bluuebunny/crossref_metadata_2025 huggingface dataset:
GB virus C/hepatitis G virus replicates in human haematopoietic cells and vascular endothelial cells
Authors: Handa Atsushi , Brown Kevin E.
Published: August 2015

A novel flavivirus, GB virus C (GBV-C)/hepatitis G virus (HGV), has been detected in chronic liver disease patients. It is known that the viral RNA can be detected in ∼5% of American blood donors. However, the implications for liver disease and the sites of virus replication remain unknown. Possible sites of virus replication were studied by using cell lines and/or primary cells derived from human lymphoid cells, myeloid cells, hepatocytes and endothelial cells. RNA was detected by virus strand-specific RT–PCR and GBV-C/HGV antigen was detected with a rabbit polyclonal anti-E2 (envelope 2) antibody by Western blot analysis. Negative-strand RNA, representative of replicating virus, was detected in lymphoid and megakaryocytoid cell lines and primary vascular endothelial cells. In addition, an increase in virus titre over time was demonstrated and viral antigen was detected, and virus could be passaged to infect fresh cells. However, viral RNA or antigen could not be detected in any of the hepatocyte lines tested. These results indicate that the replication site of GBV-C/HGV is not primarily in hepatocytes and that detection of replicating virus in hepatic tissue may reflect virus replication in haematopoietic cells and/or vascular endothelial cells present in the liver.
Interaction with CBP/p300 enables the bovine papillomavirus type 1 E6 oncoprotein to downregulate CBP/p300-mediated transactivation by p53
Authors: Zimmermann Holger , Koh Choon-Heng , Degenkolbe Roland , O’Connor Mark J. , Müller Andreas , Steger Gertrud , Chen Jason J. , Lui Yun , Androphy Elliot , Bernard Hans-Ulrich
Published: August 2015

The E6 oncoprotein of bovine papillomavirus type 1 (BPV-1) can transform cells independently of p53 degradation. The precise mechanisms underlying this transformation are not yet completely understood. Here it is shown that BPV-1 E6 interacts with CBP/p300 in the same way as described for the E6 proteins of oncogenic human papillomaviruses. This interaction results in an inhibition of the transcriptional coactivator function of CBP/p300 required by p53 and probably by other transcription factors. The comparison of the CBP/p300-binding properties of BPV-1 E6 mutants previously characterized in transcription and transformation studies suggests (i) that the E6–CBP/p300 interaction may be necessary, but not sufficient, for cell transformation, and (ii) that the transcriptional activator function, inherent to the E6 protein, is not derived from forming a complex with CBP/p300.
Characterization of the herpesvirus saimiri ORF73 gene product
Authors: Hall Kersten T. , Giles Mathew S. , Goodwin Delyth J. , Calderwood Michael A. , Markham Alexander F. , Whitehouse Adrian
Published: August 2015

The herpesvirus saimiri (HVS) gene product encoded by ORF73 shares a limited homology with the ORF73 encoded protein of Kaposi’s sarcoma-associated herpesvirus (KSHV). It has recently been shown that the KSHV ORF73 protein is expressed during a latent infection and co-localizes with host cell chromosomes, suggesting that it plays a role in episomal maintenance by tethering viral genomes to host cell chromosomes. At present the role of the HVS ORF73 gene product is unknown. However, the expression of HVS ORF73 in a stably transduced human carcinoma cell line, where the HVS genome persists as a non-integrated circular episome, has recently been shown. In this report, the characterization of the HVS ORF73 protein and the mapping of its functional domains are described. The results suggest that the HVS ORF73 gene encodes a 64 kDa nuclear protein. Moreover, the amino terminus contains two functional nuclear localization signals, whereas the carboxy terminus is required for the distinctive speckled nuclear distribution pattern as observed with both the HVS and KSHV ORF73 proteins.
The antigenic domain 1 of human cytomegalovirus glycoprotein B contains an intramolecular disulphide bond
Authors: Speckner Andrea , Kropff Barbara , Knör Susanne , Mach Michael
Published: August 2015

Glycoprotein B (gB, gpUL55) is the major antigen recognized by the neutralizing humoral immune response against human cytomegalovirus (HCMV). The immunodominant region on gB is the antigenic domain 1 (AD-1), a complex structure that requires a minimal continuous sequence of more than 75 amino acids (aa 552–635) for antibody binding. In this study, the structural requirements for antibody binding to AD-1 have been determined. The domain was expressed in prokaryotic and eukaryotic systems and analysed in immunoblots under reducing and non-reducing conditions. In addition, AD-1 was purified in an immunologically active form and the concentration of sulphydryl groups was determined. The data clearly show that the only form that is recognized by antibodies is a disulphide-linked monomer of AD-1. The disulphide bond is formed between cysteines at amino acid positions 573 and 610 of gB.
The in vivo effects of recombinant bovine herpesvirus-1 expressing bovine interferon-γ
Authors: Raggo Camilo , Habermehl Monique , Babiuk Lorne A. , Griebel Philip
Published: August 2015

