bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular intermediates of fitness gain of an RNA virus: characterization of a mutant spectrum by biological and molecular cloning
Authors: Arias Armando , Lázaro Ester , Escarmís Cristina , Domingo Esteban
Published: August 2015

The mutant spectrum of a virus quasispecies in the process of fitness gain of a debilitated foot-and-mouth disease virus (FMDV) clone has been analysed. The mutant spectrum was characterized by nucleotide sequencing of three virus genomic regions (internal ribosome entry site; region between the two AUG initiation codons; VP1-coding region) from 70 biological clones (virus from individual plaques formed on BHK-21 cell monolayers) and 70 molecular clones (RT–PCR products cloned inE. coli). The biological and molecular clones provided statistically indistinguishable definitions of the mutant spectrum with regard to the distribution of mutations among the three genomic regions analysed and with regard to the types of mutations, mutational hot-spots and mutation frequencies. Therefore, the molecular cloning procedure employed provides a simple protocol for the characterization of mutant spectra of viruses that do not grow in cell culture. The number of mutations found repeated among the clones analysed was higher than expected from the mean mutation frequencies. Some components of the mutant spectrum reflected genomes that were dominant in the prior evolutionary history of the virus (previous passages), confirming the presence of memory genomes in virus quasispecies. Other components of the mutant spectrum were genomes that became dominant at a later stage of evolution, suggesting a predictive value of mutant spectrum analysis with regard to the outcome of virus evolution. The results underline the observation that greater insight into evolutionary processes of viruses may be gained from detailed clonal analyses of the mutant swarms at the sequence level.
Binding of human and animal immunoglobulins to the IgG Fc receptor induced by human cytomegalovirus
Authors: Antonsson Annika , Johansson P. J. Hugo
Published: August 2015

Human cytomegalovirus (HCMV)-infected cells express a virus-encoded receptor that is able to bind the Fc part of IgG. Some basic binding properties of this Fc receptor (FcR) have been examined. The affinity constant (Ka) for human IgG Fc fragment in its interaction with acetone-fixed, HCMV-infected human embryonic lung fibroblasts was estimated to be around 2×108M−1and the number of binding sites was estimated to be around 2×106per cell. Of the human IgG, IgA, IgM and IgD classes, only IgG reacted with the receptor, and all four of the IgG subclasses were reactive. IgG from rabbit, hamster, cat, swine and horse exhibited binding to the HCMV FcR, in contrast to IgG from mouse, rat, guinea pig, dog, sheep, goat, cow and chicken. Immunoglobulins with and without HCMV IgG FcR-binding properties, like IgG from rabbit and mouse, can be of value in revealing the functional importance of the receptor. When the immunoglobulins were tested against herpes simplex virus type 1-induced FcR, both similarities and differences in immunoreactivity were seen relative to the HCMV FcR, which makes it unlikely that the binding sites for these two herpesvirus FcRs on the IgG molecule are identical.
Genetic heterogeneity among parapoxviruses isolated from sheep, cattle and Japanese serows (Capricornis crispus)
Authors: Inoshima Yasuo , Murakami Kenji , Yokoyama Takashi , Sentsui Hiroshi
Published: August 2015

Standard strains of four parapoxviruses and seven unclassified Japanese strains isolated from sheep, cattle and wild Japanese serows (Capricornis crispus) were compared molecularly. Restriction fragment length polymorphism (RFLP) analysis of viral DNA, indirect immunofluorescence assays using monoclonal antibodies, partial nucleotide sequencing of the envelope gene, phylogenetic analysis and PCR–RFLP were carried out. These analyses revealed that the parapoxviruses were divided into four groups and the region sequenced in this study was highly conserved within each group. Each of the Japanese isolates was classified into one of these groups. These findings also indicated that parapoxvirus infections among wild Japanese serows seem to be caused by at least two different parapoxviruses, bovine papular stomatitis virus and orf virus. The methods presented here are useful for genetic characterization and classification of parapoxviruses.
Non-structural proteins 2 and 3 interact to modify host cell membranes during the formation of the arterivirus replication complex
Authors: Snijder Eric J. , van Tol Hans , Roos Norbert , Pedersen Ketil W.
Published: August 2015

The replicase polyproteins of equine arteritis virus (EAV; family Arteriviridae, order Nidovirales) are processed by three viral proteases to yield 12 non-structural proteins (nsps). The nsp2 and nsp3 cleavage products have previously been found to interact, a property that allows nsp2 to act as a co-factor in the processing of the downstream part of the polyprotein by the nsp4 protease. Remarkably, upon infection of Vero cells, but not of BHK-21 or RK-13 cells, EAV nsp2 is now shown to be subject to an additional, internal, cleavage. In Vero cells, approximately 50% of nsp2 (61 kDa) was cleaved into an 18 kDa N-terminal part and a 44 kDa C-terminal part, most likely by a host cell protease that is absent in BHK-21 and RK-13 cells. Although the functional consequences of this additional processing step are unknown, the experiments in Vero cells revealed that the C-terminal part of nsp2 interacts with nsp3. Most EAV nsps localize to virus-induced double-membrane structures in the perinuclear region of the infected cell, where virus RNA synthesis takes place. It is now shown that, in an expression system, the co-expression of nsp2 and nsp3 is both necessary and sufficient to induce the formation of double-membrane structures that strikingly resemble those found in infected cells. Thus, the nsp2 and nsp3 cleavage products play a crucial role in two processes that are common to positive-strand RNA viruses that replicate in mammalian cells: controlled proteolysis of replicase precursors and membrane association of the virus replication complex.
Visualization by atomic force microscopy of tobacco mosaic virus movement protein–RNA complexes formed in vitro
Authors: Kiselyova O. I. , Yaminsky I. V. , Karger E. M. , Frolova O. Yu. , Dorokhov Y. L. , Atabekov J. G.
Published: August 2015

