bluuebunny/crossref_metadata_2025 huggingface dataset:
The vaccinia virus A41L protein is a soluble 30 kDa glycoprotein that affects virus virulence
Authors: Ng Aylwin , Tscharke David C. , Reading Patrick C. , Smith Geoffrey L.
Published: August 2015

Vaccinia virus (VV) gene A41L encodes an acidic protein with amino acid similarity to the 35 kDa protein of VV strain Lister, a soluble protein called vCKBP that binds CC chemokines, and to a protein from orf virus, called GIF, that binds GM-CSF and IL-2. However, despite the similarity, recombinant A41L protein was found not to bind these ligands or a variety of other chemoattractant molecules when tested using surface plasmon resonance. The A41L gene is expressed early and late during infection and encodes a 30 kDa protein that contains bothN- andO-linked carbohydrate and is secreted from the infected cell. All 16 strains of VV and 2 strains of cowpox virus that were tested express the A41L protein, implying it has an important function for orthopoxviruses. Nonetheless, a VV strain Western Reserve deletion mutant lacking the A41L gene (vΔA41L) formed normal sized plaques and replicated to the same titre as wild-type and revertant viruses. The importance of the A41L proteinin vivowas demonstrated in a mouse intradermal model in which infection with vΔA41L caused more severe lesions compared to wild-type and revertant viruses. Further examination in this model revealed that deletion of A41L enhanced clearance of infectious virus, suggesting that A41L expression reduces immunopathology. Consistent with this, histological examination of infected rabbit skin showed that the A41L protein could reduce the infiltration of inflammatory cells into the infected area. Together, these data suggest that the A41L protein constitutes a novel immunomodulatory protein.
In situ hybridization and immunolabelling study of the early replication of simian immunodeficiency virus (SIVmacJ5) in vivo
Authors: Cantó-Nogués Carmen , Jones Sue , Sangster Rebecca , Silvera Peter , Hull Robin , Cook Roger , Hall Graham , Walker Barry , Stott E. Jim , Hockley David , Almond Neil
Published: August 2015

The distribution of virus-infected cells in cynomolgus macaques was determined at 4, 7, 14 and 28 days following intravenous challenge with 1000 TCID50 of the wild-type simian immunodeficiency virus SIVmacJ5 (stock J5C). At each time-point, pairs of macaques were killed humanely and the presence of SIV was determined and quantified in blood, spleen, peripheral and mesenteric lymph nodes, thymus, lung and ileum by virus co-cultivation with C8166 cells, by quantitative DNA PCR or by in situ hybridization (ISH). At day 4 post-infection (p.i.), detection of the virus was sporadic. By day 7 p.i., however, significant SIV loads were detected in the blood and lymphoid tissues by DNA PCR and virus co-cultivation. Large numbers of cells expressing SIV RNA were detected in mesenteric lymph nodes by ISH and significantly fewer (P<0·05) in the spleen. Significant numbers of ISH-positive cells were also observed in sections of ileum. By day 14 p.i., the distribution of SIV was more even in all lymphoid tissues analysed. By day 28, most of the tissues were negative by ISH, but all remained positive by virus isolation and DNA PCR. Immunolabelling of sections of mesenteric lymph node with monoclonal antibodies specific for SIV envelope and Nef largely confirmed the observations from ISH. These results indicate that, even following intravenous challenge, a major site of the initial replication of SIV is gut-associated lymphoid tissue. Vaccines that induce protection at this site may therefore be superior, even against parenteral challenge.
Determination of the intramolecular disulfide bond arrangement and biochemical identification of the glycosylation sites of the nonstructural protein NS1 of Murray Valley encephalitis virus
Authors: Blitvich Bradley J. , Scanlon Denis , Shiell Brian J. , Mackenzie John S. , Pham Kim , Hall Roy A.
Published: August 2015