To study the biological relevance of using bovine herpesvirus-1 (BHV-1) as a vector for expressing cytokines, a BHV-1 virus that expressed bovine interferon-γ (IFN-γ) was constructed. This recombinant virus (BHV-1/IFNγ) was then used to infect the natural host in a respiratory disease model.In vitrocharacterization of the recombinant interferon-γ confirmed that the cytokine expressed in BHV-1-infected cells was biologically active. Thein vivoeffects of the recombinant IFN-γ were then analysed during a primary infection and after reactivation of a latent infection. During the primary infection, similar body temperature, clinical responses and virus shedding were observed for calves infected with either recombinant BHV-1/IFNγ or parental gC−/LacZ+virus. An analysis of cellular and humoral responses did not reveal any significant immunomodulation by BHV-1/IFNγ during the primary infection. The stability and activity of recombinant IFN-γ was also analysed following the establishment of a latent infection. The presence of recombinant IFN-γ did not significantly alter virus shedding following reactivation. The isolation of reactivated BHV-1/IFNγ virus confirmed that a functional IFN-γ gene was retained during latency. Thus, herpesviruses may provide virus vectors that retain functional genes during latency and recrudescence.
Equine infectious anaemia virus proteins with epitopes most frequently recognized by cytotoxic T lymphocytes from infected horses
Authors: McGuire Travis C. , Leib Steven R. , Lonning Scott M. , Zhang Wei , Byrne Katherine M. , Mealey Robert H.
Published: August 2015

Efficacious lentiviral vaccines designed to induce cytotoxic T lymphocytes (CTL) in outbred populations with a diverse repertoire of MHC class I molecules should contain or express multiple viral proteins. To determine the equine infectious anaemia virus (EIAV) proteins with epitopes most frequently recognized by CTL from seven horses infected for 0·5 to 7 years, retroviral vector-transduced target cells expressing viral proteins were used in CTL assays. Gag p15 was recognized by CTL from 100% of these infected horses. p26 was recognized by CTL from 86%, SU and the middle third of Pol protein were each recognized by 43%, TM by 29%, and S2 by 14%. Based on these results, it is likely that a construct expressing the 359 amino acids constituting p15 and p26 would contain epitopes capable of stimulating CTL in most horses.
Simian immunodeficiency virus resistance of macaques infused with interferon β-engineered lymphocytes
Authors: Matheux Franck , Lauret Evelyne , Rousseau Véronique , Larghero Jérôme , Boson Bertrand , Vaslin Bruno , Cheret Arnaud , De Maeyer Edward , Dormont Dominique , LeGrand Roger
Published: August 2015

To test thein vivoanti-simian immunodeficiency virus (SIV) efficacy of interferon (IFN)-β-engineered lymphocytes, peripheral blood lymphocytes harvested from two uninfected macaques were transduced with a retroviral vector carrying a constitutively expressed IFN-β gene and reinfused, resulting in approximately 1 IFN-β-transduced cell out of 1000 circulating cells. The gene-modified cells were well tolerated and could be detected for at least 74 days without causing any apparent side effects. These two animals together with three untreated control macaques were then infected with SIVmac251. The two IFN-β-infused macaques are in good health, 478 days after infection, with a reduced plasma virus load and sustained numbers of CD4+and CD8+cells. Throughout the study, the proportion of IFN-β-transduced cells has been maintained. Of the three control macaques, two were characterized by a high plasma virus load and a decrease in CD4+cells. One was moribund and was sacrificed 350 days after infection and the other now has fewer than 100 circulating CD4+cells/ml. Unexpectedly, the third control macaque, which, like the two IFN-β-infused animals, had a low plasma virus load and a maintenance of CD4+and CD8+cell number, was characterized by a permanent level of serum IFN-β, of unknown origin, already present before SIV infection. Although no definite conclusion can be made in view of the limited number of animals, these data indicate that further exploration is warranted of an IFN-β-based anti-human immunodeficiency virus gene therapy.
Construction of a full-length infectious cDNA clone of swine vesicular disease virus strain NET/1/92 and analysis of new antigenic variants derived from it
Authors: Rebel J. M. J. , Leendertse C. H. , Dekker A. , van Poelwijk F. , Moormann R. J. M.
Published: August 2015

The Dutch swine vesicular disease virus (SVDV) isolate NET/1/92 was one of the first isolates belonging to a new SVDV antigenic group. This strain was completely sequenced and was shown to have 93% similarity with the UKG/27/72 isolate. To enable antigenicity, replication, maturation and pathogenicity studies of NET/1/92, an infectious full-length cDNA clone, designated pSVD146, was prepared. The in vitro and in vivo biological properties of the virus derived from pSVD146 were studied by analysing antigenicity, plaque morphology, growth curves and virulence in pigs. The epitopes of newly prepared monoclonal antibodies were roughly mapped by fusion-PCR. Fine mapping of epitopes at the amino acid level was achieved by introducing single amino acid mutations in pSVD146. Two new amino acids important in epitope formation were located in VP1; one was mapped in the C-terminal end and the second is thought to be located in the H–I loop. Growth curve and plaque sizes in vitro were similar between virus derived from pSVD146 and the parent wild-type virus. In virulence studies in pigs, the lesions score, neutralization titres and the seroconversion rates were comparable between virus derived from pSVD146 and the parent strain. Since virus derived from pSVD146 had the same biological properties as the parent strain NET/1/92, the full-length infectious cDNA clone pSVD146 will be very useful in studies of the antigenicity, virulence, pathogenesis, maturation and replication of SVDV.
Natural recombination between Tomato yellow leaf curl virus-Is and Tomato leaf curl virus
Authors: Navas-Castillo J. , Sánchez-Campos S. , Noris E. , Louro D. , Accotto G. P. , Moriones E.
Published: August 2015