The structure of complexes formed in vitro by tobacco mosaic virus (TMV)-coded movement protein (MP) with TMV RNA and short (890 nt) synthetic RNA transcripts was visualized by atomic force microscopy on a mica surface. MP molecules were found to be distributed along the chain of RNA and the structure of MP–RNA complexes depended on the molar MP:RNA ratios at which the complexes were formed. A rise in the molar MP:TMV RNA ratio from 20:1 to 60–100:1 resulted in an increase in the density of the MP packaging on TMV RNA and structural conversion of complexes from RNase-sensitive ‘beads-on-a-string’ into a ‘thick string’ form that was partly resistant to RNase. The ‘thick string’-type RNase-resistant complexes were also produced by short synthetic RNA transcripts at different MP:RNA ratios. The ‘thick string’ complexes are suggested to represent clusters of MP molecules cooperatively bound to discrete regions of TMV RNA and separated by protein-free RNA segments.
Mutational analysis of the proteinase function of Potato leafroll virus
Authors: Sadowy Ewa , Juszczuk Marek , David Chantal , Gronenborn Bruno , Hulanicka M. Danuta
Published: August 2015

cDNA expression vectors of Potato leafroll virus (PLRV) were used to analyse specific mutations in the proteinase and replicase domains of the proteins encoded by ORF1 and ORF2. Agrobacterium-mediated DNA transfer was used to introduce a PLRV RNA expression unit, controlled by the 35S promoter of Cauliflower mosaic virus, into potato leaf cells. Expression of unmodified PLRV cDNA led to the replication of viral genomic and subgenomic RNAs and accumulation of the viral capsid protein, whereas alteration of amino acids GDD513–515 of the replicase to VHD abolished PLRV replication. Mutations in the presumed H-D-S catalytic triad of the viral proteinase abolished the formation of viral genomic and subgenomic RNAs as well as synthesis of the viral capsid protein. Co-agroinoculation of the GDD mutant along with any of the proteinase mutants restored virus replication in leaf discs, showing that these mutants are able to complement each other. Moreover, mutation of the postulated serine residue of the catalytic triad of the proteinase altered the pattern of proteins synthesized in vitro in comparison to wild-type, further supporting the relevance of the H-D-S motif.
Coordinate effects of human immunodeficiency virus type 1 protein Tat and cellular protein Purα on DNA replication initiated at the JC virus origin
Authors: Daniel Dianne C. , Wortman Margaret J. , Schiller Robin J. , Liu Hong , Gan Li , Mellen Jonathan S. , Chang Chun-F. , Gallia Gary L. , Rappaport Jay , Khalili Kamel , Johnson Edward M.
Published: August 2015

JC virus (JCV) causes progressive multifocal leukoencephalopathy, a demyelinating disease in brains of individuals with AIDS. Previous work has shown that the Tat protein, encoded by human immunodeficiency virus type 1 (HIV-1), can interact with cellular protein Purα to enhance both TAR-dependent HIV-1 transcription and JCV late gene transcription. Tat has been shown to activate JCV transcription through interaction with Purα, which binds to promoter sequence elements near the JCV origin of replication. DNA footprinting has shown that Purα and large T-antigen cooperatively interact at several binding sites in the origin and transcriptional control region. Overexpression of Purα inhibits replication initiated at the JCV origin by T-antigen. In transfected glial cells Tat reversed this inhibition and enhanced DNA replication. In an in vitro replication system maximal activation by Tat, more than sixfold the levels achieved with T-antigen alone, was achieved in the presence of Purα. Effects of mutant Tat proteins on both activation of replication and binding to Purα have revealed that Cys22 exerts a conformational effect that affects both activities. The origin of an archetypal strain of JCV was less susceptible to activation of replication by Tat relative to the rearranged Mad-1 strain. These results have revealed a previously undocumented role for Tat in DNA replication and have indicated a regulatory role for JCV origin auxiliary sequences in replication and activation by Tat.
The carboxyl terminus of the human cytomegalovirus UL37 immediate-early glycoprotein is conserved in primary strains and is important for transactivation
Authors: Hayajneh Wail A. , Contopoulos-Ioannidis Despina G. , Lesperance Marci M. , Venegas Ana M. , Colberg-Poley Anamaris M.
Published: August 2015

The human cytomegalovirus (HCMV) UL37 exon 3 (UL37x3) open reading frame (ORF) encodes the carboxyl termini of two immediate-early glycoproteins (gpUL37 and gpUL37M). UL37x3 homologous sequences are not required for mouse cytomegalovirus (MCMV) growth in vitro; yet, they are important for MCMV growth and pathogenesis in vivo. Similarly, UL37x3 sequences are dispensable for HCMV growth in culture, but their requirement for HCMV growth in vivo is not known. To determine this requirement, we directly sequenced the complete UL37x3 gene in multiple HCMV primary strains. A total of 63 of the 310 amino acids in the UL37x3 ORF differ non-conservatively in one or more HCMV primary strains. The HCMV UL37x3 genetic diversity is non-random: the N-glycosylation (46/186 aa) and basic (9/15 aa) domains have the highest proportion of non-conservative variant amino acids. Nonetheless, most (15/17 signals) of the N-glycosylation signals are retained in all HCMV primary strains. Moreover, new N-glycosylation signals are encoded by 5/20 primary strains. In sharp contrast, the UL37x3 transmembrane (TM) ORF completely lacks diversity in all 20 HCMV sequenced primary strains, and only 1 of 28 cytosolic tail residues differs non-conservatively. To test the functional significance of the conserved carboxyl terminus, gpUL37 mutants lacking the TM and/or cytosolic tail were tested for transactivating activity. The gpUL37 carboxyl-terminal mutants are partially defective in hsp70 promoter transactivation even though they trafficked similarly to the wild-type protein into the endoplasmic reticulum and to mitochondria. From these results, we conclude that N-glycosylated gpUL37, particularly its TM and cytosolic domains, is important for HCMV growth in humans.
The degree of attenuation of tick-borne encephalitis virus depends on the cumulative effects of point mutations
Authors: Gritsun T. S. , Desai A. , Gould E. A.
Published: August 2015

