bluuebunny/crossref_metadata_2025 huggingface dataset:
Mutagenesis of hepatitis C virus E1 protein affects its membrane-permeabilizing activity
Authors: Ciccaglione A. R. , Costantino A. , Marcantonio C. , Equestre M. , Geraci A. , Rapicetta M.
Published: August 2015

The E1 glycoprotein of hepatitis C virus is a transmembrane glycoprotein with a C-terminal anchor domain. When expressed inEscherichia coli, E1 induces a change in membrane permeability that is toxic to the bacterial cell. The C-terminal hydrophobic region (aa 331–383) of E1 is mainly responsible for membrane association and for inducing changes in membrane permeability. These observed changes are similar to those produced inE. coliby influenza virus M2, human immunodeficiency virus gp41 and poliovirus 3AB proteins, whose hydrophobic domains are thought to cause pore formation in biological membranes. To further characterize the activity of E1 at a molecular level, the membrane-permeabilizing ability of a second internal hydrophobic region (aa 262–291) was examined by expressing different deletion mutants of E1 in anE. colisystem that is widely used for analysing membrane-active proteins from other animal viruses. Moreover, highly conserved amino acids in the C-terminal hydrophobic region were mutated to identify residues that are critical for inducing changes in membrane permeability. Analysis of cell growth curves of recombinant cultures and membrane-permeability assays revealed that synthesis of this fragment increased the flux of small compounds through the membrane and caused progressive cell lysis, suggesting that this domain has membrane-active properties. Furthermore, analysis of C-terminal mutants indicated that the conserved amino acids Arg339, Trp368and Lys370play a critical role in protein function, as both cell lysis and changes in membrane permeability induced by the wild-type clone could be blocked by substitutions in these positions.
Expression and localization of LEF-11 in Autographa californica nucleopolyhedrovirus-infected Sf9 cells
Authors: Lin Guangyun , Slack Jeffrey M. , Blissard Gary W.
Published: August 2015

The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) lef-11 gene was found previously to be necessary to support optimal levels of transient expression from an AcMNPV late promoter. The lef-11 gene is unusual in that it overlaps both upstream (orf38) and downstream (pp31) genes. In this study, the expression and cellular localization of LEF-11 were examined. The lef-11 transcripts were detected from 4 to 36 h post-infection (p.i.). The 1·5 kb lef-11 mRNA initiates 196 nt upstream of the lef-11 translation initiation codon, within the upstream orf38 gene. This relatively long 5′ upstream region encodes a potential small upstream open reading frame (ORF) of 58 amino acids that overlaps the lef-11 ORF. The 3′ end of the lef-11 mRNA was mapped as co-terminal with mRNAs from the downstream pp31 gene. Using affinity purified anti-LEF-11 antibodies, levels of LEF-11 expression were found to be maximal between approximately 8 and 24 h p.i., although LEF-11 could be detected as late as 72 h p.i. Using immunofluorescence microscopy, it was determined that LEF-11 localized to dense regions of infected cell nuclei, consistent with its role as a possible late transcription factor.
Phylogenetic analysis of conserved genes within the ecdysteroid UDP-glucosyltransferase gene region of the slow-killing Adoxophyes orana granulovirus
Authors: Wormleaton S. L. , Winstanley D.
Published: August 2015

A physical map of the genome of Adoxophyes orana granulovirus (AoGV) was constructed for the restriction enzymes BamHI, BglII, EcoRI, PstI and SacI using restriction endonuclease analysis and DNA hybridization techniques. This enabled the size of the AoGV genome to be estimated at 100·9 kbp. A plasmid library covering 99·9% of the AoGV genome was constructed using five restriction enzymes. The ecdysteroid UDP-glucosyltransferase gene (egt) was located by hybridization with the egt gene of Cydia pomonella granulovirus. The sequence of 6000 bp of the egt region is presented and compared to the equivalent area in other GVs. Database searches showed that this region contained eight open reading frames (ORFs) similar to the baculovirus genes egt, granulin, pk-1, me53 and four ORFs of Xestia c-nigrum granulovirus (ORF 178, ORF 2, ORF 7 and ORF 8). The egt gene was shown to encode an active EGT using an EGT assay. Phylogenetic trees of the granulovirus genes egt, granulin, pk-1 and me53 were constructed using maximum parsimony and distance analyses. These analyses indicated that AoGV genes may be more closely related to other tortricid-infecting GVs than to GVs that infect other lepidopteran families.
Evolutionary trends of the first hypervariable region of the hepatitis C virus E2 protein in individuals with differing liver disease severity
Authors: Curran Rebecca , Jameson Claire L. , Craggs Joanna K. , Grabowska Anna M. , Thomson Brian J. , Robins Adrian , Irving William L. , Ball Jonathan K.
Published: August 2015

Hepatitis C virus (HCV) exists as a complex swarm of genetically related viruses known as a quasispecies. Recent work has shown that quasispecies complexity and evolutionary rates are associated with the outcome of acute infection. Knowledge of how the virus population evolves at different stages of chronic infection is less clear. We have studied rates of evolution of the first hypervariable region (HVR1) of the E2 envelope protein in six individuals with disparate liver disease severity. These data show that virus populations present in individuals with mild non-progressive liver disease evolve in a typical Darwinian fashion, with a consistent accumulation of non-synonymous (amino acid-changing) substitutions. By contrast, the virus population remains relatively static in individuals with severe progressive liver disease. Possible mechanisms for this disparity are discussed.
Detection of quantitative trait loci for resistance/susceptibility to pseudorabies virus in swine
Authors: Reiner Gerald , Melchinger Elke , Kramarova Marcela , Pfaff Eberhardt , Büttner Matthias , Saalmüller Armin , Geldermann Hermann
Published: August 2015