The 12 cysteine residues in the flavivirus NS1 protein are strictly conserved, suggesting that they form disulfide bonds that are critical for folding the protein into a functional structure. In this study, we examined the intramolecular disulfide bond arrangement of NS1 of Murray Valley encephalitis virus and elucidated three of the six cysteine-pairing arrangements. Disulfide linkages were identified by separating tryptic-digested NS1 by reverse-phase high pressure liquid chromatography and analysing the resulting peptide peaks by protein sequencing, amino acid analysis and/or electrospray mass spectrometry. The pairing arrangements between the six amino-terminal cysteines were identified as follows: Cys4–Cys15, Cys55–Cys143 and Cys179–Cys223. Although the pairing arrangements between the six carboxy-terminal cysteines were not determined, we were able to eliminate several cysteine-pairing combinations. Furthermore, we demonstrated that all three putative N-linked glycosylation sites of NS1 are utilized and that the Asn207 glycosylation site contains a mannose-rich glycan.
Coat protein gene-mediated resistance to Potato virus A in transgenic plants is suppressed following infection with another potyvirus
Authors: Savenkov E. I. , Valkonen J. P. T.
Published: August 2015

High levels of resistance to Potato virus A (PVA, genus Potyvirus), indicated by absence of detectable infection in inoculated leaves, were attained in Nicotiana benthamiana transformed with a construct expressing the PVA 5′-untranslated region fused with the coat protein (CP)-encoding sequence. Low steady-state levels of the transgene transcripts were detected. Resistance was PVA-specific and did not protect the plants against infection with Potato virus Y (PVY, genus Potyvirus). Consequently, the steady-state levels of the CP-transgene mRNA were greatly elevated in the plants infected with PVY, and plants became susceptible to infection with PVA. These data show that virus resistance obtained by expressing regions of a plant virus genome in transgenic plants may be suppressed following infection with another virus that evades the virus-specific resistance.
Adverse effects of feline IL-12 during DNA vaccination against feline infectious peritonitis virus
Authors: Glansbeek Harrie L. , Haagmans Bart L. , te Lintelo Eddie G. , Egberink Herman F. , Duquesne Véronique , Aubert André , Horzinek Marian C. , Rottier Peter J. M.
Published: August 2015

Cell-mediated immunity is thought to play a decisive role in protecting cats against feline infectious peritonitis (FIP), a progressive and lethal coronavirus disease. In view of the potential of DNA vaccines to induce cell-mediated responses, their efficacy to induce protective immunity in cats was evaluated. The membrane (M) and nucleocapsid (N) proteins were chosen as antigens, because antibodies to the spike (S) protein of FIP virus (FIPV) are known to precipitate pathogenesis. However, vaccination by repeated injections of plasmids encoding these proteins did not protect kittens against challenge infection with FIPV. Also, a prime–boost protocol failed to afford protection, with priming using plasmid DNA and boosting using recombinant vaccinia viruses expressing the same coronavirus proteins. Because of the role of IL-12 in initiating cell-mediated immunity, the effects of co-delivery of plasmids encoding the feline cytokine were studied. Again, IL-12 did not meet expectations – on the contrary, it enhanced susceptibility to FIPV challenge. This study shows that DNA vaccination failed to protect cats against FIP and that IL-12 may yield adverse effects when used as a cytokine adjuvant.
Antibody-sensitive and antibody-resistant cell-to-cell spread by vaccinia virus: role of the A33R protein in antibody-resistant spread
Authors: Law Mansun , Hollinshead Ruth , Smith Geoffrey L.
Published: August 2015

The roles of vaccinia virus (VV) intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV) and their associated proteins in virus spread were investigated. The plaques made by VV mutants lacking individual IEV- or EEV-specific proteins (vΔA33R, vΔA34R, vΔA36R, vΔA56R, vΔB5R, vΔF12L and vΔF13L) were compared in the presence of IMV- or EEV-neutralizing antibodies (Ab). Data presented show that for long-range spread, the comet-shaped plaques of VV were caused by the unidirectional spread of EEV probably by convection currents, and for cell-to-cell spread, VV uses a combination of Ab-resistant and Ab-sensitive pathways. Actin tails play a major role in the Ab-resistant pathway, but mutants such as vΔA34R and vΔA36R that do not make actin tails still spread from cell to cell in the presence of Ab. Most strikingly, the Ab-resistant pathway was abolished when the A33R gene was deleted. This effect was not due to alterations in the efficiency of neutralization of EEV made by this mutant, nor due to a deficiency in IMV wrapping to form IEV, which was indispensable for EEV formation by vΔA33R and vΔA34R. We suggest a role for A33R in promoting Ab-resistant cell-to-cell spread of virus. The roles of the different virus forms in the VV life-cycle are discussed.
An investigation of incorporation of cellular antigens into vaccinia virus particles
Authors: Krauss Oliver , Hollinshead Ruth , Hollinshead Michael , Smith Geoffrey L.
Published: August 2015