An infectious clone (pGGVs) of the tick-borne encephalitis complex virus Vasilchenko (Vs) was constructed previously. Virus recovered from pGGVs produced slightly smaller plaques than the Vs parental virus. Sequence analysis demonstrated five nucleotide differences between the original Vs virus and pGGVs; four of these mutations resulted in amino acid substitutions, while the fifth mutation was located in the 3′ untranslated region (3′UTR). Two mutations were located in conserved regions and three mutations were located in variable regions of the virus genome. Reverse substitutions from the conserved regions of the genome, R496→H in the envelope (E) gene and C10884→T in the 3′UTR, were introduced both separately and together into the infectious clone and their biological effect on virus phenotype was evaluated. The engineered viruses with R496 in the E protein produced plaques of smaller size than viruses with H496 at this position. This mutation also affected the growth and neuroinvasiveness of the virus. In contrast, the consequence of a T10884→C substitution within the 3′UTR was noticeable only in cytotoxicity and neuroinvasiveness tests. However, all virus mutants engineered by modification of the infectious clone, including one with two wild-type mutations, H496 and T10884, showed reduced neuroinvasiveness in comparison with the Vs parental virus. Therefore, although the H496→R and T10884→C substitutions clearly reduce virus virulence, the other mutations within the variable regions of the capsid (I45→F) and the NS5 (T2688→A and M3385→I) genes also contribute to the process of attenuation. In terms of developing flavivirus vaccines, the impact of accumulating apparently minor mutations should be assessed in detail.
Interaction between molecules of hantavirus nucleocapsid protein
Authors: Kaukinen Pasi , Koistinen Vesa , Vapalahti Olli , Vaheri Antti , Plyusnin Alexander
Published: August 2015

Intermolecular interactions of Tula hantavirus N (nucleocapsid) protein were detected in the yeast two-hybrid system, prompting further attempts to study this phenomenon. Using chemical cross-linking and immunoblotting it was shown that the N protein from purified virus and from infected cell lysates as well as recombinant protein produced in a baculovirus expression system are capable of forming dimers, trimers and multimers, thus confirming the capacity of the protein molecules to interact with each other. An ELISA format was developed in which molecules of the recombinant N protein were shown to associate non-covalently, via electrostatic interactions. Divalent cations (Ca2+, Mn2+, Mg2+, Ba2+) enhanced the process 3- to 8-fold suggesting that adequate folding of the N protein is crucial for the association. Based on these data a model for hantavirus nucleocapsid assembly is proposed, in which N molecules first trimerize around the viral RNA molecule, and then the trimers gradually assemble forming longer multimers.
Establishment of persistent infection with non-cytopathic bovine viral diarrhoea virus in cattle is associated with a failure to induce type I interferon
Authors: Charleston B. , Fray M. D. , Baigent S. , Carr B. V. , Morrison W. I.
Published: August 2015

The establishment of persistent infections with non-cytopathic bovine virus diarrhoea virus (ncpBVDV) is crucial for the maintenance of BVDV in cattle populations. Also, super-infection of persistently infected individuals with antigenically homologous cytopathic BVDV (cpBVDV) results in fatal mucosal disease. Persistent infection with ncpBVDV is established by infection of the foetus during the first trimester of pregnancy. It has been shown previously that foetal infection with cpBVDV does not result in persistent infection. Infection of cellsin vitrohas demonstrated that cpBVDV induces type I interferon (IFN), whereas ncpBVDV fails to induce IFN. In this study we demonstrate that foetal challenge with cpBVDV results in IFN production, whereas ncpBVDV does not. These findings strongly suggest that the ability of ncpBVDV to inhibit the induction of type I IFN has evolved to enable the virus to establish persistent infection in the early foetus.
Role of conserved residues in the activity of adenovirus preterminal protein
Authors: Botting Catherine H. , Hay Ronald T.
Published: August 2015

Preterminal protein (pTP) is a component of the preinitiation complex which forms at the adenovirus origin of DNA replication and acts as the protein primer during DNA synthesis. In order to determine the role of various regions of the molecule a series of 18 mutations was introduced into conserved motifs of pTP which were predicted to be surface exposed, and the mutants expressed in insect cells using a baculovirus expression system. Their ability to initiate DNA replication was assessed and the effect the mutations have on the individual interactions which contribute to the formation of the pre-initiation complex was determined. Classes of mutants could be identified which were unable to bind DNA or interact with the adenovirus DNA polymerase, but one class of mutants retained these activities and yet failed to initiate DNA replication. These mutants therefore identify regions of pTP required for different aspects of adenovirus DNA replication.
Alteration of amino acids in VP2 of very virulent infectious bursal disease virus results in tissue culture adaptation and attenuation in chickens
Authors: van Loon A. A. W. M. , de Haas N. , Zeyda I. , Mundt E.
Published: August 2015