The complete genome sequences (2791 and 2793 nt) of isolates ofTomato yellow leaf curl virus-Is (TYLCV-Is) from Spain (SP72/97) and Portugal (Port2/95) were determined. These isolates are closely related to TYLCV-Is isolates reported in Japan (Japan-A and Japan-S) and Israel (Israel/Mild). Comparison of all sequenced isolates of TYLCV-Is showed that part of the genome comprising the intergenic region and the 5′-end of therepgene of the Iran and Israel isolates was not closely related to that of other isolates. Phylogenetic analyses suggest that the Israel and Iran isolates may have chimeric genomes that have arisen by recombination between TYLCV-Is-like and tomato leaf curl virus (ToLCV)-like ancestors. The TYLCV-Is donors of the Iran and the Israel genomes were closely related to each other and to other known TYLCV-Is isolates. However, the ToLCV donors differed from each other, although both were related to ToLCV isolates from India (Bangalore-2 and Bangalore-4).
PrPC expression in the peripheral nervous system is a determinant of prion neuroinvasion
Authors: Glatzel Markus , Aguzzi Adriano
Published: August 2015

Transmissible spongiform encephalopathies are often propagated by extracerebral inoculation. The mechanism of spread from peripheral portals of entry to the central nervous system (neuroinvasion) is complex: while lymphatic organs typically show early accumulation of prions, and B-cells and follicular dendritic cells are required for efficient neuroinvasion, actual entry into the central nervous system occurs probably via peripheral nerves and may utilize a PrPC-dependent mechanism. This study shows that transgenic mice overexpressing PrPCundergo rapid and efficient neuroinvasion upon intranerval and footpad inoculation of prions. These mice exhibited deposition of the pathological isoform of the prion protein (PrPSc) and infectivity in specific portions of the central and peripheral sensory pathways, but almost no splenic PrPScaccumulation. In contrast, wild-type mice always accumulated splenic PrPSc, and had widespread deposition of PrPScthroughout the central nervous system even when prions were injected directly into the sciatic nerve. These results indicate that a lympho-neural sequence of spread occurs in wild-type mice even upon intranerval inoculation, while overexpression of PrPCleads to substantial predilection of intranerval over lymphoreticular spread. The rate of transport of infectivity in peripheral nerves was ca. 0·7 mm per day, and prion infectivity titres of sciatic nerves were much higher intga20than in wild-type mice, suggesting that overexpression of PrPCmodulates the capacity for intranerval transport.
Functional analysis of an epitope in the S2 subunit of the murine coronavirus spike protein: involvement in fusion activity
Authors: Taguchi Fumihiro , Shimazaki Yohko K.
Published: August 2015

The monoclonal antibody (MAb) 5B19.2, which has virus-neutralizing and fusion inhibition activities, binds to an epitope (S2A) consisting of nine hydrophobic amino acids in the S2 subunit of the mouse hepatitis virus (MHV) spike (S) protein. This suggests that the S2A epitope may be involved in binding the virus to the MHV receptor and/or in virus–cell fusion. Co-immunoprecipitation analyses demonstrated that while the binding of virus to the receptor was blocked by anti-S1 MAbs, it was not blocked by the S2A antiserum, indicating that S2A was not involved in receptor-binding. The S proteins prepared in this study with mutations in the S2A epitope were either fusogenic or non-fusogenic and their fusogenicity did not correlate with the hydrophobic feature of the S2A epitope. All of these wt and mutated S proteins were similarly transported onto the cell membrane independent of their fusogenicity capability. These results suggest that S2A may mediate the fusion activity of the MHV S protein during virus entry into cells.
Identification of a novel variant of hepatitis E virus in Austria: sequence, phylogenetic and serological analysis
Authors: Worm Harald C. , Schlauder George G. , Wurzer Herbert , Mushahwar Isa K.
Published: August 2015

We isolated a novel hepatitis E virus (HEV-Au1) variant from a patient in Austria suffering from acute viral hepatitis, who had no known risk factors for acquiring hepatitis E. The clinical presentation and initial serological findings have been reported previously. In this paper we report the results of sequence and phylogenetic analysis of HEV products from viral RNA isolated from acute phase serum. The results show that HEV-Au1 is significantly divergent from other HEV isolates. The nucleotide identity of analysed fragments from the novel isolate ranges from 76·6 to 78·4% when compared to isolates from endemic regions and 84·6 to 87·9% when compared to isolates from non-endemic regions. Divergent results were obtained when serum samples taken from the convalescent phase of disease were tested with three different immunoassays (EIAs). An EIA based on United States isolate-specific peptides showed enhanced reactivity whereas EIAs based on recombinant proteins derived from prototype HEV strains from Burma and Mexico were unable to detect antibodies to HEV (anti-HEV) in late phase serum. The findings verify the presence of an additional HEV variant in an industrialized country and provide information about possible problems in detecting anti-HEV.
T-cell line adaptation of human immunodeficiency virus type 1 strain SF162: effects on envelope, vpu and macrophage-tropism
Authors: Dejucq Nathalie , Simmons Graham , Clapham Paul R.
Published: August 2015

Changes in co-receptor-use by human immunodeficiency virus type 1 (HIV-1) strains are relatively rarein vivo. Here we describe two variants derived from the CCR5-using strain SF162, selected for replication in the C8166 T-cell line. Amino acid substitutions in the V3 loop conferred CXCR4-use; however, the loss of macrophage-tropism by one variant was due to a single mutation in the start codon ofvpu. We discuss how V3 loop andvpumutations acquired by replication in T-cell linesin vitrocorrelate with similar changes reported for primary isolates and HIV-1 sequencesin vivo.
T-tropic human immunodeficiency virus (HIV) type 1 Nef protein enters human monocyte–macrophages and induces resistance to HIV replication: a possible mechanism of HIV T-tropic emergence in AIDS
Authors: Alessandrini Laura , Santarcangelo Anna Claudia , Olivetta Eleonora , Ferrantelli Flavia , d’Aloja Paola , Pugliese Katherina , Pelosi Elvira , Chelucci Cristiana , Mattia Gianfranco , Peschle Cesare , Verani Paola , Federico Maurizio
Published: August 2015

