bluuebunny/crossref_metadata_2025 huggingface dataset:
A subpopulation of RNA 1 of Cucumber mosaic virus contains 3′ termini originating from RNAs 2 or 3
Authors: Canto Tomas , Choi Seung Kook , Palukaitis Peter
Published: August 2015

Tobacco plants transgenic for RNA 1 of Cucumber mosaic virus and inoculated with transcript of RNAs 2 and 3 regenerated viral RNA 1 from the transgenic mRNA, and the plants became systemically infected by the reconstituted virus. cDNA fragments corresponding to the 3′ non-coding region (NCR) of viral RNA 1 were amplified, cloned and sequenced. In some clones the termini of the 3′ NCR corresponded to those of viral RNAs 2 or 3. This suggested that in some cases RNA 1 may have been regenerated during replication by a template switching mechanism between the inoculated transcript RNAs and the mRNA. However, encapsidated, recombinant RNA 1 with the 3′ NCR ends originating from RNAs 2 or 3 also was found in virus samples that had been passaged exclusively through non-transgenic plants. Thus, these chimeras occur naturally due to recombination between wild-type viral RNAs, and they are found encapsidated in low, but detectable amounts.
Persistent expression of a newly characterized Hyposoter didymator polydnavirus gene in long-term infected lepidopteran cell lines
Authors: Volkoff Anne-Nathalie , Rocher Janick , Cérutti Pierre , Ohresser Marc C. P. , d’Aubenton-Carafa Yves , Devauchelle Gérard , Duonor-Cérutti Martine
Published: August 2015

An Hyposoter didymator ichnovirus (HdIV) gene was stably maintained and efficiently transcribed in lepidopteran cell lines more than 3 years after HdIV infection. This K-gene had two introns and the fully spliced cDNA, named K19, comprised a short open reading frame and a long 3′-untranslated region with 13 imperfectly repeated sequences (44 to 102 nt). Transcripts related to the K-gene were detected in several long-term infected cell lines (Sf9, Spodoptera littoralis haemocytes, Trichoplusia ni). Conversely, no transcripts related to seven other viral cDNAs were detected, suggesting that the K-related DNA is selectively retained in long-term infected Sf9 cells. The function of the K-gene product and its association with stably transformed insect cell lines remains to be investigated.
Phylogenetic analyses confirm the high prevalence of hepatitis C virus (HCV) type 4 in the Seine-Saint-Denis district (France) and indicate seven different HCV-4 subtypes linked to two different epidemiological patterns
Authors: Morice Yoann , Roulot Dominique , Grando Véronique , Stirnemann Jérome , Gault Elyanne , Jeantils Vincent , Bentata Michelle , Jarrousse Bernard , Lortholary Olivier , Pallier Coralie , Dény Paul
Published: August 2015

Hepatitis C virus (HCV) has been classified into six clades as a result of high genetic variability. In the Seine-Saint-Denis district of north-east Paris, the prevalence of HCV-4, which usually infects populations from Africa or the Middle East, is twice as high as that recorded for the whole of continental France (10·2 versus 4·5%). Although the pathogenicity of HCV-4 remains unknown, resistance of HCV-4 to therapy appears to be similar to that observed for HCV-1. In order to characterize the epidemiology of HCV-4 in Paris, sequences of the non-structural 5B gene (332 bp) were obtained from 38 HCV-4-infected patients. Extensive phylogenetic analyses indicated seven different HCV-4 subtypes. Moreover, phylogenetic tree topologies clearly distinguished two epidemiological profiles. The first profile (52·6% of patients) reflects the intra-suburban emergence of two distinct HCV-4 subclades occurring mainly among intravenous drug users (65% of patients). The second profile shows six subclades [HCV-4a, -4f, -4h, -4k, -4a(B) and a new sequence] and accounts for patients from Africa (Egypt and sub-Saharan countries) who have unknown risk factors (77·8% of patients) and in whom no recent diffusion of HCV-4 is evident. This study indicates the high diversity of HCV-4 and the extension of HCV-4a and -4d subclades among drug users in France.
Roles of the H-2D b and H-K b genes in resistance to persistent Theiler’s murine encephalomyelitis virus infection of the central nervous system
Authors: Azoulay-Cayla Arièle , Syan Sylvie , Brahic Michel , Bureau Jean-François
Published: August 2015

Theiler’s murine encephalomyelitis virus, a member of thePicornaviridaefamily, persists in the spinal cord of susceptible strains of mice. Resistant strains of mice, such as theH-2bstrain, clear the virus infection after an acute encephalomyelitis. TheH-2Dlocus, but not theH-2Klocus, has a major effect on this resistance, although both loci code for MHC class I molecules with similar general properties. For the present work, we rendered susceptibleH-2qFVB/N mice transgenic for either theH-2Dbgene, theH-2Kbgene or a chimericH-2Db/Kbgene in which the exons encoding the peptide-binding groove of theH-2Kbgene have been replaced by those of theH-2Dbgene. Mice transgenic for either theH-2Dbgene or the chimericH-2Db/Kbgene were significantly more resistant to persistent virus infection than mice transgenic for theH-2Kbgene, suggesting that the difference in the effects of theH-2Dbgene and theH-2Kbgene are due to the nature of the peptides presented by these class I molecules.
A complex human immunodeficiency virus type 1 A/G/J recombinant virus isolated from a seronegative patient with AIDS from Benin, West Africa
Authors: Baldrich-Rubio E. , Anagonou S. , Stirrups K. , Lafia E. , Candotti D. , Lee H. , Allain J. P.
Published: August 2015