Reverse genetics technology offers the possibility to study the influence of particular amino acids of infectious bursal disease virus (IBDV) on adaptation to tissue culture. Genomic segments A and B of the very virulent (vv) IBDV field strain UK661 were completely cloned and sequenced, and the strain was rescued from full-length cDNA copies of both segments (UK661rev). Using site-directed mutagenesis, alteration of a single amino acid in the segment A-encoded VP2 (A284T) resulted in a limited capacity of UK661 to replicate in tissue culture. Additional alteration of a second amino acid (Q253H) increased replication efficiency in tissue culture. The second mutant (UK661-Q253H-A284T) was used to infect chickens and results were compared with UK661 and UK661rev. Whereas UK661 and UK661rev induced 100% morbidity and 50–80% mortality, UK661-Q253H-A284T proved to be strikingly attenuated, producing neither morbidity nor mortality. Moreover, UK661-Q253H-A284T-infected animals were protected from challenge infection. Thus, alteration of two specific amino acids in the VP2 region of IBDV resulted in tissue culture adaptation and attenuation in chickens of vvIBDV. The data demonstrate that VP2 plays a decisive role in pathogenicity of IBDV.
Monoclonal antibodies and human sera directed to the secreted glycoprotein G of herpes simplex virus type 2 recognize type-specific antigenic determinants
Authors: Liljeqvist Jan-Åke , Trybala Edward , Hoebeke Johan , Svennerholm Bo , Bergström Tomas
Published: August 2015

Glycoprotein G-2 (gG-2) of herpes simplex virus type 2 (HSV-2) is cleaved to a secreted amino-terminal portion (sgG-2) and to a cell-associated carboxy-terminal portion which is furtherO-glycosylated to constitute the mature gG-2 (mgG-2). In contrast to mgG-2, which is known to elicit a type-specific antibody response in the human host, information on the immunogenic properties of sgG-2 is lacking. Here the sgG-2 protein was purified on a heparin column and used for production of monoclonal antibodies (mAbs). Four anti-sgG-2 mAbs were mapped using a Pepscan technique and identified linear epitopes which localized to the carboxy-terminal part of the protein. One additional anti-sgG-2 mAb, recognizing a non-linear epitope, was reactive to three discrete peptide stretches where the most carboxy-terminally located stretch was constituted by the amino acids320RRAL323.Although sgG-2 is rapidly secreted into the cell-culture medium after infection, the anti-sgG-2 mAbs identified substantial amounts of sgG-2 in the cytoplasm of HSV-2-infected cells. All of the anti-sgG-2 mAbs were HSV-2 specific showing no cross-reactivity to HSV-1 antigen or to HSV-1-infected cells. Similarly, sera from 50 HSV-2 isolation positive patients were all reactive to sgG-2 in an enzyme immunoassay whilst no reactivity was seen in 25 sera from HSV-1 isolation positive patients or in 25 serum samples from HSV-negative patients suggesting that sgG-2 is a novel antigen potentially suitable for type-discriminating serodiagnosis.
Characterization of Campoletis sonorensis ichnovirus segment I genes as members of the repeat element gene family
Authors: Hilgarth Roland S. , Webb Bruce A.
Published: August 2015

Campoletis sonorensis ichnovirus (CsIV) is a symbiotic virus associated with the endoparasitic wasp C. sonorensis. The virus is injected into the wasp’s host, Heliothis virescens, during oviposition. One CsIV gene has been identified as a repeat element (rep) gene and encodes a ubiquitous imperfectly conserved 540 bp sequence. We report the sequencing and mapping of a rep-containing segment, segment I, that hybridizes to a known rep sequence from segment O1. Analysis of this 8·6 kbp segment identified three ORFs having high similarity to the 540 bp rep sequence. All three rep sequence ORFs were expressed in parasitized H. virescens as well as in C. sonorensis tissues. Two of these rep genes, I 0.9 and I 1.1, have single copies of the 540 bp repeat sequence, while the third rep gene, I 1.2, has two imperfect copies, which are more similar to each other than to sequences on the segment I single-motif genes. Like the CsIV BHv 0.9 rep gene, the segment I rep genes lack introns and a signal peptide, suggesting that they are not secreted. Based on their similarity in nucleotide sequence, predicted amino acid sequence and gene structure, the three segment I repeat-containing genes, I 0.9, I 1.1 and I 1.2, are new members of the rep gene family.
Evidence of recombination in the capsid-coding region of type A foot-and-mouth disease virus
Authors: Tosh Chakradhar , Hemadri Divakar , Sanyal Aniket
Published: August 2015

Recombination is one of the factors that contribute to genetic diversity in foot-and-mouth disease virus (FMDV). Similarity and bootscan analyses have provided evidence of recombination in the capsid-coding (P1) region of the virus. In the present study, of the 14 subtype A22 field isolates that were distributed in three previously described genotypes (IV, VI and VII) based on the 1D (VP1-encoding) gene sequence (Tosh et al., 2002), one isolate (IND 170/88) was found to be a hybrid of genotypes VI and VII in the P1 region. VP1, VP4, the 5′ region of VP2 and the 3′ region of VP3 of this virus were characteristic of genotype VI, whereas the remaining 3′ region of VP2 and the 5′ region of VP3 were characteristic of genotype VII. No insertion or deletion was observed in the recombinant virus. Recombination in the P1 region may provide an escape mechanism for the virus.
Structure of antigenic sites on the haemagglutinin molecule of H5 avian influenza virus and phenotypic variation of escape mutants
Authors: Kaverin Nikolai V. , Rudneva Irina A. , Ilyushina Natalia A. , Varich Natalia L. , Lipatov Aleksandr S. , Smirnov Yuri A. , Govorkova Elena A. , Gitelman Asya K. , Lvov Dmitri K. , Webster Robert G.
Published: August 2015