This study describes genetic differences in resistance/susceptibility to pseudorabies virus (PrV) between European Large White and Chinese Meishan pigs, with a mapping of quantitative trait loci (QTL) obtained from a genome-wide scan in F2animals. Eighty-nine F2pigs were challenged intranasally at 12 weeks with 105p.f.u. of the wild-type PrV strain NIA-3. For QTL analysis, 85 microsatellite markers, evenly spaced on the 18 porcine autosomes and on the pseudoautosomal region of the X chromosome, were genotyped. All pigs developed clinical signs, i.e. fever, from 3 to 7 days p.i. The pure-bred Large White pigs, the F1and three-quarters of the F2animals, but none of the Meishan pigs, developed neurological symptoms and died or were euthanized. QTLs for appearance/non-appearance of neurological symptoms were found on chromosomes 9, 5, 6 and 13. They explained 10·6–17·9% of F2phenotypic variance. QTL effects for rectal temperature after PrV challenge were found on chromosomes 2, 4, 8, 10, 11 and 16. Effects on chromosomes 9, 10 and 11 were significant on a genome-wide level. The results present chromosomal regions that are associated with presence/absence of neurological symptoms as well as temperature course after intranasal challenge with NIA-3. The QTLs are in proximity to important candidate genes that are assumed to play crucial roles in host defence against PrV.
Expansion in scid mice of Epstein–Barr virus-associated post-transplantation lymphoproliferative disease biopsy material
Authors: Johannessen Ingolfur , Perera Sunimali M. , Gallagher Alice , Hopwood Paul A. , Thomas J. Alero , Crawford Dorothy H.
Published: August 2015

Post-transplant lymphoproliferative disease (PTLD) biopsy material is rarely available in adequate quantity for research. Therefore, the present study was designed to expand biopsy material in scid mice. Epstein–Barr virus (EBV)+ve PTLD samples from five transplant patients were established in scid mice. PCR analysis of immunoglobulin gene rearrangements demonstrated that four of the five biopsies (80%) gave rise to scid tumours which represented the original tumour cell clones. Immunophenotyping showed that these four biopsies (and all scid tumours) expressed all EBV latent genes and a B lymphoblast phenotype; ≤26% T cells were found in the biopsy material whereas scid tumours showed a paucity of T lymphocytes. RT–PCR analysis revealed expression of IL-2, -4, -6, -10 and IFN-γ in all tumour material, suggesting key roles for these factors in tumour growth. The results show that EBV+ve PTLD material can be expanded in scid mice giving rise to quantities of homogeneous malignant tissue sufficient for research studies.
Latent nuclear antigen of Kaposi’s sarcoma herpesvirus/human herpesvirus-8 induces and relocates RING3 to nuclear heterochromatin regions
Authors: Mattsson Karin , Kiss Csaba , Platt Georgina M. , Simpson Guy R. , Kashuba Elena , Klein George , Schulz Thomas F. , Szekely Laszlo
Published: August 2015

LANA, the major latency-associated nuclear antigen of Kaposi’s sarcoma herpesvirus/human herpesvirus-8 (KSHV/HHV-8), binds RING3 protein, one of five human homologues of thefsh(female sterile homeotic) gene product ofDrosophila. In KSHV/HHV-8-infected cells LANA and the viral episomes accumulate in heterochromatin-associated nuclear bodies. Here we show that in several KSHV/HHV-8-negative cell lines derived from carcinomas, sarcomas and lymphomas, RING3 was expressed at low levels, primarily localized to the euchromatin, and dissociated from the chromosomes during mitosis. In contrast, in KSHV/HHV-8-infected body cavity lymphoma cells the bulk of RING3 localizes to the LANA nuclear bodies and remains associated with the chromosomes during cell division. KSHV/HHV-8-infected body cavity lymphoma cells expressed RING3 at much higher levels than cells without the virus. Transfection of full-length LANA, but not the C terminus alone, greatly induced RING3 gene expression, and LANA and RING3 co-localized even in the transfected cells, in the absence of KSHV/HHV-8 viral DNA. High levels of LANA expression led to the disappearance of heterochromatin in both human and mouse cells. We suggest that LANA and RING3 may create a local euchromatic microenvironment around the viral episomes that are anchored to the heterochromatin.
Characterization of a vaccinia-like virus isolated in a Brazilian forest
Authors: da Fonseca Flávio G. , Trindade Giliane S. , Silva Ricardo L. A. , Bonjardim Cláudio A. , Ferreira Paulo C. P. , Kroon Erna G.
Published: August 2015

The SPAn232 virus (SPAnv) was isolated from sentinel mice in the forest of Cotia, São Paulo, Brazil. It was grouped originally as a Cotia virus (CV) sample due to serological cross-reaction with the latter. However, SPAnv presented genetic characteristics that differed from CV and indicated that SPAnv is a member of the vaccinia virus (VV) subgroup. SPAnv showed a HindIII-digested DNA pattern similar to those of the WR and Lister strains of VV. Also, SPAnv presented genes homologous to the vaccinia growth factor, thymidine kinase and A-type inclusion (ATI) genes from VV. RFLP analysis of the SPAnv ATI homologous gene indicated that the virus belongs to the VV group. Nucleotide sequences from SPAnv genes showed up to 99% similarity with the same genes from VV. Such a relationship was confirmed visually through the drawing of phylogenetic trees. The results point out the occurrence of a VV strain that is possibly in active circulation in the forests of Southeast Brazil.
Sequence analysis of the GP, NP, VP40 and VP24 genes of Ebola virus isolated from deceased, surviving and asymptomatically infected individuals during the 1996 outbreak in Gabon: comparative studies and phylogenetic characterization
Authors: Leroy Eric M. , Baize Sylvain , Mavoungou Elie , Apetrei Cristian
Published: August 2015