Increasing interest has been devoted to the role that monocyte–macrophages play in the pathogenesis of AIDS. The hypothesis of an involvement in AIDS pathogenesis of human/simian immunodeficiency virus (HIV/SIV) Nef also is currently under evaluation by many investigators. The original basis of this hypothesis came from evidence that monkeys infected with a nef-deleted SIV strain failed to develop simian AIDS. Here, we show that treatment of human monocyte-derived macrophages (MDM) with recombinant HIV-1 Nef protein (rNef) induces a strong inhibition of the replication of either macrophage (M-) or dual-tropic HIV-1 strains. Through cytofluorimetric analyses, we detected internalization of FITC-conjugated rNef in MDM as early as 6 h after treatment. Confocal microscope observations demonstrated that the intracellular distribution of internalized rNef was identical to that of endogenously produced Nef. Down-regulation of the CD4 HIV receptor detected upon rNef treatment of MDM suggested that the rNef-induced HIV inhibition occurred at the virus entry step. This deduction was strengthened by the observation that CD4-independent infection was totally insensitive to rNef treatment. The specificity of all observed effects was demonstrated by immunodepletion of rNef. Finally, we showed that the resistance to HIV replication induced by rNef treatment in MDM favours the spread of T-tropic over M-tropic HIV strains in doubly infected CD4+ lymphocyte–MDM co-cultures. We propose that extracellular Nef contributes to AIDS pathogenesis by inducing resistance to M-tropic HIV replication in MDM, thereby facilitating the switching from M- to T-tropic HIV prevalence that correlates frequently with AIDS progression.
Use of a TT virus ORF1 recombinant protein to detect anti-TT virus antibodies in human sera
Authors: Ott Catherine , Duret Laurent , Chemin Isabelle , Trépo Christian , Mandrand Bernard , Komurian-Pradel Florence
Published: August 2015

TT virus (TTV), isolated initially from a Japanese patient with hepatitis of unknown aetiology, has since been found to infect both healthy and diseased individuals and numerous prevalence studies have raised questions about its role in unexplained hepatitis. In order to determine the prevalence of ongoing TTV infection as well as resolved infection, a serological study was performed with a recombinant protein generated from the open reading frame 1 (ORF1) sequence isolated from a French patient infected by TTV. The C-terminal end of the ORF1 protein, containing a particularly hydrophilic region, was retained to be used as antigen to detect the presence of anti-TTV antibodies in serum samples by a Western blot analysis. For this purpose, the C-terminal ORF1 region was expressed in fusion with a hexahistidine tail in E. coli and purified by metal-chelate affinity chromatography. The serological screening of 70 human sera, including 30 patients with hepatitis of unknown aetiology, 30 blood donors and 10 healthy children, allowed the immune response of infected hosts to be identified by the detection of TTV-specific antibodies, with a very high prevalence of 98·6% in the human sera tested. In contrast, TTV DNA was detected by PCR in only 76·1% of the tested sera. The use of the ORF1 C-terminal recombinant protein thereby provided a diagnostic tool to follow the immune response of the host against TTV.
Molecular variants of human papillomavirus types 16 and 18 preferentially associated with cervical neoplasia
Authors: Villa Luisa L. , Sichero Laura , Rahal Paula , Caballero Otavia , Ferenczy Alex , Rohan Tom , Franco Eduardo L.
Published: August 2015

In order to determine geographically related intratypic variation in human papillomavirus (HPV) type 16 and 18 isolates that could be associated with lesion development, data were analysed from an ongoing cohort study of the natural course of infection of HPVs and cervical neoplasia. Testing for HPVs was carried out by PCR and molecular variants of these HPVs were characterized by sequence analysis of the long control region and by dot blot hybridization of the E6 and L1 genes. Tests for HPV were done in multiple first-year specimens from 1690 women enrolled in a cancer screening program from 1993 to 1997. Subjects were followed-up by cytology and cervicography for detection of cervical lesions. Seven variants of HPV-16 and four of HPV-18 were detected in one or more specimens from 65 subjects. The same variant was found in specimens taken on different visits from each case of persistent infection. Overall, non-European variants tended to persist more frequently [odds ratio (OR)=4·5; 95% confidence interval (CI), 1·6–12·4] than European (E) variants (OR=2·5; 95% CI, 1·3–4·9), relative to the risk of persistence for non-oncogenic HPVs. In addition, non-E variants were more strongly associated with risk of both prevalent (age- and race-adjusted OR=172·2; 95% CI, 47·1–630·1) and incident [relative risk (RR)=22·5; 95% CI, 6·0–83·9] high-grade lesions than E variants (prevalent lesions OR=46·3; 95% CI, 15·5–138·0 and incident lesons RR=6·1; 95% CI, 1·3–27·4), relative to the risk for HPV-negative women. Although consistent, the latter differences were not statistically significant. If confirmed in other populations, measurement of intratypic variation of HPV-16 and -18 has the potential to serve as an ancillary tool in cervical cancer screening.
Phylogenetic position of the Diadromus pulchellus ascovirus DNA polymerase among viruses with large double-stranded DNA genomes
Authors: Stasiak Karine , Demattei Marie-Véronique , Federici Brian A. , Bigot Yves
Published: August 2015