To elucidate the structure of the antigenic sites of avian H5 influenza virus haemagglutinin (HA) we analysed escape mutants of a mouse-adapted variant of the H5N2 strain A/Mallard/Pennsylvania/10218/84. A panel of five anti-H5 monoclonal antibodies (mAbs) was used to select 16 escape mutants. The mutants were tested by ELISA and haemagglutination inhibition with this panel of anti-H5 mAbs and the HA genes of the mutants were sequenced. The sequencing demonstrated that the amino acid changes were grouped in two antigenic sites. One corresponded to site A in the H3 HA. The other contained areas that are separated in the amino acid sequence but are topographically close in the three-dimensional structure and partially overlap in the reactions with mAbs. This site corresponds in part to site B in the H3 structure; it also includes a region not involved in site B that partially overlaps site Sa in the H1 HA and an antigenic area in H2 HA. Mutants with the amino acid change K152N, as well as those with the change D126N, showed reduced lethality in mice. The substitution D126N, creating a new glycosylation site, was accompanied by an increase in the sensitivity of the mutants to normal mouse serum inhibitors. Several amino acid changes in the H5 escape mutants occurred at the positions of reported changes in H2 drift variants. This coincidence suggests that the antigenic sites described and analysed here may be important for drift variation if H5 influenza virus ever appears as a pathogen circulating in humans.
PrPCWD lymphoid cell targets in early and advanced chronic wasting disease of mule deer
Authors: Sigurdson Christina J. , Barillas-Mury Carolina , Miller Michael W. , Oesch Bruno , van Keulen Lucien J. M. , Langeveld Jan P. M. , Hoover Edward A.
Published: August 2015

Up to 15% of free-ranging mule deer in northeastern Colorado and southeastern Wyoming, USA, are afflicted with a prion disease, or transmissible spongiform encephalopathy (TSE), known as chronic wasting disease (CWD). CWD is similar to a subset of TSEs including scrapie and variant Creutzfeldt–Jakob disease in which the abnormal prion protein isoform, PrPCWD, accumulates in lymphoid tissue. Experimental scrapie studies have indicated that this early lymphoid phase is an important constituent of prion replication interposed between mucosal entry and central nervous system accumulation. To identify the lymphoid target cells associated with PrPCWD, we used triple-label immunofluorescence and high-resolution confocal microscopy on tonsils from naturally infected deer in advanced disease. We detected PrPCWDprimarily extracellularly in association with follicular dendritic and B cell membranes as determined by frequent co-localization with antibodies against membrane bound immunoglobulin and CD21. There was minimal co-localization with cytoplasmic labels for follicular dendritic cells (FDC). This finding could indicate FDC capture of PrPCWD, potentially in association with immunoglobulin or complement, or PrPCconversion on FDC. In addition, scattered tingible body macrophages in the germinal centre contained coarse intracytoplasmic aggregates of PrPCWD, reflecting either phagocytosis of PrPCWDon FDC processes, apoptotic FDC or B cells, or actual PrPCWDreplication within tingible body macrophages. To compare lymphoid cell targets in early and advanced disease, we also examined: (i) PrPCWDdistribution in lymphoid cells of fawns within 3 months of oral CWD exposure and (ii) tonsil biopsies from preclinical deer with naturally acquired CWD. These studies revealed that the early lymphoid cellular distribution of PrPCWDwas similar to that in advanced disease, i.e. in a pattern suggesting FDC association. We conclude that in deer, PrPCWDaccumulates primarily extracellularly and associated with FDCs and possibly B cells – a finding which raises questions as to the cells responsible for pathological prion production.
Truncated gp120 envelope glycoprotein of human immunodeficiency virus 1 elicits a broadly reactive neutralizing immune response
Authors: Jeffs S. A. , Shotton C. , Balfe P. , McKeating J. A.
Published: August 2015

Removal of the V1–V3 loops from IIIB gp120 results in a protein, PR12, with altered immunogenicity compared to the full-length protein. Polyclonal immune sera raised in rats using PR12 as immunogen recognizes envelope glycoproteins of clades A, B, C, E, F and G and can neutralize chimeric human immunodeficiency virus type 1 (HIV-1) HXB2 viruses expressing envelopes from primary HIV-1 clades B, C, E and F. These data suggest that the immune response to PR12 is directed toward conserved epitopes expressed by viral glycoproteins of diverse genotypes. Five monoclonal antibodies (mAb) derived from PR12-immunized rats were unable to neutralize virus infectivity; hence the epitopes responsible for the induction of this cross-clade neutralizing activity remain to be elucidated. However, PR12 immune sera were able to compete with the human neutralizing mAb 2G12 for gp120 binding, implying that this epitope may be immunogenic when expressed in the context of this truncated protein.
The R1 subunit of herpes simplex virus ribonucleotide reductase protects cells against apoptosis at, or upstream of, caspase-8 activation
Authors: Langelier Yves , Bergeron Stéphane , Chabaud Stéphane , Lippens Julie , Guilbault Claire , Sasseville A. Marie-Josée , Denis Stéphan , Mosser Dick D. , Massie Bernard
Published: August 2015