A human immunodeficiency virus type 1 (HIV-1B76) originating from Benin (West Africa) was isolated and characterized. The patient had severe clinical AIDS and presented an unusual serological profile. Only one out of five different detection assays was able to demonstrate the presence of antibodies to HIV, whereas confirmatory assays remained indeterminate. In contrast, both plasma viral load and p24 antigen level were unusually high. HIV-1 infection was proved by viral RNA and proviral DNA amplification. HIV-1B76 partially purified lysate reacted strongly with all anti-HIV-1-positive sera from the region but B76 plasma did not react with subtype A control viral antigen. This patient is likely to have had severe acquired immune dysfunction explaining her lack of immunological reactivity. Phylogenetic analysis of the genome identified a complex HIV-1 A/G/J recombinant. The gag and pol genes, and the majority of nef,are characteristic of subtype A; the gag/pol junction, the 3′ end of pol, vpu and env genes were characteristic of subtype G; vif, vpr and the 5′ end of nef were subtype J. In addition, part of the HIV-1B76 genome had considerable sequence similarity with the previously described CRF06 cpx (BFP90) isolate. HIV-1B76 did not exhibit any remarkable replication properties or cell tropism in vitro.
A mutational analysis of the vaccinia virus B5R protein
Authors: Mathew Elizabeth C. , Sanderson Christopher M. , Hollinshead Ruth , Smith Geoffrey L.
Published: August 2015

A mutational analysis of the vaccinia virus (VV) B5R protein is presented. This protein is related to the regulators of complement activation (RCA) superfamily, has four short consensus repeats (SCRs) that are typical of this superfamily and is present on extracellular enveloped virus (EEV) particles. Here we have constructed VV mutants in which the cytoplasmic tail (CT) of the B5R protein is progressively truncated, and domains of the B5R protein [the SCR (short consensus repeat) domains, the transmembrane anchor region or the CT] are substituted by corresponding domains from the VV haemagglutinin (HA), another EEV protein. Analysis of these mutant viruses showed that loss of the B5R CT did not affect the formation of intracellular enveloped virus (IEV), actin tails, EEV or virus plaque size. However, if the SCR domains of the B5R protein were replaced by the corresponding region of the HA, the virus plaque size was diminished, the formation of actin tails was decreased severely and the titre of infectious EEV released from cells was reduced approximately 25-fold compared to wild-type virus and 5-fold compared to a virus lacking the entire B5R gene. Thus the linkage of HA to the B5R transmembrane and CT is deleterious for the formation and release of EEV and for cell-to-cell virus spread. In contrast, deletion or substitution of the B5R CT did not affect virus replication, although the amount of cell surface B5R was reduced compared to control.
Virus-specific spatial differences in the interference with silencing of the chs-A gene in non-transgenic petunia
Authors: Teycheney Pierre-Yves , Tepfer Mark
Published: August 2015

Potyviruses, such as potato virus Y and tobacco etch virus, as well as cucumber mosaic cucumovirus, interfere with post-transcriptional gene silencing (PTGS). When RedStar-type Petunia hybrida cultivars, whose flowers have alternating white and pigmented sectors, were infected with these viruses, each virus induced a different pattern of restoration of floral anthocyanin pigmentation. Local reversion to coloured phenotypes in the white sectors, which occurred through interference with PTGS of the chalcone synthase A (chs-A) gene, was correlated with locally increased levels of chs-A mRNA and virus concentration. Our results show that virus infection can interfere with PTGS of a native plant gene, and that this can have profound effects on symptom expression.
Identification of an immunodominant epitope in the C terminus of glycoprotein 5 of porcine reproductive and respiratory syndrome virus
Authors: Rodriguez María Jose , Sarraseca Javier , Fominaya Jesús , Cortés Elena , Sanz Antonio , Casal J. Ignacio
Published: August 2015

Glycoprotein 5 (GP5) is the major glycoprotein of porcine reproductive and respiratory syndrome virus (PRRSV). Expression of GP5 has been improved by removing the transmembrane regions. Vectors were constructed encoding complete GP5 plus three mutants: GP5 ΔNs (residues 28–201), GP5[30–67] (residues 30–67) and GP5[30–201] (residues 30–67/130–201). The three deletion mutants were expressed at levels 20–30 times higher than complete GP5. GP5[30–201] was well recognized in ELISA or immunoblotting by a collection of pig sera. All the fragments were tested for the generation of MAbs, but only the polyhistidine-tagged fragment GP5[30–201]H elicited an antibody response sufficient to produce MAbs. The two MAbs were positive for PRRSV in ELISA and immunoblotting, but negative for virus neutralization. MAb 4BE12 reacted with residues 130–170 and MAb 3AH9 recognized residues 170–201. This region was recognized strongly in immunoblotting by a collection of infected-pig sera. These results indicate diagnostic potential for this epitope.
Infectious RNA transcribed in vitro from a cDNA copy of the human coronavirus genome cloned in vaccinia virus
Authors: Thiel Volker , Herold Jens , Schelle Barbara , Siddell Stuart G.
Published: August 2015