Vaccinia virus (VV) infection produces several types of virus particle called intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). Some cellular antigens are associated with EEV and these vary with the cell type used to grow the virus. To investigate if specific cell antigens are associated with VV particles, and to address the origin of membranes used to envelope IMV and IEV/CEV/EEV, we have studied whether cell antigens and foreign antigens expressed by recombinant VVs are incorporated into VV particles. Membrane proteins that are incorporated into the endoplasmic reticulum (ER), intermediate compartment (IC),cis/medial-Golgi,trans-Golgi network (TGN) or plasma membrane were not detected in purified IMV particles. In contrast, proteins present in the TGN or membrane compartments further downstream in the exocytic pathway co-purify with EEV particles when analysed by immunoblotting. Immunoelectron microscopy found only low levels of these proteins in IEV, CEV/EEV. The incorporation of foreign antigens into VV particles was not affected by loss of individual IEV or EEV-specific proteins or by redirection of B5R to the ER. These data suggest that (i) host cell antigens are excluded from the lipid envelope surrounding the IMV particle and (ii) membranes of the ER, IC andcis/medial-Golgi are not used to wrap IMV particles to form IEV. Lastly, the VV haemagglutinin was absent from one-third of IEV and CEV/EEV particles, whereas other EEV antigens were present in all these virions.
Epstein–Barr virus nuclear antigen-1 is highly colocalized with interphase chromatin and its newly replicated regions in particular
Authors: Ito Sayuri , Gotoh Eisuke , Ozawa Shigeru , Yanagi Kazuo
Published: August 2015

Epstein–Barr virus (EBV) nuclear antigen-1 (EBNA-1), which binds to both the EBV origin of replication (oriP) and metaphase chromosomes, is essential for the replication/retention and segregation/partition oforiP-containing plasmids. Here the chromosomal localization of EBNA-1 fused to green fluorescent protein (GFP–EBNA-1) is examined by confocal microscopy combined with a ‘premature chromosome condensation’ (PCC) procedure. Analyses show that GFP–EBNA-1 expressed in living cells that lackoriPplasmids is associated with cellular chromatin that has been condensed rapidly by the PCC procedure into identifiable forms that are unique to each phase of interphase as well as metaphase chromosomes. Studies of cellular chromosomal DNAs labelled with BrdU or Cy3-dUTP indicate that GFP–EBNA-1 colocalizes highly with the labelled, newly replicated regions of interphase chromatin in cells. These results suggest that EBNA-1 is associated not only with cellular metaphase chromosomes but also with condensing chromatin/chromosomes and probably with interphase chromatin, especially with its newly replicated regions.
Characterization of a novel envelope protein (VP281) of shrimp white spot syndrome virus by mass spectrometry
Authors: Huang Canhua , Zhang Xiaobo , Lin Qingsong , Xu Xun , Hew Choy(-)L.
Published: August 2015