The Ascoviridae is a family of large double-stranded (ds) DNA insect viruses that contains four species, the Spodoptera frugiperda (SfAV1), Trichoplusia ni (TnAV2), Heliothis virescens (HvAV3) and Diadromus pulchellus (DpAV4) ascoviruses. These are unique among insect viruses in that the primary means of transmission among their lepidopteran hosts is generally by being vectored mechanically by hymenopteran parasitoids. Ascoviruses are similar in virion structure, but their relationships with their parasitoid vectors vary from being opportunistic to obligate. Little is known, however, about the relatedness of these viruses to one another or to other large dsDNA viruses. We therefore cloned and sequenced the δ DNA polymerase gene of DpAV4, characterized it and compared it to 59 eukaryotic and viral δ and ϵ DNA polymerases. Phylogenetic analyses based on these genes revealed that the ascoviruses DpAV4 and SfAV1 formed a group of virus species distinct from, but closely related to, species of the family Iridoviridae. Detailed analyses of the relatedness of ascovirus species based on conserved δ DNA polymerase motifs showed two groups within the family Ascoviridae, one containing DpAV4 and the other containing SfAV1, TnAV2 and HvAV3, which was consistent with their host–vector relationships. Despite significant differences in capsid symmetry between ascoviruses and iridoviruses, these results suggest that these viruses may have originated from a common ancestral virus.
Early accumulation of PrPSc in gut-associated lymphoid and nervous tissues of susceptible sheep from a Romanov flock with natural scrapie
Authors: Andréoletti Olivier , Berthon Patricia , Marc Daniel , Sarradin Pierre , Grosclaude Jeanne , van Keulen Lucien , Schelcher François , Elsen Jean-Michel , Lantier Frédéric
Published: August 2015

The immune system is known to be involved in the early phase of scrapie pathogenesis. However, the infection route of naturally occurring scrapie and its spread within the host are not entirely known. In this study, the pathogenesis of scrapie was investigated in sheep of threePrPgenotypes, from 2 to 9 months of age, which were born and raised together in a naturally scrapie-affected Romanov flock. The kinetics of PrPScaccumulation in sheep organs were determined by immunohistochemistry. PrPScwas detected only in susceptible VRQ/VRQ sheep, from 2 months of age, with an apparent entry site at the ileal Peyer’s patch as well as its draining mesenteric lymph node. At the cellular level, PrPScdeposits were associated with CD68-positive cells of the dome area and B follicles before being detected in follicular dendritic cells. In 3- to 6-month-old sheep, PrPScwas detected in most of the gut-associated lymphoid tissues (GALT) and to a lesser extent in more systemic lymphoid formations such as the spleen or the mediastinal lymph node. All secondary lymphoid organs showed a similar intensity of PrPSc-immunolabelling at 9 months of age. At this time-point, PrPScwas also detected in the autonomic myenteric nervous plexus and in the nucleus parasympathicus nervi X of the brain stem. These data suggest that natural scrapie infection occurs by the oral route via infection of the Peyer’s patches followed by replication in the GALT. It may then spread to the central nervous system through the autonomic nervous fibres innervating the digestive tract.
Replication of Aleutian mink disease parvovirus in mink lymph node histocultures
Authors: Jensen Klaus T. , Wolfinbarger James B. , Aasted Bent , Bloom Marshall E.
Published: August 2015

Aleutian mink disease parvovirus (ADV), causes an immune disorder with a persistent infection of lymphoid organs in adult mink. We studied replication of ADV in gel-supported histocultures prepared from adult mink mesenteric lymph node (MLN). Evidence of virus replication in the histocultures was first observed by indirect immunofluorescence 72 h after incubation with virus. Cells resembling lymphocytes and macrophages contained both ADV capsid (VP2) and nonstructural (NS1 and NS2) proteins, and were present in a distribution suggestive of infected cells within germinal centres. ADV replicative form and encapsidated virion DNA were also detected in infected histocultures at time-points after 72 h. In addition, we were able to passage ADV-Utah to a new round of histocultures. These results suggested that the infected cells were actual target cells for ADV replication and that productive ADV-Utah replication, complete with the generation of virus, was occurring in the histocultures. The mink MLN histocultures provide a system to study the replication and molecular pathogenesis of ADV in target tissues.
Monocyte-derived dendritic cells are permissive to the complete replicative cycle of human cytomegalovirus
Authors: Riegler S. , Hebart H. , Einsele H. , Brossart P. , Jahn G. , Sinzger C.
Published: August 2015

The susceptibility of monocyte-derived immature dendritic cells (DC) to infection by various strains of human cytomegalovirus (HCMV) was analysed. Immature DC were generated by incubation of peripheral blood monocytes with interleukin-4 and granulocyte–macrophage colony-stimulating factor for 7 days and were characterized by a CD1a+/CD40+/CD80+/CD86+/HLA-DR+/CD14− phenotype. Viral antigen expression and production of infectious progeny virus were analysed in infected immature DC cultures. Immature DC were 80–90 % susceptible to HCMV strains that had been propagated in endothelial cell culture, whereas the infection rate was negligible with fibroblast-adapted HCMV strains. Immature DC infection resulted in expression of viral immediate early, early and late genes. Productive infection was proven by the detection of infectious virus in single-step growth curves and in infectious centre assays. It is concluded that HCMV might interfere with the host immune reaction by permissive, lytic infection of immature DC.
A putative latency promoter/enhancer (PLAT2) region of pseudorabies virus contains a virulence determinant
Authors: Boldogköi Zsolt , Erdélyi Ferenc , Fodor István
Published: August 2015