The coronavirus genome is a positive-strand RNA of extraordinary size and complexity. It is composed of approximately 30000 nucleotides and it is the largest known autonomously replicating RNA. It is also remarkable in that more than two-thirds of the genome is devoted to encoding proteins involved in the replication and transcription of viral RNA. Here, a reverse-genetic system is described for the generation of recombinant coronaviruses. This system is based upon the in vitro transcription of infectious RNA from a cDNA copy of the human coronavirus 229E genome that has been cloned and propagated in vaccinia virus. This system is expected to provide new insights into the molecular biology and pathogenesis of coronaviruses and to serve as a paradigm for the genetic analysis of large RNA virus genomes. It also provides a starting point for the development of a new class of eukaryotic, multi-gene RNA vectors that are able to express several proteins simultaneously.
Induction of apoptosis in bone marrow neutrophil-lineage cells by classical swine fever virus
Authors: Summerfield Artur , Zingle Katy , Inumaru Shigeki , McCullough Kenneth C.
Published: August 2015

The pathogenesis of bone marrow atrophy during classical swine fever (CSF) was investigatedin vitroby using CSF virus (CSFV) infection of bone marrow haematopoietic cells (BMHC). The monocytic lineage had the highest susceptibility to CSFV infection, whereas the more mature SWC8+granulocytic cells were not directly susceptible to infection. However, myelomonocytic precursors were targets for CSFV infection and continued to differentiate into SWC8+granulocytic cells, which remained infected. This explains the occurrence of infected peripheral blood granulocytes during CSF. The infection of BMHC resulted in increased apoptosis and necrosis, mainly within the granulocytic lineage. Caspases 3 and 9 were particularly activated, relating to the mitochondrial pathway of apoptosis. Interestingly, the majority of infected cells were non-apoptotic, the apoptotic cells being primarily non-infected. This indicated an indirect mechanism for induction of apoptosis, but no role could be identified for bone marrow stroma cells such as macrophages or fibroblastoid cells. Furthermore, soluble factors including cytokines and reactive oxygen species were not primarily responsible. In contrast, contact between infected and non-infected BMHC was critical for increasing apoptosis in the latter. Taken together, these resultsin vitrorelate to and help to explain further the apoptosis of BMHC that occursin vivoduring CSF. This experimental system will also be particularly useful for the study of CSFV gene products involved in leukocyte apoptosis.
Sabin attenuated LSc/2ab strain of poliovirus spreads to the spinal cord from a peripheral nerve in bonnet monkeys (Macaca radiata)
Authors: Ponnuraj Esther M. , John T. Jacob , Levin Myron J. , Simoes Eric A. F.
Published: August 2015

Vaccine-associated paralytic poliomyelitis is a serious concern while using the live attenuated oral polio vaccine for the eradication of poliomyelitis. The bonnet monkey model of poliovirus central nervous system (CNS) infection following experimental inoculation into the ulnar nerve allows the comparative study of wild-type and attenuated poliovirus invasiveness. Dosages ⩾104 TCID50 of Mahoney strain of poliovirus type 1 [PV1(M)] result in paralysis. In contrast, even with 107 TCID50 of Sabin attenuated strain of poliovirus type 1 (LSc/2ab), no paralysis occurs, but virus spreads into the CNS where viral RNA is found in spinal cord neurons. While wild-type PV1(M) viral RNA replicates in neurons (and possibly in glial cells) and in cells around vessel walls, which may be mononuclear or endothelial cells, attenuated viral RNA is detected only in neurons. Systemic viraemia and gastrointestinal virus shedding occurs only in PV1(M)-infected animals. While a systemic serologic response is detected in both groups of animals, cerebrospinal fluid antibodies are detected only in animals infected with PV1(M). Both the PV1(M) and LSc/2ab strains spread to the cervical spinal cord and then to the lumbar spinal cord following ulnar nerve inoculation. Neuronophagia and neuronal loss are only seen in PV1(M)-infected monkeys in whom clinical paralysis is observed. Infection with LSc/2ab does not result in neuronophagia, neuronal loss or clinical paralysis. Spread of attenuated poliovirus in spinal cord neurons without causing paralysis following inoculation into the ulnar nerve is an important finding.
Retroviral recombination is temperature dependent
Authors: Li Ting , Zhang Jiayou
Published: August 2015

Two conflicting in vitro observations suggest that retroviral recombinations are temperature dependent. Ouhammouch & Brody (Nucleic Acids Research 20, 5443–5450, 1992) suggested that retroviral recombination rates should increase as temperature increases. However, Shimomaye & Salvato (Gene Analysis Techniques 6, 25–28, 1989) and Brooks et al. (Biotechniques 19, 806–812, 814–815, 1985) found that at low temperature the tightly folded structure of RNAs may hinder reverse transcription proceeding along the RNA template, which increases its chance of dissociating from the template; therefore, raising the reaction temperature was the simplest way to overcome template secondary structure and prevent premature termination of cDNA synthesis. In this report, two vectors based on murine leukaemia virus (MLV) were constructed. The first contained two mutated gfp genes in tandem positions. The upstream gfp gene encoded a mutation at its 3′ end, while the downstream gfp gene encoded a mutation at its 5′ end. The recombination that occurred between the two mutated gfp genes restored a functional gfp gene. The cells that contained the functional gfp gene were green when observed under a fluorescence microscope. The second MLV vector contained a functional gfp gene with two identical sequences flanking either end. A recombination that occurred between the two identical sequences resulted in deletion of the gfp gene. Cells containing the vector with the gfp deletion were colourless or clear when observed under the microscope. Using these two vectors, we have demonstrated that retroviral recombination is temperature dependent and the rate of recombination decreases as temperature is raised from 31 to 43 °C.
Role of Epstein–Barr virus-encoded latent membrane protein 2A on virus-induced immortalization and virus activation
Authors: Konishi Kazuya , Maruo Seiji , Kato Hiroyuki , Takada Kenzo
Published: August 2015