The primary structure of a novel envelope protein from shrimp white spot syndrome virus (WSSV) was characterized using a combination of SDS–PAGE and mass spectrometry. The resulting amino acid sequence matched an open reading frame (ORF), ORF1050, of the WSSV genome ORF database. ORF1050 contained 843 nt, encoding 281 aa, and was termed the vp281 gene. Computer-assisted analysis showed that both the vp281 gene and its product shared no significant homology with other known viruses. However, they shared striking identity/similarity with another WSSV structural protein, VP292, at both the nucleotide and amino acid sequence level, suggesting that vp281 and vp292 might have evolved by gene duplication from a common ancestral gene. WSSV VP281 cDNA was cloned into a pET32a(+) expression vector containing a T7 RNA polymerase promoter to produce (His)6-tagged fusion proteins in Escherichia coli strain BL21. Specific mouse antibodies were raised using the purified fusion protein (His)6-VP281. Western blot analysis showed that the mouse anti-(His)6-VP281 antibodies bound specifically to VP281 of WSSV, without cross-reactivity with VP292. The transmission electron microscope immunogold-labelling method was used to localize VP281 in the WSSV virion as an envelope protein. The cell attachment ‘Arg–Gly–Asp’ motif in VP281 indicated that this protein might play an important role in mediating WSSV infectivity.
Sequence of the 3′-terminal end (8·1 kb) of the genome of porcine haemagglutinating encephalomyelitis virus: comparison with other haemagglutinating coronaviruses
Authors: Sasseville A. Marie-Josée , Boutin Martine , Gélinas Anne-Marie , Dea Serge
Published: August 2015

A cytopathogenic coronavirus, serologically identified as porcine haemagglutinating encephalomyelitis virus (HEV), has recently been associated with acute outbreaks of wasting and encephalitis in nursing piglets from pig farms in southern Québec and Ontario, Canada. The 3′-terminal end of the genome of the prototype HEV-67N strain and that of the recent Québec IAF-404 field isolate, both propagated in HRT-18 cells, were sequenced. Overall, sequencing data indicated that HEV has remained antigenically and genetically stable since its first isolation in North America in 1962. Compared with the prototype strain of bovine enteropathogenic coronavirus (BCoV), HEV, as well as the human respiratory coronavirus (HCoV-OC43) showed a major deletion in their ORF4 gene. Deduced amino acid sequences for both HEV strains revealed 89/88, 80, 93/92 and 95/94% identities with the structural proteins HE, S, M and N of BCoV and HCoV-OC43, respectively. Major variations were observed in the S1 portion of the S gene of both HEV strains, with only 73/71% amino acid identities compared with those of the two other haemagglutinating coronaviruses.
In vitro analysis of factors involved in the disassembly of Sindbis virus cores by 60S ribosomal subunits identifies a possible role of low pH
Authors: Wengler Gerd , Wengler Gisela
Published: August 2015

Disassembly of alphavirus cores early in infection involves interaction of the core with 60S ribosomal subunits. This interaction might be subjected to regulatory processes. We have established an in vitro system of core disassembly in order to identify cellular proteins involved in the regulation of disassembly. No evidence for the existence of such proteins was found, but it became apparent that certain organic solvents and detergents or a high proton concentration (pH 6·0) stimulated core disassembly. Alphaviruses infect cells by an endosomal pathway. The low pH in the endosome activates a fusion activity of the viral surface protein E1 and leads to fusion of the viral membrane with the endosomal membrane, followed by release of the core into the cytoplasm. Since the presence of the E1 protein in the plasma membrane of infected cells leads to increased membrane permeability at low pH, our findings indicate that disassembly of alphavirus cores could be regulated by the proton concentration. We propose that the viral membrane proteins present in the endosomal membrane after fusion form a pore, which allows the flow of protons from the endosome into the cytoplasm. This process would generate a region of low pH in the cytoplasm at the correct time and place to allow the efficient disassembly of the incoming viral core by 60S subunits.
Detection of measles virus genome in bone-marrow aspirates from adults
Authors: Sonoda Satomi , Kitahara Mitsuo , Nakayama Tetsuo
Published: August 2015