Contradictory data have recently been reported on the role of the unique long–internal repeat junction area of pseudorabies (Aujeszky’s disease) virus (PrV) genome in the virulence of the virus. To investigate the basis of the difference, four recombinant PrVs mutated at the outer region of inverted repeats that involved a putative latency promoter (PLAT2) were constructed in this study. Propagation characteristics of mutant viruses in cultured cells were similar to those of the wild-type virus. However, a 757 bp deletion at this location caused significant reduction in the virulence of PrV after intraperitoneal inoculation of mice and a moderate decrease in the virulence after intracranial inoculation. These results indicate that the PLAT2 region is an important virulence determinant that may be implicated in the neuroinvasive capability of the virus.
Interactions of bovine viral diarrhoea virus glycoprotein Erns with cell surface glycosaminoglycans
Authors: Iqbal Munir , Flick-Smith Helen , McCauley John W.
Published: August 2015

Recombinant Ernsglycoprotein of bovine viral diarrhoea virus (BVDV) has been tagged with a marker epitope or linked to an immunoglobulin Fc tail and expressed in insect and mammalian cell lines. The product was shown to be functional, both having ribonuclease activity and binding to a variety of cells that were permissive and non-permissive for replication of BVDV. Addition of soluble Ernsto the medium blocked replication of BVDV in permissive cells. Binding of epitope-tagged Ernsto permissive calf testes (CTe) cells was abolished and virus infection was reduced when cells were treated with heparinases I or III. Ernsfailed to bind to mutant Chinese hamster ovary (CHO) cells that lacked glycosaminoglycans (pgsA-745 cells) or heparan sulphate (pgsD-677 cells) but bound to normal CHO cells. Ernsalso bound to heparin immobilized on agarose and could be eluted by heparin and by a high concentration of salt. Flow cytometric analysis of Ernsbinding to CTe cell cultures showed that glycosaminoglycans such as heparin, fucoidan and dermatan sulphate all inhibit binding but dextran sulphate, keratan sulphate, chondroitin sulphate and mannan fail to inhibit binding. The low molecular mass polysulphonated inhibitor suramin also inhibited binding to CTe cells but poly-l-lysine did not. Furthermore, suramin, the suramin analogue CPD14, fucoidan and pentosan polysulphate inhibited the infectivity of virus. It is proposed that binding of Ernsto cells is through an interaction with glycosaminoglycans and that BVDV may bind to cells initially through this interaction.
Agnoprotein 1a and agnoprotein 1b of avian polyomavirus are apoptotic inducers
Authors: Johne Reimar , Jungmann Annett , Müller Hermann
Published: August 2015

Avian polyomavirus (APV) causes an acute fatal disease in a variety of avian species. DNA laddering indicating apoptosis was demonstrated in APV-infected chicken embryo (CE) cells. DNA laddering, however, was not observed in Vero cells infected with mammalian polyomavirus simian virus 40. Expression of APV agnoprotein 1a and agnoprotein 1b induced apoptosis in insect cells and CE cells. An APV full-length plasmid transfected in CE cells induced apoptosis, and infectious virus was produced. After transfection of CE cells with a plasmid containing a mutated initiation codon for agnoprotein 1a and agnoprotein 1b, however, a considerably lower number of apoptotic cells was observed, and no infectious progeny was produced.
Foot-and-mouth disease virus is a ligand for the high-affinity binding conformation of integrin α5β1: influence of the leucine residue within the RGDL motif on selectivity of integrin binding
Authors: Jackson Terry , Blakemore Wendy , Newman John W. I. , Knowles Nick J. , Mould A. Paul , Humphries Martin J. , King Andrew M. Q.
Published: August 2015

Field isolates of foot-and-mouth disease virus (FMDV) use RGD-dependent integrins as receptors for internalization, whereas strains that are adapted for growth in cultured cell lines appear to be able to use alternative receptors like heparan sulphate proteoglycans (HSPG). The ligand-binding potential of integrins is regulated by changes in the conformation of their ectodomains and the ligand-binding state would be expected to be an important determinant of tropism for viruses that use integrins as cellular receptors. Currently, αvβ3 is the only integrin that has been shown to act as a receptor for FMDV. In this study, a solid-phase receptor-binding assay has been used to characterize the binding of FMDV to purified preparations of the human integrin α5β1, in the absence of HSPG and other RGD-binding integrins. In this assay, binding of FMDV resembled authentic ligand binding to α5β1 in its dependence on divalent cations and specific inhibition by RGD peptides. Most importantly, binding was found to be critically dependent on the conformation of the integrin, as virus bound only after induction of the high-affinity ligand-binding state. In addition, the identity of the amino acid residue immediately following the RGD motif is shown to influence differentially the ability of FMDV to bind integrins α5β1 and αvβ3 and evidence is provided that α5β1 might be an important FMDV receptorin vivo.
Germinal centre localization of bovine viral diarrhoea virus in persistently infected animals
Authors: Fray M. D. , Supple E. A. , Morrison W. I. , Charleston B.
Published: August 2015

Immunohistochemical analysis of peripheral lymph nodes from gnotobiotic calves persistently infected with bovine viral diarrhoea virus (BVDV) revealed extensive deposition of Ernsand localization of the viral genome in the light zone of germinal centres. Viral antigen co-localized with immunoglobulin in the germinal centres and was shown to be extracellular. Despite the presence of viral antigen in germinal centres, circulating anti-BVDV antibody was not detected. These findings provide evidence that calves persistently infected with BVDV, in the absence of adventitious infection, can generate a B cell response to the persisting virus. The nature of the tolerance in calves persistently infected with BVDV is discussed in light of these findings.
The complete sequence of hepatitis E virus genotype 4 reveals an alternative strategy for translation of open reading frames 2 and 3
Authors: Wang Youchun , Zhang Huayuan , Ling Roger , Li Hemin , Harrison Tim J.
Published: August 2015