To quantitatively evaluate the role of Epstein-Barr virus (EBV)-encoded latent membrane protein 2A (LMP2A) in immortalization of peripheral B-lymphocytes, we used the Akata cell system to generate an EBV recombinant in which the first exon of the LMP2A gene was disrupted. The results indicated that deletion of the LMP2A gene did not affect the immortalization efficiency of EBV in B-lymphocytes. Deletion of the LMP2A gene made EBV-transformed lymphocytes more permissive for virus replication in response to surface immunoglobulin cross-linking. On the other hand Akata cells, in which LMP2A expression was much lower than in EBV-transformed lymphocytes, were equally permissive for virus replication whether they were infected with wild EBV or LMP2A-knockout EBV. The results raise a question as to the role of LMP2A in inhibition of disruption of virus latency in vivo, where LMP2A expression has been expected to be low as in Akata cells.
The ORF0 product of Potato leafroll virus is indispensable for virus accumulation
Authors: Sadowy Ewa , Maasen Anna , Juszczuk Marek , David Chantal , Zagórski-Ostoja Wlodzimierz , Gronenborn Bruno , Hulanicka M. Danuta
Published: August 2015

Using a cDNA expression cassette in combination with agroinoculation of potato leaf discs we have investigated the role the protein encoded by ORF0 ofPotato leafroll virus(PLRV) and have shown its importance for virus accumulation. Two mutations introduced into ORF0 by site-directed mutagenesis prevented expression of the corresponding protein and completely abolished virus accumulation in plant cells. They did not, however, affect translation of ORF1 and ORF2. We therefore conclude that ORF0 of PLRV produces a protein essential for virus accumulation, a hitherto undescribed finding.
Evidence for structural differences in the S domain of L in comparison with S protein of hepatitis B virus
Authors: Clayton Reginald F. , Owsianka Ania , Patel Arvind H.
Published: August 2015

The structures of the large (L), middle (M) and small (S) versions of the envelope proteins of hepatitis B virus remain poorly characterized due to the complex nature of their conformations. Several groups have proposed transmembrane topological models depicting the lumenally and cytosolically disposed regions of these proteins. Recently, post-translational topological changes in L have been described. However, no overall differences in the topology of the S domains of the L or M, to the S protein are predicted. In this report, we investigated a previously uncharacterized anti-S monoclonal antibody (MAb), 6B1, which recognizes a conformation-sensitive epitope in S. Unlike other anti-S MAbs tested, this MAb did not recognize its epitope in the S domain of L protein. Interestingly, however, the M protein was efficiently recognized. This unique characteristic of MAb 6B1 has allowed us to study the intracellular distribution of L and S proteins. In cells expressing both L and S, L re-localized from the endoplasmic reticulum–Golgi intermediate compartment (ERGIC) to the membrane-associated distribution of S protein indicating that L and S interact with each other. This was confirmed by immunoprecipitation assays, which also showed that the interaction between L and S results in the secretion of L protein from cells. Overall, the ability of MAb 6B1 to selectively recognize S and M, but not L, strongly points to the existence of significant topological differences in the S domain of L. The availability of this important reagent should help further our understanding of the structure of HBV surface antigens.
Evaluation of the primary effect of brefeldin A treatment upon herpes simplex virus assembly
Authors: Dasgupta Anindya , Wilson Duncan W.
Published: August 2015

Addition of the drug brefeldin A (BFA) to cells infected by herpes simplex virus (HSV) type 1 is known to result in a complex pattern of defects in particle assembly. BFA-treated, infected cells accumulate perinuclear enveloped virions and non-enveloped (‘naked’) cytoplasmic capsids, and it has been difficult to interpret these data in terms of the assembly pathway of HSV and the known effects of BFA on the secretory apparatus. Since BFA is a cytotoxic drug, and earlier studies commonly examined the effects of long-term BFA incubations on infected cells, it was hypothesized that the drug could have pleiotropic and indirect effects on HSV assembly. To test this, use was made of an HSV synchronized assembly assay, in which cells are infected with the virus mutant tsProt.A and maintained at 39 °C to induce reversible accumulation of a population of procapsids. By first adding BFA and then shifting these cells to 31 °C for 3 h to allow the accumulated procapsids to mature, it was possible to test the effect of short-term BFA treatment on only those HSV assembly events that are downstream of procapsid maturation. Under these conditions, it was found that procapsids matured and packaged the viral genome normally, but remained non-enveloped and failed to exit the nucleus. It is concluded that the primary effect of BFA on HSV replication is to inhibit budding at the inner nuclear membrane.
The inhibition of cAMP-dependent protein kinase by full-length hepatitis C virus NS3/4A complex is due to ATP hydrolysis
Authors: Aoubala Mustapha , Holt John , Clegg Roger A. , Rowlands David J. , Harris Mark
Published: August 2015