We investigated the presence of the measles virus genome in order to identify asymptomatic infections in the adult population. Bone-marrow aspirates were obtained from 179 patients, 20–96 years of age, for the diagnosis of malignant diseases (29 with malignant lymphoma, 28 with acute leukaemia, 21 with myelodysplastic syndrome, five with multiple myeloma and 96 with other diseases). The measles virus genome was detected in 17 (9·5%) of 179 individuals by RT–PCR and 28 (15·6%) through hybridization. The genomes detected in bone marrow were all in the same cluster, D5, the strain circulating during the study period, and no evidence of persistent infection was obtained. We conclude that asymptomatic infections of measles virus are common in adults and the presence of the measles virus genome would not be related to the pathogenesis of illness.
Selection of antigenic variants in maedi–visna virus infection
Authors: Andrésdóttir Valgerdur , Skraban Robert , Matthíasdóttir Sigrídur , Lutley Roger , Agnarsdóttir Gudrún , Thorsteinsdóttir Hólmfrídur
Published: August 2015

In order to analyse the pattern of sequence variation in maedi–visna virus (MVV) in persistently infected sheep and to answer the question of whether antigenic variants are selected in a long-term MVV infection, an 87 bp variable region in theenvgene of ten antigenic variants and 24 non-variants was sequenced. Nine of the ten antigenic variants had mutations in this region, comprising 24 point mutations and a deletion of 3 bp. Twenty-three of the point mutations (96%) were non-synonymous. There was only a single mutation in this region in the 24 non-variants. A type-specific neutralizing antibody response appeared in all the sheep 2–5 months post-infection, and in most sheep more broadly reacting neutralizing antibodies appeared up to 4 years later. All the antigenic variants were neutralized by the broadly reacting sera. It is noteworthy that the antigenic variants were isolated at a time when only the type-specific antibodies were acting, before the broadly reacting antibodies appeared. The same picture emerged when molecularly cloned virus was used for infection. Three sheep were infected with a molecularly cloned virus, and of six virus isolates, one was an antigenic variant. This variant arose in the absence of broadly reacting antibodies. The results indicate that there is selection for mutants that escape neutralization.
The viral envelope is a major determinant for the induction of lymphoid and myeloid tumours by avian leukosis virus subgroups A and J, respectively
Authors: Chesters P. M. , Howes K. , Petherbridge L. , Evans S. , Payne L. N. , Venugopal K.
Published: August 2015

Among the six envelope subgroups of avian leukosis virus (ALV) that infect chickens, subgroups A (ALV-A) and J (ALV-J) are the most pathogenic and widespread among commercial chicken populations. While ALV-A is predominantly associated with lymphoid leukosis (LL) and less frequently with erythroblastosis (EB), ALV-J mainly induces tumours of the myeloid lineage. In order to examine the basis for the lineage specificity of tumour induction by these two ALV subgroups, we constructed two chimeric viruses by substituting the env genes into the reciprocal proviral clones. The chimeric HPRS-103(A) virus carrying the subgroup A env gene is identical to ALV-J prototype virus HPRS-103 except for the env gene, and the chimeric RCAS(J) virus carrying the subgroup J env gene is identical to the parent replication-competent ALV-A vector RCAS except for the env gene. In experimentally inoculated chickens, HPRS-103(A) virus induced LL and EB similar to ALV-A isolates such as RAV-1, while RCAS(J) virus induced myeloid leukosis (ML) and EB, similar to ALV-J, suggesting that the env gene is the major determinant for the lineage-specific oncogenicity. There were genetic differences in susceptibility to tumour induction between line 0 and line 15I chickens, indicating that in addition to the env gene, other viral or host factors could also serve as determinants for oncogenicity. Induction of both LL and ML by the two chimeric viruses occurred through the activation of c-myc, while the EB tumours were induced by activation of the c-erbB oncogene.
Characterization of the receptor-destroying enzyme activity from infectious salmon anaemia virus
Authors: Kristiansen Marianne , Frøystad Marianne K. , Rishovd Anne Lise , Gjøen Tor
Published: August 2015