The R1 subunit of herpes simplex virus (HSV) ribonucleotide reductase, which in addition to its C-terminal reductase domain possesses a unique N-terminal domain of about 400 amino acids, is thought to have an additional, as yet unknown, function. Here, we report that the full-length HSV-2 R1 has an anti-apoptotic function able to protect cells against death triggered by expression of R1(Δ2–357), an HSV-2 R1 subunit with its first 357 amino acids deleted. We further substantiate the R1 anti-apoptotic activity by showing that its accumulation at low level could completely block apoptosis induced by TNF-receptor family triggering. Activation of caspase-8 induced either by TNF or by Fas ligand expression was prevented by the R1 protein. As HSV R1 did not inhibit cell death mediated by several agents acting via the mitochondrial pathway (Bax overexpression, etoposide, staurosporine and menadione), it is proposed that it functions to interrupt specifically death receptor-mediated signalling at, or upstream of, caspase-8 activation. The N-terminal domain on its own did not exhibit anti-apoptotic activity, suggesting that both domains of R1 or part(s) of them are necessary for this new function. Evidence for the importance of HSV R1 in protecting HSV-infected cells against cytokine-induced apoptosis was obtained with the HSV-1 R1 deletion mutants ICP6Δ andhrR3. These results show that, in addition to its ribonucleotide reductase function, which is essential for virus reactivation, HSV R1 could contribute to virus propagation by preventing apoptosis induced by the immune system.
Nucleotide sequence and genomic organization of an ophiovirus associated with lettuce big-vein disease
Authors: van der Wilk F. , Dullemans A. M. , Verbeek M. , van den Heuvel J. F. J. M.
Published: August 2015

The complete nucleotide sequence of an ophiovirus associated with lettuce big-vein disease has been elucidated. The genome consisted of four RNA molecules of approximately 7·8, 1·7, 1·5 and 1·4 kb. Virus particles were shown to contain nearly equimolar amounts of RNA molecules of both polarities. The 5′- and 3′-terminal ends of the RNA molecules are largely, but not perfectly, complementary to each other. The virus genome contains seven open reading frames. Database searches with the putative viral products revealed homologies with the RNA-dependent RNA polymerases of rhabdoviruses and Ranunculus white mottle virus, and the capsid protein of Citrus psorosis virus. The gene encoding the viral polymerase appears to be located on the RNA segment 1, while the nucleocapsid protein is encoded by the RNA3. No significant sequence similarities were observed with other viral proteins. In spite of the morphological resemblance with species in the genus Tenuivirus, the ophioviruses appear not to be evolutionary closely related to this genus nor any other viral genus.
Herpes simplex virus type 1 mutants containing the KOS strain ICP34.5 gene in place of the McKrae ICP34.5 gene have McKrae-like spontaneous reactivation but non-McKrae-like virulence
Authors: Perng Guey-Chuen , Mott Kevin R. , Osorio Nelson , Yukht Ada , Salina Susan , Nguyen Quynh-Hoa , Nesburn Anthony B. , Wechsler Steven L.
Published: August 2015

Herpes simplex virus type 1 (HSV-1) strain McKrae is neurovirulent in rabbits infected by the ocular route, causing fatal encephalitis in approximately 50% of the animals, and has a high-level spontaneous reactivation phenotype, with 10% of rabbit eyes containing reactivated virus at any given time. In contrast, HSV-1 strain KOS is completely avirulent (no rabbits die) and has a completely negative spontaneous reactivation phenotype. Mutations of the ICP34.5 gene can reduce the neurovirulence of HSV-1 strains McKrae and 17syn+ by up to 100000-fold. ICP34.5 mutants also have reduced spontaneous reactivation phenotypes. To determine whether differences in the ICP34.5 gene might be involved in the reduced neurovirulence and spontaneous reactivation phenotypes of KOS compared with McKrae, we constructed chimeric viruses containing the KOS ICP34.5 gene in place of the McKrae ICP34.5 gene. Rabbits ocularly infected with the chimeric viruses had a high spontaneous reactivation phenotype indistinguishable from McKrae. In contrast, neurovirulence of the chimeric viruses was decreased compared with McKrae. Thus, one or more ‘defects’ in the KOS ICP34.5 gene appeared to be at least partially responsible for the reduced neurovirulence of KOS compared with McKrae. However, there appeared to be no ‘defect′ in the KOS ICP34.5 function required for efficient spontaneous reactivation.
The latency-related gene of bovine herpesvirus-1 can inhibit the ability of bICP0 to activate productive infection
Authors: Geiser Vicki , Inman Melissa , Zhang Yange , Jones Clinton
Published: August 2015

Transfection of bovine cells with bovine herpesvirus-1 genomic DNA yields low levels of infectious virus. Cotransfection with the bICP0 gene enhances productive infection and virus yield because bICP0 can activate viral gene expression. Since the latency-related (LR) gene overlaps and is antisense to bICP0, the effects of LR gene products on productive infection were tested. The intact LR gene inhibited productive infection in a dose-dependent fashion but LR protein expression was not required. Further studies indicated that LR gene sequences near the 3′ terminus of the LR RNA are necessary for inhibiting productive infection. When cotransfected with the bICP0 gene, the LR gene inhibited bICP0 RNA and protein expression in transiently transfected cells. Taken together, these results suggest that abundant LR RNA expression in sensory neurons is one factor that has the potential to inhibit productive infection and consequently promote the establishment and maintenance of latency.
Characterization of pif, a gene required for the per os infectivity of Spodoptera littoralis nucleopolyhedrovirus
Authors: Kikhno Iryna , Gutiérrez Serafín , Croizier Liliane , Croizier Guy , Ferber Miguel López
Published: August 2015