The aims of this study were to determine if the clinical outcome of Ebola virus (EBOV) infection is associated with virus genetic structure and to document the genetic changes in the Gabon strains of EBOV by sequencing the GP, NP, VP40 and VP24 genes from deceased and surviving symptomatic and asymptomatic individuals. GP and NP sequences were identical in the three groups of patients and only one silent substitution occurred in the VP40 and VP24 genes in asymptomatic individuals. A strain from an asymptomatic individual had a reverse substitution to the Gabon-94 sequence, indicating that minor virus variants may cocirculate during an outbreak. These results suggest that the different clinical outcomes of EBOV infection do not result from virus mutations. Phylogenetic analysis confirmed that Gabon-96 belonged to the Zaire subtype of EBOV and revealed that synonymous substitution rates were higher than nonsynonymous substitution rates in the GP, VP40 and VP24 genes. In contrast, nonsynonymous substitutions predominated over synonymous substitutions in the NP gene of the two Gabon strains, pointing to divergent evolution of these strains and to selective pressures on this gene.
Induction of anti-simian immunodeficiency virus cellular and humoral immune responses in rhesus macaques by peptide immunogens: correlation of CTL activity and reduction of cell-associated but not plasma virus load following challenge
Authors: Vogel Thorsten U. , Beer Brigitte E. , zur Megede Jan , Ihlenfeldt Hans-Georg , Jung Günther , Holzammer Silke , Watkins David I. , Altman John D. , Kurth Reinhard , Norley Stephen
Published: August 2015

Lipopeptides which carry the N-terminal moiety tripalmitoyl-S-glyceryl-cysteinyl-seryl-seryl (P3CSS) have been shown to have effective adjuvant and transmembrane carrier properties. To test the ability of these constructs to immunize against simian immunodeficiency virus [(SIV)mac] infection, rhesus macaques, prescreened for expression of the Mamu-A*01 MHC class I molecule, were immunized at regular intervals with lipopeptides corresponding to known SIVmacCTL epitopes alone or in combination with multiple antigenic peptides corresponding to neutralizing epitopes. Both humoral and CTL responses were elicited and the monkeys, along with non-immunized control animals, were challenged intravenously with 20 MID50of the homologous, uncloned SIVmac251-32Hgrown in rhesus monkey PBMC. Although none of the monkeys were protected from infection, most demonstrated an anamnestic CTL response with epitope-specific CTL precursor frequencies reaching as high as 1 in 20 total PBMC as measured by limiting dilution CTL assay or 25% of all CD8+T-cells using tetrameric MHC-I/peptide complexes. A significant inverse correlation between the levels of CTLp and the number of infected cells in circulation was observed. However, no such correlation with the plasma viral load (RNA copies/ml) was evident.
Therapeutic vaccination with vhs− herpes simplex virus reduces the severity of recurrent herpetic stromal keratitis in mice
Authors: Keadle Tammie L. , Laycock Keith A. , Morris Jessica L. , Leib David A. , Morrison Lynda A. , Pepose Jay S. , Stuart Patrick M.
Published: August 2015

Virion host shutoff (vhs)-deficient herpes simplex virus (HSV) was tested as a therapeutic vaccine in a mouse model of UV light-induced recurrent herpetic stromal keratitis. Four weeks after primary corneal infection, mice were vaccinated intraperitoneally with vhs−vaccine or control. Four weeks after vaccination, the eyes of latently infected mice were UV-B irradiated to induce recurrent virus shedding and disease. Post-irradiation corneal opacity in latently infected, vhs−-vaccinated mice was significantly reduced compared to control-vaccinated mice (P=0·007 to 0·035). The incidence and duration of recurrent virus shedding were the same in both groups. Antibody titres were increased (P=0·05) and delayed type hypersensitive responses were unaffected by vhs−vaccination. Combined with studies using different vaccination timing and vhs−genotypes, these data suggest that deletion of vhs is a useful strategy in the development of a therapeutic HSV vaccine, and that temporal and genetic factors influence vaccination outcome.
Genomic and phylogenetic analyses of an adenovirus isolated from a corn snake (Elaphe guttata) imply a common origin with members of the proposed new genus Atadenovirus
Authors: Farkas Szilvia L. , Benkő Mária , Élő Péter , Ursu Krisztina , Dán Ádám , Ahne Winfried , Harrach Balázs
Published: August 2015

Approximately 60% of the genome of an adenovirus isolated from a corn snake (Elaphe guttata) was cloned and sequenced. The results of homology searches showed that the genes of the corn snake adenovirus (SnAdV-1) were closest to their counterparts in members of the recently proposed new genus Atadenovirus. In phylogenetic analyses of the complete hexon and protease genes, SnAdV-1 indeed clustered together with the atadenoviruses. The characteristic features in the genome organization of SnAdV-1 included the presence of a gene homologous to that for protein p32K, the lack of structural proteins V and IX and the absence of homologues of the E1A and E3 regions. These characteristics are in accordance with the genus-defining markers of atadenoviruses. Comparison of the cleavage sites of the viral protease in core protein pVII also confirmed SnAdV-1 as a candidate member of the genus Atadenovirus. Thus, the hypothesis on the possible reptilian origin of atadenoviruses (Harrach, Acta Veterinaria Hungarica 48, 484–490, 2000) seems to be supported. However, the base composition of DNA sequence (>18 kb) determined from the SnAdV-1 genome showed an equilibrated GC content of 51%, which is unusual for an atadenovirus.
Molecular epidemiology of Rabbit haemorrhagic disease virus
Authors: Moss S. R. , Turner S. L. , Trout R. C. , White P. J. , Hudson P. J. , Desai A. , Armesto M. , Forrester N. L. , Gould E. A.
Published: August 2015