Infectious salmon anaemia virus (ISAV) infects cells via the endocytic pathway and, like many other enveloped viruses, ISAV contains a receptor-destroying enzyme. We have analysed this acetylesterase activity with respect to substrate specificity, enzyme kinetics, inhibitors, temperature and pH stability. The ISAV acetylesterase was inhibited by di-isopropyl fluorophosphate (DFP) in a dose-dependent fashion but not by other known hydrolase inhibitors, suggesting that a serine residue is part of the active site. The pH optimum of the enzyme was in the range 7·5–8·0 and the enzymatic activity was lessened at temperatures above 40 °C. The effect of DFP on agglutination/elution of erythrocytes by ISAV demonstrated that the acetylesterase activity is the bona fide receptor-destroying enzyme. A haemadsorption assay was used to analyse whether the esterase was active on the surface of infected cells or not.
Rapid replacement of endemic measles virus genotypes
Authors: Santibanez Sabine , Tischer Annedore , Heider Alla , Siedler Anette , Hengel Hartmut
Published: August 2015

Although vaccination campaigns have significantly reduced the number of measles cases worldwide, endemic transmission of measles virus (MV) continues to occur in several continents, including Europe. To obtain current information on measles incidence and molecular data on circulating MVs in Germany, a nationwide measles sentinel was established. Phylogenetic analysis based on the variable part of the N gene from 80 MVs isolated between November 1999 and October 2001 revealed the presence of at least six distinct MV genotypes: B3, C2, D4, D6, G2 and a new variant of D7. Both the incidence and the pattern of MV genotypes differed markedly between the former East and West Germany. In the eastern part, few measles cases, mainly caused by genotypes originating from other countries (B3, D4, G2), were detected. In the western and southern parts, genotypes C2, D6 and D7 were associated with endemic transmission. Surprisingly, the indigenous genotypes predominant during the 1990s – C2 and D6 – disappeared simultaneously over the period of observation coinciding with the emergence and the wide spread of D7 viruses. While the incidence of measles remained constant, all MVs isolated in 2001 were assigned to D7. We note that the haemagglutinin (H) sequence of D7 viruses shows distinct exchanges of certain amino acids in the stem and propeller domain compared to C2, D6 and the MV vaccine strains used. This raises the possibility of a selective advantage of D7 viruses transmitted in the presence of H-specific antibodies.
Scrapie epidemic in a fully PrP-genotyped sheep flock
Authors: Baylis M. , Goldmann W. , Houston F. , Cairns D. , Chong A. , Ross A. , Smith A. , Hunter N. , McLean A. R.
Published: August 2015

In scrapie-affected sheep flocks, host PrP genotype plays a vital role in determining which sheep will succumb to scrapie and the incubation period. Consequently, within-flock scrapie dynamics is best understood within the context of the genotype profile of the flock. Here we describe a 17 month epidemic of scrapie in a commercially farmed flock of 230 genotyped Texel sheep. At the start of the study, 70% of the sheep were of three genotypes only: ARR/ARQ, ARH/ARQ and ARQ/ARQ. Only 15% of sheep encoded the disease-associated VRQ allele and only a single sheep (0·4%) was of the most susceptible VRQ/VRQ genotype. For susceptible genotypes there was a marked deficit (P<0·025) of older animals (⩾3 years), implying that some cases of scrapie had occurred previously. In the ensuing 17 months, 18 sheep of known genotype were confirmed positive for the disease: seven VRQ/ARQ, six VRQ/ARH, two VRQ/ARR, three ARQ/ARQ. Median ages at death were 2·7, 2·8, 4·2 and 3·8 years respectively. Mortality rates were 55, 86, 13 and 3% respectively. Survival analysis revealed a highly significant effect of genotype on survivorship, but no difference between VRQ/ARQ and VRQ/ARH, or between VRQ/ARR and ARQ/ARQ. There was no difference in the survivorship of middle- and older-age cohorts of susceptible sheep. Scrapie risk group (as defined by PrP genotype) was not associated with submission as a scrapie suspect but later found to be negative, or with dying of unknown causes on the farm.
Genetic variation and immunohistochemical differences among geographic isolates of Taura syndrome virus of penaeid shrimp
Authors: Robles-Sikisaka Refugio , Hasson Kenneth W. , Garcia Denise K. , Brovont Katherine E. , Cleveland Karyn D. , Klimpel Kurt R. , Dhar Arun K.
Published: August 2015