During plaque purification of Spodoptera littoralis nucleopolyhedrovirus in S. littoralis Sl52 cell culture, a deletion mutant virus was isolated. Analysis of the biological properties of this mutant virus revealed an absence of per os infectivity of the occluded virus. Infectivity by injection of the non-occluded (budded) virus is not different between the wild-type and the deleted virus. Restriction analysis of the mutant virus genome revealed a 4·5 kb deletion within the NotI D fragment. The observed phenotype was mapped to the deleted region by rescue experiments. The deletion was characterized and the equivalent DNA fragment on the wild-type virus was sequenced. By co-transfecting the DNA of the deleted virus with plasmids derived from the wild-type virus, it was possible to determine that ORF 7 in this fragment is responsible for the observed phenotype. ORF 7, called pif (per os infectivity factor), is homologous to ORF 119 of Autographa californica nucleopolyhedrovirus. Similar ORFs are present in all sequenced baculoviruses. The product of this gene is an occlusion body-derived virion structural protein required only for the first steps of larva infection, as viruses being produced in cells expressing the gene but not containing it in their genomes are able to produce successful infections.
Studies on the different conditions for rabies virus neutralization by monoclonal antibodies #1-46-12 and #7-1-9
Authors: Irie Takashi , Kawai Akihiko
Published: August 2015

Virus-neutralizing activity of two monoclonal antibodies (mAbs), #7-1-9 and #1-46-12, against rabies virus glycoprotein (G) was compared. Although these mAbs affected the virion’s ability to bind to host cells similarly, a big difference was found in the titres of virus neutralization (1:7132 and 1:32 for mAbs #1-46-12 and #7-1-9, respectively, at a concentration of 10 μg protein/ml). Although no big difference in virion-binding affinity between the two mAbs was found, the number of antibodies required for virus neutralization was very low, ⩽20 molecules for mAb #1-46-12 and ⩾250 molecules for mAb #7-1-9. In the latter case, the mAbs cover a major part of the virion surface and cause steric hindrance of viral receptor-binding activity. The infectivity of an epitope-preserved escape mutant virus (R-61) was not affected by the binding of high numbers of mAb #1-46-12 to the virion, which implies that mAb binding does not mask the receptor-binding site of the viral spikes. Based on these results, it is hypothesized that mAb #1-46-12 affected virus infectivity by a mechanism different from covering the virion spikes. Possible virus-neutralizing mechanisms by low numbers of mAb #1-46-12 in comparison to that of mAb #7-1-9 are discussed.
Cauliflower mosaic virus is preferentially acquired from the phloem by its aphid vectors
Authors: Palacios Itziar , Drucker Martin , Blanc Stéphane , Leite Silvia , Moreno Aranzazu , Fereres Alberto
Published: August 2015

Cauliflower mosaic virus (CaMV) is transmitted in a non-circulative manner by aphids following the helper strategy. Helper proteins P2 and P3 act as a bridge between virions and the aphid cuticle. Electronic monitoring of aphid stylet activities (EPG technique), transmission tests and electron microscopy showed that CaMV is preferentially acquired from the phloem by its most common aphid vectors, Brevycorine brassicae and Myzus persicae. We also found that CaMV is semipersistently transmitted and that the rate of acquisition does not follow a typical bimodal curve. Instead, the virus could be acquired from non-phloem tissues at a low and fairly constant rate after one or more intracellular punctures within a few minutes, but the probability of acquisition rose significantly when aphids reached the phase of committed ingestion from the phloem. The acquisition rate of CaMV did not increase with increasing number of intracellular punctures, but the total duration of intracellular puncture was one of the variables selected by the stepwise logistic regression model used to fit the data that best explained acquisition of CaMV. Furthermore, aphids reaching the phloem faster had a higher probability of acquiring the virus. Our results support the hypothesis that multiple intracellular punctures of epidermal and mesophyll cells result in loading aphids with the CaMV-encoded aphid transmission factor (P2), and that aphids, in most cases, subsequently acquire CaMV particles during phloem sap ingestion. Consistently, immunoelectron microscopy showed that P3–virions are frequently found in the sieve element lumen, whereas P2 could not be detected.
Systemic movement of a movement-deficient strain of Cucumber mosaic virus in zucchini squash is facilitated by a cucurbit-infecting potyvirus
Authors: Choi Seung Kook , Yoon Ju Yeon , Ryu Ki Hyun , Choi Jang Kyung , Palukaitis Peter , Park Won Mok
Published: August 2015

Zucchini squash (Cucurbita pepo) is a systemic host for most strains of the cucumovirus Cucumber mosaic virus (CMV), although the long-distance movement of the M strain of CMV (M-CMV) is inhibited in some cultivars. However, co-infection of zucchini plants with M-CMV and the potyvirus Zucchini yellow mosaic virus strain A (ZYMV-A) allowed M-CMV to move systemically, as demonstrated by tissue-print analysis. These doubly infected plants exhibited severe synergism in pathology. Infection of zucchini squash by M-CMV and an attenuated strain of ZYMV (ZYMV-AG) showed a milder synergy in pathology, in which ZYMV-AG also facilitated the long-distance movement of M-CMV similar to that promoted by ZYMV-A. Variation in the extent of synergy in pathology by the two strains of ZYMV did not correlate with differences in levels of accumulation of either virus. Thus, the extent of synergy in pathology is at least in part independent of the resistance-neutralizing function of the potyvirus.
Topors, a p53 and topoisomerase I binding protein, interacts with the adeno-associated virus (AAV-2) Rep78/68 proteins and enhances AAV-2 gene expression
Authors: Weger Stefan , Hammer Eva , Heilbronn Regine
Published: August 2015