Millions of domestic and wild European rabbits (Oryctolagus cuniculus) have died in Europe, Asia, Australia and New Zealand during the past 17 years following infection by Rabbit haemorrhagic disease virus (RHDV). This highly contagious and deadly disease was first identified in China in 1984. Epidemics of RHDV then radiated across Europe until the virus apparently appeared in Britain in 1992. However, this concept of radiation of a new and virulent virus from China is not entirely consistent with serological and molecular evidence. This study shows, using RT–PCR and nucleotide sequencing of RNA obtained from the serum of healthy rabbits stored at 4 °C for nearly 50 years, that, contrary to previous opinions, RHDV circulated as an apparently avirulent virus throughout Britain more than 50 years ago and more than 30 years before the disease itself was identified. Based on molecular phylogenetic analysis of British and European RHDV sequences, it is concluded that RHDV has almost certainly circulated harmlessly in Britain and Europe for centuries rather than decades. Moreover, analysis of partial capsid sequences did not reveal significant differences between RHDV isolates that came from either healthy rabbits or animals that had died with typical haemorrhagic disease. The high stability of RHDV RNA is also demonstrated by showing that it can be amplified and sequenced from rabbit bone marrow samples collected at least 7 weeks after the animal has died.
Full-length genome analysis of natural isolates of vesicular stomatitis virus (Indiana 1 serotype) from North, Central and South America
Authors: Rodriguez Luis L. , Pauszek Steven J. , Bunch Thomas A. , Schumann Kate R.
Published: August 2015

Most studies on the molecular biology and functional analysis of vesicular stomatitis virus Indiana 1 serotype (VSV-IN1) are based on the only full-length genomic sequence currently deposited in GenBank. This sequence is a composite of several VSV-IN1 laboratory strains passaged extensively in tissue culture over the years and it is not certain that this sequence is representative of strains circulating in nature. We describe here the complete genomic sequence of three natural isolates, each representing a distinct genetic lineage and geographical origin: 98COE (North America), 94GUB (Central America) and 85CLB (South America). Genome structure and organization were conserved, with a 47 nucleotide 3′ leader, five viral genes – N, P, M, G and L – and a 59 nucleotide 5′ trailer. The most conserved gene was N, followed by M, L and G, with the most variable being P. Sequences containing the polyadenylation and transcription stop and start signals were completely conserved among all the viruses studied, but changes were found in the non-transcribed intergenic nucleotides, including the presence of a trinucleotide at the M–G junction of the South American lineage isolate. A 102–189 nucleotide insertion was present in the 5′ non-coding region of the G gene only in the viruses within a genetic lineage from northern Central America. These full-length genomic sequences should be useful in designing diagnostic probes and in the interpretation of functional genomic analyses using reverse genetics.
Antigenic relationships between six genotypes of the small hydrophobic protein gene of mumps virus
Authors: Örvell Claes , Tecle Tesfaldet , Johansson Bo , Saito Hiroyuki , Samuelson Agneta
Published: August 2015

Six different genotypes of mumps virus, A, C, D, G, H and I, genotyped on the basis of the small hydrophobic protein gene sequence, were subjected to antigenic comparison. Monoclonal antibodies directed against the haemagglutinin–neuraminidase protein of the SBL-1 strain of genotype A were used in immunofluorescence tests with different mumps virus strains. In addition, the six virus genotypes were compared by cross-neutralization tests with human post-vaccination sera after vaccination with the Jeryl Lynn (JL) strain of mumps virus and with rabbit hyperimmune sera directed against the A or D genotypes of mumps virus. Genotypes C, D, G, H and I could not be antigenically separated. In contrast, three different virus strains of genotype A, SBL-1, JL and Kilham, were distinct and were found to represent three different serotypes within the A genotype of mumps virus. Vaccination of Swedish children with the JL strain of mumps virus resulted in clearly lower neutralization titres against the SBL-1 strain, which is endemic in Sweden, compared to the homologous vaccine titres.
Initiation of hepatitis delta virus (HDV) replication: HDV RNA encoding the large delta antigen cannot replicate
Authors: Sheu Gwo-Tarng
Published: August 2015

The hepatitis delta virus (HDV) nucleocapsid consists of a genomic-length RNA of 1·7 kb and approximately equimolar amounts of the small and large forms of the hepatitis delta antigen (S-HDAg and L-HDAg, respectively). Since HDV RNA particles contain not only a genomic RNA species encoding S-HDAg but also an RNA species encoding L-HDAg, which is produced by an RNA-editing process, the question arises as to whether RNAs encoding either L-HDAg or S-HDAg can initiate replication. To study this, two cDNA-free transfection methods were employed: HDV RNA cotransfected with either the S-HDAg-encoding mRNA species or the ribonucleocapsid protein complex, comprising HDV RNA and recombinant S-HDAg. Results showed that the genomic-sense RNA encoding S-HDAg could promote HDV replication, whereas the L-HDAg-encoding RNA species was unable to replicate under the same conditions. The antigenomic RNA species encoding either S-HDAg or L-HDAg could not replicate by either of these procedures. In addition, L-HDAg alone could not promote replication of the genomic RNA but, by supplementing an equal amount of S-HDAg, replication occurred. These data indicate that L-HDAg-encoding RNA species are probably not involved in the initiation of HDV RNA synthesis; instead, their main function may be to serve as template for producing L-HDAg, which regulates HDV RNA synthesis and virion assembly. These results suggest that the genomic RNA species encoding S-HDAg is the only functional genome for HDV infection and explain why the presence of the edited HDV RNA encoding L-HDAg does not interfere with HDV infection.
Evidence for diversifying selection in Potato virus Y and in the coat protein of other potyviruses
Authors: Moury Benoît , Morel Caroline , Johansen Elisabeth , Jacquemond Mireille
Published: August 2015