Hepatitis C virus (HCV) is an important cause of chronic liver disease, but the molecular mechanisms of viral pathogenesis remain to be established. The HCV non-structural protein NS3 complexes with NS4A and has three enzymatic activities: a proteinase and a helicase/NTPase. Recently, catalytically inactive NS3 fragments containing an arginine-rich motif have been reported to interact with, and inhibit, the catalytic subunit of cAMP-dependent protein kinase (PKA C-subunit). Here we demonstrate that full-length, catalytically active NS3/4A, purified from recombinant baculovirus-infected insect cells, is also able to inhibit PKA C-subunitin vitro. This inhibition was abrogated by mutation of either the arginine-rich motif or the conserved helicase motif II, both of which also abolished NTPase activity. As PKA C-subunit inhibition was also enhanced by poly(U) (an activator of NS3 NTPase activity), we hypothesized that PKA C-subunit inhibition could be due to NS3/4A-mediated ATP hydrolysis. This was confirmed by experiments in which a constant ATP concentration was maintained by addition of an ATP regeneration system – under these conditions PKA C-subunit inhibition was not observed. Interestingly, the mutations also abrogated the ability of wild-type NS3/4A to inhibit the PKA-regulated transcription factor CREB in transiently transfected hepatoma cells. Our data are thus not consistent with the previously proposed model in which the arginine-rich motif of NS3 was suggested to act as a pseudosubstrate inhibitor of PKA C-subunit. However,in vivoeffects of NS3/4A suggest that ATPase activity may play a role in viral pathology in the infected liver.
Identification of conformational neutralizing epitopes on the capsid protein of canine calicivirus
Authors: Matsuura Yuichi , Tohya Yukinobu , Mochizuki Masami , Takase Kozo , Sugimura Takaaki
Published: August 2015

Two neutralizing monoclonal antibodies (MAbs) against canine calicivirus (CaCV), which has a distinct antigenicity from feline calicivirus (FCV), were obtained. Both MAbs recognized conformational epitopes on the capsid protein of CaCV and were used to identify these epitopes. Neutralization-resistant variants of CaCV were selected in the presence of individual MAbs in a cell culture. Cross-neutralization tests using the variants indicated that the MAbs recognized functionally independent epitopes on the capsid protein. Recombinantly expressed ORF2 products (capsid precursors) of the variants showed no reactivity to the MAbs used for the selection, suggesting that the resistance was induced by a failing in binding of the MAbs to the variant capsid proteins. Several nucleotide changes resulting in amino acid substitutions in the capsid protein were found by sequence analysis. Reactivities of the MAbs to the revertant ORF2 products produced from each variant ORF2 by site-directed mutagenesis identified a single amino acid substitution in each variant capsid protein responsible for the failure of MAb binding. The amino acid residues related to forming the conformational neutralizing epitopes were located in regions equivalent to the 5′ and 3′ hypervariable regions of the FCV capsid protein, where antigenic sites were demonstrated in previous studies. The recombinant ORF2 products expressed in bacteria failed to induce neutralizing antibody, suggesting that neutralizing antibodies were only generated when properly folded capsid protein was used as an antigen. In CaCV, the conformational epitopes may play a more important role in neutralization than do linear epitopes.
Naked RNA immunization with replicons derived from poliovirus and Semliki Forest virus genomes for the generation of a cytotoxic T cell response against the influenza A virus nucleoprotein
Authors: Vignuzzi Marco , Gerbaud Sylvie , van der Werf Sylvie , Escriou Nicolas
Published: August 2015

The potential of RNA-based vaccines was evaluated for the generation of a protective immune response in the mouse model of influenza type A virus infection using the internal nucleoprotein (NP) as antigen. This antigen is of particular interest, since it has the potential to elicit protective cytotoxic T lymphocytes (CTL) against heterologous strains of influenza A virus. In view of the short half-life of RNA, self-replicating RNAs or replicons of the positive-stranded genomes of Semliki Forest virus (SFV) and poliovirus were engineered to synthesize the influenza A virus NP in place of their structural proteins. NP expression was demonstrated by immunoprecipitation after transfection of cells with RNA from the SFV (rSFV-NP) and poliovirus (rΔP1-E-NP) genome-derived replicons transcribedin vitro. C57BL/6 mice were injected intramuscularly with these synthetic RNAs in naked form. Both replicons, rSFV-NP and rΔP1-E-NP, induced antibodies against the influenza virus NP, but only mice immunized with the rSFV-NP replicon developed a CTL response against the immunodominant H-2Dbepitope NP366. Finally, the protective potential of the CTL response induced by immunization of mice with rSFV-NP RNA was demonstrated by the reduction of virus load in the lungs after challenge infection with mouse-adapted influenza A/PR/8/34 virus and was comparable to the protective potential of the response induced by plasmid DNA immunization. These results demonstrate that naked RNA immunization with self-replicating molecules can effectively induce both humoral and cellular immune responses and constitutes an alternative strategy to DNA immunization.
Phylogenetic relationships of flaviviruses correlate with their epidemiology, disease association and biogeography
Authors: Gaunt Michael W. , Sall Amadou A. , Lamballerie Xavier de , Falconar Andrew K. I. , Dzhivanian Tatyana I. , Gould Ernest A.
Published: August 2015