The adeno-associated virus type 2 (AAV-2) Rep proteins are essential for AAV DNA replication and regulation of AAV gene expression. We have identified a cellular protein interacting with Rep78 and Rep68 in yeast two-hybrid analysis and in GST pull-down assays. This protein has recently been described as both a p53 (p53BP3) and a topoisomerase I interacting protein (Topors). It contains an arginine/serine-rich domain, a RING finger domain and five PEST sequences. A minimal sequence sufficient for interaction with Rep was mapped to Topors amino acids 871 to 917. We show that the same region is also involved in the interaction with p53. Rep sequences involved in interaction with Topors were mapped to Rep amino acids 172 to 481. Overexpression of Topors stimulated AAV gene expression in the absence of helper virus, suggesting a function of Topors as a transcriptional regulator.
Caveolin-1 is incorporated into mature respiratory syncytial virus particles during virus assembly on the surface of virus-infected cells
Authors: Brown Gaie , Aitken James , Rixon Helen W. McL. , Sugrue Richard J.
Published: August 2015

We have employed immunofluorescence microscopy and transmission electron microscopy to examine the assembly and maturation of respiratory syncytial virus (RSV) in the Vero cell line C1008. RSV matures at the apical cell surface in a filamentous form that extends from the plasma membrane. We observed that inclusion bodies containing viral ribonucleoprotein (RNP) cores predominantly appeared immediately below the plasma membrane, from where RSV filaments form during maturation at the cell surface. A comparison of mock-infected and RSV-infected cells by confocal microscopy revealed a significant change in the pattern of caveolin-1 (cav-1) fluorescence staining. Analysis by immuno-electron microscopy showed that RSV filaments formed in close proximity to cav-1 clusters at the cell surface membrane. In addition, immuno-electron microscopy showed that cav-1 was closely associated with early budding RSV. Further analysis by confocal microscopy showed that cav-1 was subsequently incorporated into the envelope of RSV filaments maturing on the host cell membrane, but was not associated with other virus structures such as the viral RNPs. Although cav-1 was incorporated into the mature virus, it was localized in clusters rather than being uniformly distributed along the length of the viral filaments. Furthermore, when RSV particles in the tissue culture medium from infected cells were examined by immuno-negative staining, the presence of cav-1 on the viral envelope was clearly demonstrated. Collectively, these findings show that cav-1 is incorporated into the envelope of mature RSV particles during egress.
Respiratory syncytial virus matrix protein associates with nucleocapsids in infected cells
Authors: Ghildyal R. , Mills J. , Murray M. , Vardaxis N. , Meanger J.
Published: August 2015

Little is known about the functions of the matrix (M) protein of respiratory syncytial virus (RSV). By analogy with other negative-strand RNA viruses, the M protein should inhibit the viral polymerase prior to packaging and facilitate virion assembly. In this study, localization of the RSV M protein in infected cells and its association with the RSV nucleocapsid complex was investigated. RSV-infected cells were shown to contain characteristic cytoplasmic inclusions. Further analysis showed that these inclusions were localization sites of the M protein as well as the N, P, L and M2-1 proteins described previously. The M protein co-purified with viral ribonucleoproteins (RNPs) from RSV-infected cells. The transcriptase activity of purified RNPs was enhanced by treatment with antibodies to the M protein in a dose-dependent manner. These data suggest that the M protein is associated with RSV nucleocapsids and, like the matrix proteins of other negative-strand RNA viruses, can inhibit virus transcription.
Whole genome analysis of the Epiphyas postvittana nucleopolyhedrovirus
Authors: Hyink Otto , Dellow Ross A. , Olsen Michael J. , Caradoc-Davies Katherine M. B. , Drake Kylie , Herniou Elisabeth A. , Cory Jennifer S. , O’Reilly David R. , Ward Vernon K.
Published: August 2015

The nucleotide sequence of the Epiphyas postvittana nucleopolyhedrovirus (EppoMNPV) genome has been determined and analysed. The circular dsDNA genome contains 118584 bp, making it the smallest group I NPV sequenced to date. The genome has a G+C content of 40·7% and encodes 136 predicted open reading frames (ORFs), five homologous repeat regions and one unique repeat region. Of the genome, 92·9% encodes predicted ORFs and 2·2% is in repeat regions; the remaining 4·9% of the genome comprises nonrepeat intergenic regions. EppoMNPV encodes homologues of 126 Orgyia pseudotsugata MNPV (OpMNPV) ORFs and 120 Autographa californica MNPV ORFs, with average identities of 64·7 and 53·5%, respectively. Between the four sequenced group I NPVs, 117 ORFs are conserved, whereas 86 ORFs are conserved between all fully sequenced NPVs. A total of 62 ORFs is present in all baculoviruses sequenced to date, with EppoMNPV lacking a homologue of the superoxide dismutase (sod) gene, which has been found in all other fully sequenced baculoviruses. Whole genome phylogenetic analyses of the ten fully sequenced baculoviruses using the sequences of the 62 shared genes, gene content and gene order data sets confirmed that EppoMNPV clusters tightly with OpMNPV in the group I NPVs. The main variation between EppoMNPV and OpMNPV occurs where extra clusters of genes are present in OpMNPV, with sod occurring in one such cluster. EppoMNPV encodes one truncated baculovirus repeated ORF (bro) gene. The only repeated ORFs are the four iap genes. Eight, randomly distributed, unique ORFs were identified on EppoMNPV, none of which show any significant homology to genes in GenBank.