The modes of evolution of the proteins ofPotato virus Ywere investigated with a maximum-likelihood method based on estimation of the ratio between non-synonymous and synonymous substitution rates. Evidence for diversifying selection was obtained for the 6K2 protein (one amino acid position) and coat protein (24 amino acid positions). Amino acid sites in the coat proteins of other potyviruses (Bean yellow mosaic virus,Yam mosaic virus) were also found to be under diversifying selection. Most of the sites belonged to the N-terminal domain, which is exposed to the exterior of the virion particle. Several of these amino acid positions in the coat proteins were shared between some of these three potyviruses. Identification of diversifying selection events in these different proteins will help to unravel their biological functions and is essential to an understanding of the evolutionary constraints exerted on the potyvirus genome. The hypothesis of a link between evolutionary constraints due to host plants and occurrence of diversifying selection is discussed.
Low genetic variation between isolates of Citrus leaf blotch virus from different host species and of different geographical origins
Authors: Vives María C. , Rubio Luis , Galipienso Luis , Navarro Luis , Moreno Pedro , Guerri José
Published: August 2015

The population structure and genetic diversity of Citrus leaf blotch virus (CLBV) were estimated by single-strand conformation polymorphism and nucleotide sequence analyses of two genomic regions located within the replicase (R) and the coat protein (C) genes. Analysis of 30 cDNA clones of each genomic region from two CLBV isolates showed that both isolates contained a predominant haplotype and others closely related. Analysis of 37 CLBV Spanish field isolates showed low genetic diversity (0·0041 and 0·0018 for genomic regions R and C, respectively). Comparison of 14 CLBV isolates from Spain, Japan, USA, France and Australia showed genetic diversities of 0·0318 (R) and 0·0209 (C), respectively. No correlation was found between genetic distance and geographical origin or host species of the isolates. The ratio between nonsynonymous and synonymous substitutions was the lowest found in a plant virus, indicating a strong negative selective pressure in both genomic regions.
Molecular structures of viruses from Raman optical activity
Authors: Blanch Ewan W. , Hecht Lutz , Syme Christopher D. , Volpetti Vito , Lomonossoff George P. , Nielsen Kurt , Barron Laurence D.
Published: August 2015

A vibrational Raman optical activity (ROA) study of a range of different structural types of virus exemplified by filamentous bacteriophage fd, tobacco mosaic virus, satellite tobacco mosaic virus, bacteriophage MS2 and cowpea mosaic virus has revealed that, on account of its sensitivity to chirality, ROA is an incisive probe of their aqueous solution structures at the molecular level. Protein ROA bands are especially prominent from which, as we have shown by comparison with the ROA spectra of proteins with known structures and by using a pattern recognition program, the folds of the major coat protein subunits may be deduced. Information about amino acid side-chain conformations, exemplified here by the determination of the sign and magnitude of the torsion angle χ2,1 for tryptophan in fd, may also sometimes be obtained. By subtracting the ROA spectrum of the empty protein capsid (top component) of cowpea mosaic virus from those of the intact middle and bottom-upper components separated by means of a caesium chloride density gradient, the ROA spectrum of the viral RNA was obtained, which revealed that the RNA takes up an A-type single-stranded helical conformation and that the RNA conformations in the middle and bottom-upper components are very similar. This information is not available from the X-ray crystal structure of cowpea mosaic virus since no nucleic acid is visible.
Various morphological aspects of Escherichia coli lysis by two distinct RNA bacteriophages
Authors: Nishihara Tohru
Published: August 2015

Transmission electron micrographs of Escherichia coli cells induced by cloned lysis genes from RNA bacteriophages GA (group A-II) and SP (group B-IV) revealed various morphological aspects of intermediates of lysing cells. Cells induced by the SP lysis gene became stretched and also tapered in shape and fragmentation of parts of the cells had also occurred. Cells induced by the GA lysis gene showed many ballooning structures on the cell surfaces and others leaked material through the cell wall. Some balloon-like structures also appeared on the surfaces of cells induced by the cloned lysis gene of RNA phage SP and material also appeared to be leaking through the cell wall in the photographs. The lysing cells observed by transmission electron microscopy showed various morphological aspects of intermediates of the lysing process.
Localization of classical swine fever virus in male gonads during subclinical infection
Authors: Choi Changsun , Chae Chanhee
Published: August 2015

In an experiment using ten boars, the distribution of classical swine fever virus (CSFV) was determined in the male reproductive tract by in situ hybridization over a period of 120 days after intranasal inoculation. CSFV was detected in the testicular tissue of infected boars. Viral nucleic acid was localized to spermatogonia, spermatocytes and spermatids but was not detected in the epithelia of the prostate, epididymis or bulbourethral gland. Sections from control, CSFV-negative, pigs showed no hybridization signals for CSFV. The demonstration that CSFV infects the spermatogonia (and their progeny) suggests that this may serve as a primary reservoir for the venereal spread of CSFV.
Modification of primary and recurrent genital herpes in guinea pigs by passive immunization
Authors: Bourne Nigel , Pyles Richard B. , Bernstein David I. , Stanberry Lawrence R.
Published: August 2015