Phylogenetic analysis of theFlavivirusgenus, using either partial sequences of the non-structural 5 gene or the structural envelope gene, revealed an extensive series of clades defined by their epidemiology and disease associations. These phylogenies identified mosquito-borne, tick-borne and no-known-vector (NKV) virus clades, which could be further subdivided into clades defined by their principal vertebrate host. The mosquito-borne flaviviruses revealed two distinct epidemiological groups: (i) the neurotropic viruses, often associated with encephalitic disease in humans or livestock, correlated with theCulexspecies vector and bird reservoirs and (ii) the non-neurotropic viruses, associated with haemorrhagic disease in humans, correlated with theAedesspecies vector and primate hosts. Thus, the tree topology describing the virus–host association may reflect differences in the feeding behaviour betweenAedesandCulexmosquitoes. The tick-borne viruses also formed two distinct groups: one group associated with seabirds and the other, the tick-borne encephalitis complex viruses, associated primarily with rodents. The NKV flaviviruses formed three distinct groups: one group, which was closely related to the mosquito-borne viruses, associated with bats; a second group, which was more genetically distant, also associated with bats; and a third group associated with rodents. Each epidemiological group within the phylogenies revealed distinct geographical clusters in either the Old World or the New World, which for mosquito-borne viruses may reflect an Old World origin. The correlation between epidemiology, disease correlation and biogeography begins to define the complex evolutionary relationships between the virus, vector, vertebrate host and ecological niche.
Human antibodies isolated from plasma by affinity chromatography increase the coxsackievirus B4-induced synthesis of interferon-α by human peripheral blood mononuclear cells in vitro
Authors: Chehadeh Wassim , Bouzidi Ahmed , Alm Gunar , Wattré Pierre , Hober Didier
Published: August 2015

Coxsackievirus B4 (CVB4) can be found in circulating blood of patients; however, the interaction of CVB4 with peripheral blood mononuclear cells (PBMCs) is poorly understood. CVB4 induced low levels of IFN-α synthesis in PBMCs from healthy donors. In contrast, preincubation of infectious CVB4 with plasma from these donors containing anti-CVB4 antibodies strongly enhanced the synthesis of IFN-α. IgG obtained from plasma by chromatography formed immune complexes with CVB4 and increased significantly the CVB4-induced production of IFN-α by PBMCs. These antibodies did not have a neutralizing effect on CVB4 infection of Hep-2 cells. The role of CVB and adenovirus receptor (CAR), FcγRII and FcγRIII in the increased synthesis of IFN-α induced by CVB4 preincubated with IgG was shown by inhibition with specific antibodies. The major interferon-α-producing cells in response to CVB4–IgG complexes were CD14+cells and monocyte-enriched PBMCs. With the latter, detection of IFN-α by immunostaining was positive whereas in monocyte-depleted PBMCs it was not. This study shows that CVB4-induced synthesis of IFN-α by PBMCs can be enhanced by an antibody-dependent mechanism through interactions between the virus, non-neutralizing antivirus antibodies, FcγRII and III and CAR.
Inhibition of transcription-regulating properties of nonstructural protein 1 (NS1) of parvovirus minute virus of mice by a dominant-negative mutant form of NS1
Authors: Deleu Laurent , Pujol Aurora , Nüesch Jürg P. F. , Rommelaere Jean
Published: August 2015

Nonstructural protein 1 (NS1) of minute virus of mice is involved in viral DNA replication, transcriptional regulation and cytotoxic action in the host cell. Viral DNA replication is dependent on the ability of NS1 to form homo-oligomers. To investigate whether oligomerization is required for NS1 transcriptional activities, a functionally impaired mutant derivative of NS1 that was able to interact with the wild-type (wt) protein and inhibit its activity in a dominant-negative manner was designed. This mutant provided evidence that transactivation of the parvoviral P38 promoter and transinhibition of a heterologous promoter by NS1 were both affected by the co-expression of the wt and the dominant-negative mutant form of NS1. These results indicate that additional functions of NS1, involved in promoter regulation, require oligomer formation.
Mutational analysis of the major heparan sulfate-binding domain of herpes simplex virus type 1 glycoprotein C
Authors: Mårdberg Kristina , Trybala Edward , Glorioso Joseph C. , Bergström Tomas
Published: August 2015

Heparan sulfate (HS) has been identified as a receptor molecule for numerous microbial pathogens, including herpes simplex virus type 1 (HSV-1). To further define the major HS-binding domain of the HSV-1 attachment protein, i.e. glycoprotein C (gC), virus mutants carrying alterations of either two neighbouring basic amino acid residues or a single hydrophobic amino acid residue within the N-terminal domain of the protein (residues 26–227) were constructed. In addition, a mutant lacking the Asn148 glycosylation site was included in the study. Binding of purified mutated gC proteins to isolated HS chains showed that viruses with mutations at residues Arg(129,130), Ile142, Arg(143,145), Arg(145,147), Arg(151,155) and Arg(155,160) had significantly impaired HS binding, in contrast to the other mutations, including Asn148. Impairment of the HS-binding activity of gC by these mutations had profound consequences for virus attachment and infection of cells in which amounts of HS exposed on the cell surface had been reduced. It is suggested that basic and hydrophobic residues localized at the Cys127–Cys144 loop of HSV-1 gC constitute a major HS-binding domain, with the most active amino acids situated near the C-terminal region of the two cysteines.
Multimeric humanized varicella-zoster virus antibody fragments to gH neutralize virus while monomeric fragments do not
Authors: Drew P. D. , Moss M. T. , Pasieka T. J. , Grose C. , Harris W. J. , Porter A. J. R.
Published: August 2015