Isolates of hepatitis E virus (HEV) have recently been described from China that are distinct from Burmese, Mexican and US viruses and constitute a novel genotype (genotype 4). Here, the complete genomic sequence of a representative isolate of genotype 4 HEV, amplified directly from the stool of an acutely infected patient, is presented. Analysis of the entire sequence confirms our previous conclusion, based upon partial sequence data, that these Chinese isolates belong to a novel genotype. Typical of genetic variation in HEV, most nucleotide substitutions occur in the third base of the codon and do not affect the amino acid sequence. The genotype 4 virus is unusual in that a single nucleotide insertion in the ORF 3 region changes the initiation of ORF 3, and perhaps also ORF 2. The consequences of these changes are discussed.
Caspases are not involved in the cleavage of translation initiation factor eIF4GI during picornavirus infection
Authors: Roberts Lisa O. , Boxall Angela J. , Lewis Louisa J. , Belsham Graham J. , Kass George E. N.
Published: August 2015

Infection of cells by many picornaviruses results in the rapid inhibition of cellular protein synthesis due to cleavage of the translation initiation factor eIF4G. The poliovirus (PV) 2A and foot-and-mouth disease virus (FMDV) L proteases are each sufficient to mediate this cleavage, but the cleavage mechanism may be indirect, involving an unidentified cellular protease(s). eIF4G is also targetted for cleavage by caspase-3 during apoptosis. Here, it is shown that caspase inhibitors do not inhibit the cleavage of eIF4GI during PV or FMDV infection. Similarly, in transient-expression studies, the cleavage of eIF4GI induced by PV 2A or FMDV L was unaffected by these inhibitors. Furthermore, the cleavage of eIF4GI was observed in PV-infected MCF-7 cells lacking caspase-3. These data, and the fact that induction of apoptosis yields different eIF4GI cleavage fragments, indicate that caspases do not have a major role in the cleavage of eIF4GI during PV or FMDV infection.
Human cytomegalovirus UL37 immediate-early regulatory proteins traffic through the secretory apparatus and to mitochondria
Authors: Colberg-Poley Anamaris M. , Patel Mital B. , Erezo Darwin P. P. , Slater Jay E.
Published: August 2015

The human cytomegalovirus (HCMV) UL36–38 immediate-early (IE) locus encodes the UL37 exon 1 (pUL37x1) and UL37 (gpUL37) regulatory proteins, which have anti-apoptotic activities. pUL37x1 shares its entire sequence, including a hydrophobic leader and an acidic domain, with the exception of one residue, with the amino terminus of gpUL37. gpUL37 has, in addition, uniqueN-linked glycosylation, transmembrane and cytosolic domains. A rabbit polyvalent antiserum was generated against residues 27–40 in the shared amino-terminal domain and a mouse polyvalent antiserum was generated against the full-length protein to study trafficking of individual UL37 proteins in human cells that transiently expressed gpUL37 or pUL37x1. Co-localization studies by confocal laser scanning microscopy detected trafficking of gpUL37 and pUL37x1 from the endoplasmic reticulum to the Golgi apparatus in permissive U373 cells and in human diploid fibroblasts (HFF). Trafficking of gpUL37 to the cellular plasma membrane was detected in unfixed HFF cells. FLAG-tagged gpUL37 trafficked similarly through the secretory apparatus to the plasma membrane. By using confocal microscopy and immunoblotting of fractionated cells, gpUL37 and pUL37x1 were found to co-localize with mitochondria in human cells. This unconventional dual trafficking pattern through the secretory apparatus and to mitochondria is novel for herpesvirus IE regulatory proteins.
Characterization of the Epstein–Barr virus BRRF1 gene, located between early genes BZLF1 and BRLF1
Authors: Segouffin-Cariou Carine , Farjot Géraldine , Sergeant Alain , Gruffat Henri
Published: August 2015

The switch from latency to a productive cycle in Epstein–Barr virus (EBV)-infected B cells proliferating in vitro is thought to be due to the transcriptional activation of two viral genes, BZLF1 and BRLF1, encoding two transcription factors called EB1 and R respectively. However, a third gene, BRRF1 is contained in the BZLF1/BRLF1 locus, overlapping with BRLF1 but in inverse orientation. We have characterized the 5′ end of the BRRF1 mRNA and the promoter, PNa, at which BRRF1 pre-mRNA is initiated. We show that although a single BRRF1 mRNA species is induced by 12-O-tetradecanoylphorbol 13-acetate/sodium butyrate in several EBV-infected B cell lines, in Akata cells treated with anti-IgG two BRRF1 mRNAs can be detected. Transcription initiated at the BRRF1 promoter was activated by EB1 but not by R, and EB1-binding sites which contribute to the EB1-activated transcription have been mapped to between positions −469 and +1. A 34 kDa protein could be translated from the BRRF1 mRNA both in vitro and in vivo, and was found predominantly in the nucleus of HeLa cells transfected with a BRRF1 expression vector. Thus there are three promoters in the region of the EBV chromatin containing the BZLF1/BRLF1 genes, two of which, PZ and PNa, potentially share regulatory elements.
Herpesvirus papio encodes a functional homologue of the Epstein–Barr virus apoptosis suppressor, BHRF1
Authors: Meseda Clement A. , Arrand John R. , Mackett Mike
Published: August 2015