Taura syndrome virus (TSV) is an important virus infecting penaeid shrimp in the western hemisphere. Genetic variation and immunohistochemical differences of 20 TSV isolates collected from the USA, Taiwan, Mexico and Nicaragua were compared. Capsid protein genes CP1 (546 bp) and CP2 (584 bp) were amplified by RT–PCR and the cDNAs were sequenced. Pairwise comparison of nucleotide sequences showed a 0–2·4% difference in CP1 and a 0–3·5% difference in CP2. Phylogenetic analyses clustered the TSV isolates into two groups: one contained USA, Taiwan and some Mexican isolates, the other contained Mexican isolates only. Immunohistochemical analysis using a TSV-specific monoclonal antibody produced positive results for the USA and Taiwan isolates but negative results for the Mexican and Nicaraguan isolates. Molecular and immunohistochemical data suggest the existence of at least two TSV strains, one of which might have evolved following contact with a new penaeid host,Penaeus stylirostris.
Sequence analysis and genomic organization of Aphid lethal paralysis virus: a new member of the family Dicistroviridae
Authors: van Munster M. , Dullemans A. M. , Verbeek M. , van den Heuvel J. F. J. M. , Clérivet A. , van der Wilk F.
Published: August 2015

The complete nucleotide sequence of the genomic RNA of an aphid-infecting virus, Aphid lethal paralysis virus (ALPV), has been determined. The genome is 9812 nt in length and contains two long open reading frames (ORFs), which are separated by an intergenic region of 163 nt. The first ORF (5′ ORF) is preceded by an untranslated leader sequence of 506 nt, while an untranslated region of 571 nt follows the second ORF (3′ ORF). The deduced amino acid sequences of the 5′ ORF and 3′ ORF products respectively showed similarity to the non-structural and structural proteins of members of the newly recognized genusCripavirus(familyDicistroviridae). On the basis of the observed sequence similarities and identical genome organization, it is proposed that ALPV belongs to this genus. Phylogenetic analysis showed that ALPV is most closely related toRhopalosiphum padi virus, and groups in a cluster withDrosophila C virusandCricket paralysis virus, while the other members of this genus are more distantly related. Infectivity experiments showed that ALPV can not only infect aphid species but is also able to infect the whiteflyTrialeurodes vaporariorum, extending its host range to another family of the order Hemiptera.
Characterization of a protein from Rice tungro spherical virus with serine proteinase-like activity
Authors: Thole Vera , Hull Roger
Published: August 2015

The RNA genome of Rice tungro spherical virus (RTSV) is predicted to be expressed as a large polyprotein precursor (Shen et al., Virology 193, 621–630, 1993 ). The polyprotein is processed by at least one virus-encoded protease located adjacent to the C-terminal putative RNA polymerase which shows sequence similarity to viral serine-like proteases. The catalytic activity of this protease was explored using in vitro transcription/translation systems. Besides acting in cis, the protease had activity in trans on precursors containing regions of the 3’ half of the polyprotein but did not process a substrate consisting of a precursor of the coat proteins. The substitution mutation of Asp2735 of the RTSV polyprotein had no effect on proteolysis; however, His2680, Glu2717, Cys2811 and His2830 proved to be essential for catalytic activity and could constitute the catalytic centre and/or substrate-binding pocket of the RTSV 3C-like protease.
Stability in vitro of the 69K movement protein of Turnip yellow mosaic virus is regulated by the ubiquitin-mediated proteasome pathway
Authors: Drugeon Gabrièle , Jupin Isabelle
Published: August 2015

Plant viruses move to adjacent cells with the use of virus-encoded cell-to-cell movement proteins. Using proteins produced byin vitrotranslation, we present evidence that the ‘69K’ movement protein ofTurnip yellow mosaic virus(TYMV) is recognized as a substrate for the attachment of polyubiquitin chains and for subsequent rapid and selective proteolysis by the proteasome, the ATP-dependent proteolytic system present in reticulocyte lysate. Truncation of the 69K protein suggests the existence of two degradation signals within its sequence. We propose that selective degradation of virus movement proteins may contribute to the previously reported transient nature of their accumulation during infection.
Herpes simplex virus type 1 glycoprotein C is necessary for efficient infection of chondroitin sulfate-expressing gro2C cells
Authors: Mårdberg Kristina , Trybala Edward , Tufaro Frank , Bergström Tomas
Published: August 2015