Guinea pigs were administered antiserum 24 h (As+24) or 72 h (As+72) after intravaginal herpes simplex virus type 2 (HSV-2) challenge. Treatment at either time reduced acute virus replication in the dorsal root ganglia and the overall magnitude of replication in the genital tract. In two studies, As+24 treatment significantly reduced the severity of primary genital skin disease and the frequency of subsequent spontaneous recurrent disease. In contrast, As+72 treatment produced a modest reduction in primary disease severity but did not impact on recurrent disease. Quantitative PCR analysis of dorsal root ganglia DNA from latently infected animals showed that As+24 treatment produced a significantly reduced viral DNA burden, which appeared to correlate with the reduction in recurrent disease. The amount of DNA in the ganglia of As+72-treated animals was not significantly lower than that of controls. These observations have implications for both the dynamics of latency establishment and desirable vaccine characteristics.
Vascular endothelial growth factors encoded by Orf virus show surprising sequence variation but have a conserved, functionally relevant structure
Authors: Mercer A. A. , Wise L. M. , Scagliarini A. , McInnes C. J. , Büttner M. , Rziha H. J. , McCaughan C. A. , Fleming S. B. , Ueda N. , Nettleton P. F.
Published: August 2015

The first report of a vascular endothelial growth factor (VEGF)-like gene inOrf virusincluded the surprising observation that the genes from two isolates (NZ2 and NZ7) shared only 41·1% amino acid sequence identity. We have examined this sequence disparity by determining the VEGF gene sequence of 21 isolates ofOrf virusderived from diverse sources. Most isolates carried NZ2-like VEGF genes but their predicted amino acid sequences varied by up to 30·8% with an average amino acid identity between pairs of NZ2-like sequences of 86·1%. This high rate of sequence variation is more similar to interspecies than intraspecies variability. In contrast, only three isolates carried an NZ7-like VEGF gene and these varied from the NZ7 sequence by no more than a single nucleotide. The VEGF family are ligands for a set of tyrosine kinase receptors. The viral VEGFs are unique among the family in that they recognize VEGF receptor 2 (VEGFR-2) but not VEGFR-1 or VEGFR-3. Comparisons of the viral VEGFs with other family members revealed some correlations between conserved residues and the ability to recognize specific VEGF receptors. Despite the sequence variations, structural predictions for the viral VEGFs were very similar to each other and to the structure determined by X-ray crystallography for human VEGF-A. Structural modelling also revealed that a groove seen in the VEGF-A homodimer and believed to play a role in its binding to VEGFR-1 is blocked in the viral VEGFs. This may contribute to the inability of the viral VEGFs to bind VEGFR-1.
Stability and circularization of herpes simplex virus type 1 genomes in quiescently infected PC12 cultures
Authors: Su Ying-Hsiu , Moxley Michael J. , Ng Alan K. , Lin Judy , Jordan Robert , Fraser Nigel W. , Block Timothy M.
Published: August 2015

Herpes simplex virus type 1 (HSV-1) DNA has been shown to exist as a linear, double-stranded molecule in the virion and as a non-linear (endless), episomal, nucleosomal form in latently infected trigeminal ganglia. The kinetics of the formation and appearance of endless viral genomes and the stability of linear genomes in neuronal cells are not well understood. Nerve growth factor (NGF)-differentiated PC12 cells can sustain long-term, quiescent infections with HSV-1. In this report, the structure and stability of HSV-1 viral DNA in NGF-differentiated PC12 cells was studied as a function of time following infection using both wild-type and replication-defective virus. Unexpectedly, unencapsidated linear genomes were stable in the nucleus of NGF-differentiated PC12 cells for up to 2–3 weeks following infection, beyond the period at which there is no detectable viral gene expression. However, following infection with wild-type HSV, the majority of quiescent viral genomes were in an endless form after 3–4 weeks. These data suggest that the stability and fate of HSV-1 DNA in non-mitotic neuronal-like cells is different from that in productively infected cells and thus there is a significant cellular role in this process. The relevance to the virus life-cycle in neurones in vivo is discussed.
Human cytomegalovirus infection induces cellular thymidylate synthase gene expression in quiescent fibroblasts
Authors: Gribaudo Giorgio , Riera Ludovica , Rudge Thomas L. , Caposio Patrizia , Johnson Lee F. , Landolfo Santo
Published: August 2015

Productive infection of non-proliferating cells by cytomegalovirus (CMV) requires the coordinated stimulation of host biochemical pathways that prepare cells to synthesize DNA. Here we illustrate the ability of human CMV (HCMV) to stimulate cellular thymidylate synthase (TS) gene expression in quiescent human embryonic lung fibroblasts. TS mRNA and protein levels are nearly undetectable in quiescent cells, but are greatly increased following HCMV infection. Inhibition of TS activity was shown to impair HCMV DNA synthesis, demonstrating that TS upregulation is required for efficient HCMV replication in quiescent cells. The increase in TS gene expression was due to an increase in gene transcription, since the expression of a reporter gene driven by the human TS promoter was strongly induced by HCMV infection. Deletion analysis of the human TS promoter identified two positive elements that are important for this increased transcription. We have previously shown that murine CMV (MCMV) stimulates the mouse TS promoter by a mechanism that depends on the presence of an E2F element in the promoter region. However, deletion of the two potential E2F binding sites in the human TS promoter did not prevent the virus-induced increase in TS promoter activity. Our data suggest that HCMV activates human TS gene transcription by mechanisms that are independent of E2F and different from those used by MCMV to stimulate the mouse TS promoter.
Role of G protein and protein kinase signalling in influenza virus budding in MDCK cells
Authors: Hui Eric Ka-Wai , Nayak Debi P.
Published: August 2015