Murine monoclonal antibody 206 (MAb mu206) binds to gH, the varicella-zoster virus (VZV) fusogen, neutralizing the virusin vitroin the absence of complement and inhibiting cell-to-cell spread and egress of VZV in cultured cells. We have humanized this antibody to generate MAb hu206 by complementarity determining region grafting. MAb hu206 retained binding andin vitroneutralizing activity, as well as cross-reactivity with ten different VZV strains. Single-chain antibody fragments (scAb) derived from MAb hu206 were produced inEscherichia coli. These scAb retained the binding properties of the whole antibody. However, monomeric scAb exhibited markedly reduced neutralizing activity compared to the bivalent parental MAb hu206. Shortening the peptide linker joining the VHto the Vκdomain from 14 to 5 or even 0 residues encouraged multimerization and increased neutralizing efficacy. The fact that Fab fragments enzymatically generated from whole MAb hu206 lost their neutralizing potency lent support to the proposal that valency is important for VZV neutralization at this epitope.
Studies on the role of the minor capsid protein in transport of Beet western yellows virus through Myzus persicae
Authors: Reinbold C. , Gildow F. E. , Herrbach E. , Ziegler-Graff V. , Gonçalves M. C. , van den Heuvel J. F. J. M. , Brault V.
Published: August 2015

Beet western yellows virus (BWYV), family Luteoviridae, is an icosahedral plant virus which is strictly transmitted by aphids in a persistent and circulative manner. Virions cross two cellular barriers in the aphid by receptor-based mechanisms involving endocytosis and exocytosis. Particles are first transported across intestinal cells into the haemolymph and then across accessory salivary gland cells for delivery to the plant via saliva. We identified the midgut part of the digestive tract as the site of intestinal passage by BWYV virions. To analyse the role in transmission of the minor capsid component, the readthrough (RT) protein, the fate of a BWYV RT-deficient non-transmissible mutant was followed by transmission electron microscopy in the vector Myzus persicae. This mutant was observed in the gut lumen but was never found inside midgut cells. However, virion aggregates were detected in the basal lamina of midgut cells when BWYV antiserum was microinjected into the haemolymph. The presence of virions in the haemolymph was confirmed by a sensitive molecular technique for detecting viral RNA. Thus, transport of the mutant virions through intestinal cells occurred but at a low frequency. Even when microinjected into the haemolymph, the RT protein mutant was never detected near or in the accessory salivary gland cells. We conclude that the RT protein is not strictly required for the transport of virus particles through midgut cells, but is necessary for the maintenance of virions in the haemolymph and their passage through accessory salivary gland cells.
A novel method using baculovirus-mediated gene transfer for production of recombinant adeno-associated virus vectors
Authors: Sollerbrant Kerstin , Elmén Joacim , Wahlestedt Claes , Acker Joel , Leblois-Prehaud Hélene , Latta-Mahieu Martine , Yeh Patrice , Perricaudet Michel
Published: August 2015

The baculovirus Autographa californica multiple nucleopolyhedrosis virus causes non-productive infection in mammalian cells. Recombinant baculovirus therefore has the capability to transfer and express heterologous genes in these cells if a mammalian promoter governs the gene of interest. We have investigated the possibility of using baculovirus as a tool to produce recombinant adeno-associated virus (rAAV). AAV has become increasingly popular as a vector for gene therapy and functional genomics efforts, although its use is hampered by the lack of a simple and efficient vector production method. We show here that co-infection of mammalian producer cells with three viruses – a baculovirus containing the reporter gene flanked by AAV ITRs, a baculovirus expressing the AAV rep gene and a helper adenovirus expressing the AAV cap gene – produces infectious rAAV particles. This baculovirus-based chimeric vector method may in future improve large-scale rAAV vector preparations and circumvent present-day problems associated with rAAV production.
Efficient downregulation of major histocompatibility complex class I molecules in human epithelial cells infected with cytomegalovirus
Authors: Benz Christine , Reusch Uwe , Muranyi Walter , Brune Wolfram , Atalay Ramazan , Hengel Hartmut
Published: August 2015

Liver and intestinal epithelial cells are a major target of infection by cytomegaloviruses (CMV), causing severe disease in affected organs of immunocompromised patients. CMV downregulates major histocompatibility complex class I (MHC-I) molecule expression in fibroblasts in order to avoid lysis by CD8+cytotoxic T lymphocytes. However, MHC-I expression in human cytomegalovirus (HCMV)-infected hepatic tissue was reported to be increased. As it is unclear at present whether HCMV affects MHC-I expression in epithelial cells, new cell culture models for HCMV infection of differentiated hepatobiliary cell lines were established. HCMV immediate early gene expression was achieved in 60 to 95% of cells. Progression of the HCMV replication cycle differed from prototypic infection of fibroblasts, since structural early and late proteins were produced at low levels and HCMV progeny yielded much lower titres in hepatobiliary cells. In contrast, HCMV glycoproteins, gpUS2, gpUS3, gpUS6 and gpUS11, that downregulate MHC-I expression were synthesized with temporal kinetics and in a similar quantity to that seen in fibroblasts. As a result, HCMV infection led to a drastic and selective downregulation of MHC-I expression in epithelial cells and was uniformly observed irrespective of the hepatic or biliary origin of the cells. The new models document for the first time a stealth function of HCMV in epithelial cells and indicate that the downregulation of MHC-I expression by HCMV can occur in the virtual absence of virus replication.
The UL41-encoded virion host shutoff (vhs) protein and vhs-independent mechanisms are responsible for down-regulation of MHC class I molecules by bovine herpesvirus 1
Authors: Koppers-Lalic Danijela , Rijsewijk Frans A. M. , Verschuren Sylvia B. E. , van Gaans-van den Brink Jacqueline A. M. , Neisig Anne , Ressing Maaike E. , Neefjes Jacques , Wiertz Emmanuel J. H. J.
Published: August 2015