The human tumour virus Epstein–Barr virus (EBV) encodes a 17 kDa protein, BHRF1, which is a member of the Bcl-2 family and has been shown to suppress apoptosis. The role of this gene in the life-cycle of EBV has not been fully elucidated. In order to identify motifs conserved in herpesviruses and possibly shed light on its function we isolated a BHRF1 homologue from herpesvirus papio (cercopithecine herpesvirus-12) a closely related gammaherpesvirus of baboons. The gene, hvpBHRF1, also encodes a 17 kDa protein which shares 64% identity and 79% similarity with EBV BHRF1 at the amino acid level. In biological assays, hvpBHRF1 and BHRF1 conferred similar levels of protection on human keratinocytes induced to apoptose with cis-platin.
Establishment of a cell line persistently infected with bovine herpesvirus-4 by use of a recombinant virus
Authors: Donofrio Gaetano , Cavirani Sandro , van Santen Vicky L.
Published: August 2015

Bovine herpesvirus-4 (BHV-4), a gammaherpesvirus lacking a clear disease association, productively infects multiple cell lines of various species and causes cell death. A human rhabdomyosarcoma cell line, RD-4, infected with BHV-4 produced low levels of early and late viral RNAs and infectious virus, but exhibited no cytopathic effect. Using a recombinant BHV-4 containing a neomycin-resistance gene, we established RD-4-derived cell lines persistently infected with BHV-4. The viral genome in these cells was predominantly circular. Because of drug selection, every cell contained a viral genome. In addition, all cells stained with a BHV-4-specific antiserum. Therefore, these cell lines are not carrier cultures. These cells produced infectious virus at all passages tested. Even though cells were selected and maintained at a concentration of geneticin at least 2·5 times that necessary to kill uninfected RD-4 cells, selected cells contained only approximately one viral genome per diploid host cell genome. Persistently infected cells grew more slowly than uninfected cells, even in the absence of drug. The slower growth of these cells suggests that any growth advantage conferred by multiple copies of the neomycin-gene-carrying viral genome might be offset by the detrimental effects of viral gene expression. This situation contrasts with other gammaherpesviruses, which are able to growth-transform cells.
Multiple infection, recombination and genome relationships among begomovirus isolates found in cotton and other plants in Pakistan
Authors: Sanz Ana I. , Fraile Aurora , García-Arenal Fernando , Zhou Xueping , Robinson David J. , Khalid Saif , Butt Tahir , Harrison Bryan D.
Published: August 2015

Begomoviruses occur in many plant species in Pakistan and are associated with an epidemic of cotton leaf curl disease that has developed since 1985. PCR analysis with primer pairs specific for each of four already sequenced types of DNA-A of cotton leaf curl virus (CLCuV-PK types a, 26, 72b and 804a), or for okra yellow vein mosaic virus (OYVMV), indicated that many individual naturally infected plants of cotton and other malvaceous species contained two or three begomovirus sequences. Similarly, sequence differences among overlapping fragments of begomovirus DNA-A, amplified from individual naturally infected plants, indicated much multiple infection in malvaceous and non-malvaceous species. Some cotton plants contained DNA-A sequences typical of begomoviruses from non-malvaceous species, and some non-malvaceous plants contained sequences typical of CLCuV-PK. Some DNA-A sequences were chimaeric; they each included elements typical of different types of CLCuV-PK, or of different malvaceous and/or non-malvaceous begomoviruses. Often an apparent recombination site occurred at the origin of replication. No complete CLCuV-PK DNA-A sequence was found in malvaceous or non-malvaceous species collected in Pakistan outside the area of the cotton leaf curl epidemic but chimaeric sequences, including a part that was typical of CLCuV-PK DNA-A, did occur there. We suggest that recombination among such pre-existing sequences was crucial for the emergence of CLCuV-PK. Recombination, following multiple infection, could also explain the network of relationships among many of the begomoviruses found in the Indian subcontinent, and their evolutionary divergence, as a group, from begomoviruses causing similar diseases in other geographical regions.
Polyomavirus persistence in lymphocytes: prevalence in lymphocytes from blood donors and healthy personnel of a blood transfusion centre
Authors: Dolei Antonina , Pietropaolo Valeria , Gomes Eduarda , Di Taranto Cristiana , Ziccheddu Maria , Spanu Maria A. , Lavorino Claudio , Manca Mario , Degener Anna Marta
Published: August 2015

BK and JC polyomaviruses (BKV and JCV) are widespread in humans and are thought to persist and reactivate under immune alterations. In addition to the kidney, lymphoid cells have been proposed as a site of latency. However, while this was shown to occur in immunocompromised patients, discordant data were published for healthy humans. To help to solve this issue, an extensive study (231 healthy subjects) was carried out on peripheral blood mononuclear cells (PBMC) from blood donors of two towns and from operators of a blood transfusion centre. To discriminate between past and recent infection, nested PCRs for BKV and JCV non-coding control region (NCCR) and VP1 DNA sequences were carried out. Twenty-two per cent of subjects had BKV NCCR, but only 7% also had BKV VP1, as detected by PCR assays of similar sensitivities; the latter positivity was found to decrease with age. In both towns, the BKV WW archetypal DDP strain, subtype I, was found. Only 0·9% of subjects contained JCV DNA, for both NCCR and VP1. Blood operators presented a statistically significant increased prevalence of BKV NCCR (3·0-fold) and BKV VP1 (9·4-fold) sequences with respect to blood donors of comparable ages, suggesting the possibility of occupational risk of BKV (re)infection or reactivation. Since the possibility of amplifying BKV VP1 sequences from PBMC of healthy humans is lost with age, this suggests that PBMC are not a site of polyomavirus persistence in healthy individuals and that detection of BKV VP1 DNA in PBMC is probably indicative of recent infection or reactivation.