Recently, we have shown that influenza virus budding in MDCK cells is regulated by metabolic inhibitors of ATP and ATP analogues (Hui & Nayak,Virology290, 329–341, 2001). In this report, we demonstrate that G protein signalling stimulators such as sodium fluoride, aluminium fluoride, compound 48/80 and mastoparan stimulated the budding and release of influenza virus. In contrast, G protein signalling blockers such as suramin and NF023 inhibited virus budding. Furthermore, in filter-grown lysophosphatidylcholine-permeabilized virus-infected MDCK cells, membrane-impermeable GTP analogues, such as guanosine 5’-O-(3-thiotriphosphate) or 5’-guanylylimidodiphosphate caused an increase in virus budding, which could be competitively inhibited by adding an excess of GTP. These results suggest that the G protein is involved in the regulation of influenza virus budding. We also determined the role of different protein kinases in influenza virus budding. We observed that specific inhibitors or activators of protein kinase A (H-89 and 8-bromoadenosine 3’,5’-cyclic monophosphate) or of protein kinase C (bisindolylmaleimide I and Ro-32-0432) or of phosphatidylinositol 3-kinase (LY294002 and wortmannin) did not affect influenza virus budding. However, the casein kinase 2 (CK2) inhibitor 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole decreased virus budding. We further observed an increase in the CK2 activity during the replication cycle of influenza virus, although Western blot analysis did not reveal any increase in the amount of CK2 protein in virus-infected cells. Also, in digitonin-permeabilized MDCK cells, the introduction of CK2 substrate peptides caused a down-regulation of virus budding. These results suggest that CK2 activity also regulates influenza virus budding.
Conservation of L and 3C proteinase activities across distantly related aphthoviruses
Authors: Hinton Tracey M. , Ross-Smith Natalie , Warner Simone , Belsham Graham J. , Crabb Brendan S.
Published: August 2015

The foot-and-mouth disease virus (FMDV) leader (L) proteinase is an important virulence determinant in FMDV infections. It possesses two distinct catalytic activities: (i) C-terminal processing at the L/VP4 junction; and (ii) induction of the cleavage of translation initiation factor eIF4G, an event that inhibits cap-dependent translation in infected cells. The only other member of the Aphthovirus genus, equine rhinitis A virus (ERAV), also encodes an L protein, but this shares only 32% amino acid identity with its FMDV counterpart. Another more distantly related picornavirus, equine rhinitis B virus (ERBV), which is not classified as an aphthovirus, also encodes an L protein. Using in vitro transcription and translation analysis, we have shown that both ERAV and ERBV L proteins have C-terminal processing activity. Furthermore, expression of ERAV L, but not ERBV L, in BHK-21 cells resulted in the efficient inhibition of cap-dependent translation in these cells. We have shown that the ERAV and FMDV L proteinases induce cleavage of eIF4GI at very similar or identical positions. Interestingly, ERAV 3C also induces eIF4GI cleavage and again produces distinct products that co-migrate with those induced by FMDV 3C. The ERBV L proteinase does not induce eIF4GI cleavage, consistent with its inability to shut down cap-dependent translation. We have also shown that another unique feature of FMDV L, the stimulation of enterovirus internal ribosome entry site (IRES) activity, is also shared by the ERAV L proteinase but not by ERBV L. The functional conservation of the divergent ERAV and FMDV proteinases indicates the likelihood of a similar and important role for these enzymes in the pathogenesis of infections caused by these distantly related aphthoviruses.
Implication of caspases during maedi–visna virus-induced apoptosis
Authors: Duval R. , Bellet V. , Delebassée S. , Bosgiraud C.
Published: August 2015

Maedi–visna virus (MVV) causes encephalitis, pneumonia and arthritis in sheep.In vitro, MVV infection and replication lead to strong cytopathic effects characterized by syncytia formation and subsequent cellular lysis. It was demonstrated previously that MVV infectionin vitroinduces cell death of sheep choroid plexus cells (SCPC) by a mechanism that can be associated with apoptotic cell death. Here, the relative implication of several caspases during acute infection with MVV is investigated by employing diversein vitroandin situstrategies. It was demonstrated using specific pairs of caspase substrates and inhibitors that, duringin vitroinfection of SCPC by MVV, the two major pathways of caspase activation (i.e. intrinsic and extrinsic pathways) were stimulated: significant caspase-9 and -8 activities, as well as caspase-3 activity, were detected. To study the role of caspases during MVV infectionin vitro, specific, cell-permeable, caspase inhibitors were used. First, these results showed that both z-DEVD-FMK (a potent inhibitor of caspase-3-like activities) and z-VAD-FMK (a broad spectrum caspase inhibitor) inhibit caspase-9, -8 and -3 activities. Second, both irreversible caspase inhibitors, z-DEVD-FMK and z-VAD-FMK, delayed MVV-induced cellular lysis as well as virus growth. Third, during SCPCin vitroinfection by MVV, cells were positively stained with FITC-VAD-FMK, a probe that specifically stains cells containing active caspases. In conclusion, these data suggest that MVV infectionin vitroinduces SCPC cell death by a mechanism that is strongly dependent on active caspases.
Genetic clustering of all 102 human rhinovirus prototype strains: serotype 87 is close to human enterovirus 70
Authors: Savolainen Carita , Blomqvist Soile , Mulders Mick N. , Hovi Tapani
Published: August 2015