The virion host shutoff (vhs) protein of alphaherpesviruses causes a rapid shutoff of host cell protein synthesis. We constructed a bovine herpesvirus 1 (BHV1) deletion mutant in which the putative vhs gene, UL41, has been disrupted. Whereas protein synthesis is inhibited within 3 h after infection with wild-type BHV1, no inhibition was observed after infection with the BHV1vhs−deletion mutant. These results indicate that the BHV1 UL41 gene product is both necessary and sufficient for shutoff of host cell protein synthesis at early times post-infection. Using the vhs deletion mutant, we investigated the mechanism of BHV1-induced down-regulation of MHC class I cell surface expression. In contrast to BHV1 wild-type infection, the BHV1vhs−mutant allows detection of MHC class I molecules at much later time-points after infection. This illustrates the role the vhs protein plays in MHC class I down-regulation. However, even after infection with BHV1vhs−, MHC class I cell surface expression is impaired. In BHV1vhs−-infected cells, MHC class I molecules are retained within the endoplasmic reticulum (ER). Moreover, the transporter associated with antigen presentation (TAP) is still blocked. Temporal control of viral protein expression using chemical inhibitors shows that viral protein(s) expressed within the early phase of BHV1 infection are responsible for ER retention of MHC class I molecules. These results indicate that multiple mechanisms are responsible for down-regulation of MHC class I molecules in BHV1-infected cells.
Construction and characterization of recombinant vaccinia viruses co-expressing a respiratory syncytial virus protein and a cytokine
Authors: Johnson Teresa R. , Fischer Julie E. , Graham Barney S.
Published: August 2015

Recombinant vaccinia viruses are well-characterized tools that can be used to define novel approaches to vaccine formulation and delivery. While vector co-expression of immune mediators has enormous potential for optimizing the composition of vaccine-induced immune responses, the impact on antigen expression and vector antigenicity must also be considered. Co-expression of IL-4 increased vaccinia virus vector titres, while IFN-γ co-expression reduced vaccinia virus replication in BALB/c mice and in C57BL/6 mice infected with some recombinant viruses. Protection against respiratory syncytial virus (RSV) challenge was similar in mice immunized with vaccinia virus expressing RSV G glycoprotein and IFN-γ, even though the replication efficiency of the vector was diminished. These data demonstrate the ability of vector-expressed cytokine to influence the virulence of the vector and to direct the development of selected immune responses. This suggests that the co-expression of cytokines and other immunomodulators has the potential to improve the safety of vaccine vectors while improving the immunogenicity of vaccine antigens.
Genetic diversity and molecular epidemiology of respiratory syncytial virus over four consecutive seasons in South Africa: identification of new subgroup A and B genotypes
Authors: Venter Marietjie , Madhi Shabir A. , Tiemessen Caroline T. , Schoub Barry D.
Published: August 2015

The molecular epidemiology of respiratory syncytial virus (RSV) was studied over four consecutive seasons (1997–2000) in a single tertiary hospital in South Africa: 225 isolates were subgrouped by RT–PCR and the resulting products sequenced. Subgroup A predominated in two seasons, while A and B co-circulated approximately equally in the other seasons. The nucleotide sequences of the C-terminal of the G-protein were compared to sequences representative of previously defined RSV genotypes. South African subgroup A and subgroup B isolates clustered into four and five genotypes respectively. One new subgroup A and three new subgroup B genotypes were identified. Different genotypes co-circulated in every season. Different circulation patterns were identified for group A and B isolates. Subgroup A revealed more variability and displacement of genotypes while subgroup B remained more consistent.
A measles virus glycoprotein-derived human CTL epitope is abundantly presented via the proteasomal-dependent MHC class I processing pathway
Authors: Herberts Carla A. , Stittelaar Koert J. , van der Heeft Ed , van Gaans-van den Brink Jacqueline , Poelen Martien C. M. , Roholl Paul J. M. , van Alphen Loek J. W. , Melief Cornelis J. M. , de Jong Ad P. J. M. , van Els Cécile A. C. M.
Published: August 2015

Peptides derived from measles virus (MV) are presented by MHC class I molecules at widely divergent levels, but it is currently unknown how functional these levels are. Here, for the first time, we studied the natural occurrence and the underlying processing events of a known MV CTL epitope derived from the fusion glycoprotein (MV-F) and restricted via HLA-B*2705. Using MHC–peptide elution of MV-infected cells followed by sensitive mass spectrometry we determined the naturally occurring sequence to be RRYPDAVYL, corresponding to MV-F438–446. Its level of expression was enumerated at approximately 1500 copies per cell, which is considered to be abundant, but lies within the range described for other viral CTL epitopes in human MHC class I molecules. We found that processing of the MV-F438–446epitope occurs primarily via the classic MHC class I loading pathway, since presentation to CTL depends on both the transporter associated with antigen presentation (TAP) and the proteasome. Even though it is cotranslationally inserted into the ER, a major part of MV-F is located in the cytosol, where it accumulates rapidly in the presence of proteasome inhibitors. We therefore conclude that a substantial cytosolic turnover of MV-F, together with some excellent processing features of MV-F438–446precursors, such as precise C-terminal excision by proteasomes, efficient TAP transport and strong HLA binding, dictate the abundant functional expression of the MV-F438–446CTL epitope in HLA-B*2705 at the surface of MV-infected cells.