were present. In addition, we noted that having P−/−T cells in irradiated Fas+/+hosts caused a lethal disease that was not apparent in the non-chimeric (unirradiated) P−/−(Fas+/+) mice. In another set of chimeric mice, P−/−T cells were able to limit persistent infection of B cells that expressed Fas, but not B cells that were Fas-deficient. These studies demonstrate that some degree of cytotoxicity via either perforin or Fas is essential for CD8+T cells to control this DNA virus.
Cellular prion protein status in sheep: tissue-specific biochemical signatures
Authors: Moudjou Mohammed , Frobert Yveline , Grassi Jacques , La Bonnardière Claude
Published: August 2015

Expression of the cellular prion protein PrPC is sine qua none for the development of transmissible spongiform encephalopathy and thus for the accumulation of the illness-associated conformer PrPSc. Therefore, the tissue distribution of PrPC at the protein level in both quantitative and qualitative terms was investigated. PrPC was quantified using a two-site enzyme immunometric assay which was calibrated with purified ovine recombinant prion protein (rPrP). The most PrPC-rich tissue was the brain, followed by the lungs, skeletal muscle, heart, uterus, thymus and tongue, which contained between 20- and 50-fold less PrPC than the brain. The PrPC content of these tissues seems to be comparable between sheep. Other organs, however, showed different, but low, levels of the protein depending on the animal examined. This was also the case for tissues from the gastrointestinal tract. The tissue containing the lowest concentration of PrPC was shown to be the liver, where PrPC was found to be between 564- and 16000-fold less abundant than in the brain. PrPC was concentrated from crude cellular extracts by immunoprecipitation using several monoclonal and polyclonal anti-ovine PrP antibodies. Interestingly, it was observed that the isoform profile of PrPC was tissue-specific. The most atypical electrophoretic profile of PrPC was found in the skeletal muscle, where two polypeptides of 32 and 35 kDa were detected.
Genomic and evolutionary characterization of TT virus (TTV) in tupaias and comparison with species-specific TTVs in humans and non-human primates
Authors: Okamoto Hiroaki , Nishizawa Tsutomu , Takahashi Masaharu , Tawara Akio , Peng Yihong , Kishimoto Junichi , Wang Yu
Published: August 2015

TT virus (TTV) was recovered from the sera of tupaias (Tupaia belangeri chinensis) by PCR using primers derived from the noncoding region of the human TTV genome, and its entire genomic sequence was determined. One tupaia TTV isolate (Tbc-TTV14) consisted of only 2199 nucleotides (nt) and had three open reading frames (ORFs), spanning 1506 nt (ORF1), 177 nt (ORF2) and 642 nt (ORF3), which were in the same orientation as the ORFs of the human prototype TTV (TA278). ORF3 was presumed to arise from a splicing of TTV mRNA, similar to reported human TTVs whose spliced mRNAs have been identified, and encoded a joint protein of 214 amino acids with a Ser-, Lys- and Arg-rich sequence at the C terminus. Tbc-TTV14 was less than 50% similar to previously reported TTVs of 3·4–3·9 kb and TTV-like mini viruses (TLMVs) of 2·8–3·0 kb isolated from humans and non-human primates, and known animal circoviruses. Although Tbc-TTV14 has a genomic length similar to animal circoviruses (1·8–2·3 kb), Tbc-TTV14 resembled TTVs and TLMVs with regard to putative genomic organization and transcription profile. Conserved motifs were commonly observed in the coding and noncoding regions of the Tbc-TTV14 genome and in all TTV and TLMV genomes. Phylogenetic analysis revealed that Tbc-TTV14 is the closest to TLMVs, and is closer to TTVs isolated from tamarin and douroucouli than to TTVs isolated from humans and chimpanzees. These results indicate that tupaias are naturally infected with a new TTV species that has not been identified among primates.