Human rhinoviruses (HRV), common agents of respiratory infections, comprise 102 designated serotypes. The genetic relationships of HRV prototype strains and the possibility of using genetic identification of a given HRV field strain were studied. Genomic sequences in the VP4/VP2 region were obtained from all 102 prototype strains. Phylogenetic analysis included 61 recently isolated Finnish field strains. Seventy-six out of the 102 prototype strains clustered in the HRV genetic group A and 25 in group B. Serotype 87 clustered separately and together with human enterovirus 70. The ‘percentage’ interserotypic differences were generally similar to those between different enterovirus serotypes, but for six pairs of HRV serotypes they were less than 10%. The maximum variation in genetic group A was 41% at the nucleotide level and 28% at the amino acid level, and in genetic group B 34% and 20%, respectively. Judging from the observed interserotypic differences, the 61 Finnish field isolates might represent as many as 19 different serotypes. One cluster of the field strains did not directly associate with any of the prototype strains and might represent a new serotype. However, larger numbers of field isolates of known serotype need to be characterized, possibly also in the VP1 region, to evaluate the feasibility of genetic typing of HRV strains.
Hepatitis C virus non-structural protein 3-specific cellular immune responses following single or combined immunization with DNA or recombinant Semliki Forest virus particles
Authors: Brinster C. , Chen M. , Boucreux D. , Paranhos-Baccala G. , Liljeström P. , Lemmonier F. , Inchauspé G.
Published: August 2015

The capacity of recombinant Semliki Forest virus particles (rSFV) expressing the hepatitis C virus non-structural protein 3 (NS3) to induce, in comparison or in combination with an NS3-expressing plasmid, specific cellular and humoral immune responses in murine models was evaluated.In vitrostudies indicated that both types of vaccine expressed the expected size protein, albeit with different efficacies. The use of mice transgenic for the human HLA-A2.1 molecule indicated that the rSFV-expressed NS3 protein induces, as shown previously for an NS3 DNA vaccine, NS3-specific cytotoxic lymphocytes (CTLs) targeted at one dominant HLA-A2 epitope described in infected patients. All DNA/rSFV vaccine combinations evaluated induced specific CTLs, which were detectable for up to 31 weeks after the first injection. Overall, less than 1 log difference was observed in terms of the vigour of the bulk CTL response induced and the CTL precursor frequency between all vaccines (ranging from 1:2·6×105to 1:1×106). Anti-NS3 antibodies could only be detected following a combined vaccine regimen in non-transgenic BALB/c mice. In conclusion, rSFV particles expressing NS3 are capable of inducing NS3-specific cellular immune responses targeted at a major HLA-A2 epitope. Such responses were comparable to those obtained with a DNA-based NS3 vaccine, whether in the context of single or combined regimens.
Mapping of the P1 proteinase cleavage site in the polyprotein of Wheat streak mosaic virus (genus Tritimovirus)
Authors: Choi Il-Ryong , Horken Kempton M. , Stenger Drake C. , French Roy
Published: August 2015

Monopartite members of the family Potyviridae utilize three virus-encoded proteinases to cleave the viral polyprotein into mature proteins. The amino-terminal region of the viral polyprotein is autolytically cleaved by the P1 proteinase. A domain required for P1 proteinase activity of Wheat streak mosaic virus (WSMV) was mapped using a series of templates with nested 3′-truncations or 5′-deletions to program in vitro transcription–translation reactions. The WSMV P1 proteinase cleavage site was mapped to a position downstream of amino acid residue 348 and upstream of amino acid residue 353, with the peptide bond between amino acid residues Y352 and G353 the most probable site of hydrolysis. An alignment of potyvirus polyprotein sequences in the carboxy-terminal region of the P1 domain revealed WSMV P1 contained conserved H257, D267, S303 and FIVXG325–329 residues upstream of the cleavage site that are typical of serine proteinases and shown by others to be required for P1 proteolysis in Tobacco etch virus. Insertion of the GUS reporter gene immediately downstream of the P1 cleavage site in a full-length clone of WSMV resulted in systemic infection and GUS expression upon inoculation of plants with in vitro transcripts. When cleaved by P1 at the amino terminus and NIa proteinase at a site engineered in the carboxy-terminus, active GUS protein expressed by WSMV in infected wheat had electrophoretic mobility similar to wild-type GUS protein.
Transcription and identification of an envelope protein gene (p22) from shrimp white spot syndrome virus
Authors: Zhang Xiaobo , Huang Canhua , Xu Xun , Hew Choy L.
Published: August 2015

White spot syndrome virus (WSSV) is one of the most virulent pathogens causing high mortality in shrimp. In the present study, an open reading frame (termed the p22 gene) was revealed from a WSSV cDNA library. The gene was expressed as a fusion protein with glutathione S-transferase (GST) in Escherichia coli and purified. Specific antibody was raised using the purified fusion protein (GST–P22). Temporal analysis showed that the p22 gene was a late gene. After binding between purified WSSV virions and anti-GST–P22 IgG followed by labelling with gold-labelled secondary antibody, the gold particles, under a transmission electron microscope, could be found along the outer envelope of WSSV virions. This experiment suggests that the p22 gene encodes an envelope protein of the virus.
The vaccinia virus soluble interferon-γ receptor is a homodimer
Authors: Alcamí Antonio , Smith Geoffrey L.
Published: August 2015

The vaccinia virus (VV) interferon (IFN)-γ receptor (IFN-γR) is a 43 kDa soluble glycoprotein that is secreted from infected cells early during infection. Here we demonstrate that the IFN-γR from VV, cowpox virus and camelpox virus exists naturally as a homodimer, whereas the cellular IFN-γR dimerizes only upon binding the homodimeric IFN-γ. The existence of the virus protein as a dimer in the absence of ligand may provide an advantage to the virus in efficient binding and inhibition of IFN-γ in solution.