The role of glycoprotein C (gC) for binding of herpes simplex virus type 1 (HSV-1) to cell surface chondroitin sulfate (CS) and the consequences of this interaction for virus attachment and infectivity were studied. To this end, a panel of HSV-1 gC mutants, including a gC-negative (gC−) variant, and mouse fibroblasts expressing either cell surface CS or heparan sulfate (HS) were used. Comparing gC-positive (gC+) and gC−viruses in terms of their attachment to and infection of CS-expressing cells indicated that gC was essential for both functions. Furthermore, purified gC bound efficiently to isolated CS chains. However, hypertonic NaCl disrupted this interaction more easily as compared to the binding of gC to HS. Also, native and selectively desulfated heparins were approximately 10 times more efficient at inhibiting gC binding to CS-expressing cells than binding to HS-expressing cells. Experiments with the HSV-1 gC mutants revealed that specific, positively charged and hydrophobic amino acids within the N-terminal part of the protein were responsible for efficient binding as well as infectivity in both CS- and HS-expressing cells. When the infectivity of the gC mutants in the two cell types was compared, it appeared that more residues contributed to the infection of CS-expressing cells than to infection of HS-expressing cells. Taken together, analysis of gC function in cell systems with limited expression of glycosaminoglycans revealed that gC could interact with either CS or HS and that these interactions exhibited subtle but definite differences as regards to the involved structural features of gC, ionic strength dependency as well as sensitivity to specifically desulfated heparin compounds.
Early gene m18, a novel player in the immune response to murine cytomegalovirus
Authors: Holtappels Rafaela , Grzimek Natascha K. A. , Thomas Doris , Reddehase Matthias J.
Published: August 2015

The identification of all antigenic peptides encoded by a pathogen, its T cell ‘immunome’, is a research aim for rational vaccine design. Screening of proteome-spanning peptide libraries or computational prediction is used to identify antigenic peptides recognized by CD8 T cells. Based on their high coding capacity, cytomegaloviruses (CMVs) could specify numerous antigenic peptides. Yet, current evidence indicates that the memory CD8 T cell response in a given haplotype is actually focused on a few viral proteins. CMVs actively interfere with antigen processing and presentation by the expression of immune evasion proteins. In the case of murine CMV (mCMV), these proteins are effectual in the early (E) phase of the virus replication cycle and should thus preclude the presentation of peptides derived from E proteins. Notably, the m18 gene is here added to a growing list of mCMV E genes that encode antigenic peptides in spite of the E phase immune evasion strategies of the virus.
Evidence for trafficking of Epstein–Barr virus strains between hairy leukoplakia and peripheral blood lymphocytes
Authors: Palefsky Joel M. , Berline Jennifer , Greenspan Deborah , Greenspan John S.
Published: August 2015

Hairy leukoplakia (HL), an epithelial lesion found on the side of the tongue in immunocompromised individuals, is characterized by high-level replication of Epstein–Barr virus (EBV) and multiple EBV strains. The source of these strains and their relationship to peripheral blood lymphocyte (PBL) strains has not previously been characterized. Using matched pairs of HL scrapings and PBL from 16 HIV-positive men, variation in EBV strain identity was characterized by detection of a 30 nucleotide deletion of the EBV latent membrane protein (LMP)-1 gene, variation in the LMP-1 repeat region and typing for Epstein–Barr nuclear antigen (EBNA)-2. Multiple EBV strains were found in both the HL and PBL specimens, but 13 of 16 (81%) patients showed evidence of strain identity for at least one strain and analysis of two patients suggested that EBV strains from HL could infect the PBL. Our data are consistent with active trafficking of EBV between these two compartments.
H3N2 influenza viruses from domestic chickens in Italy: an increasing role for chickens in the ecology of influenza?
Authors: Campitelli Laura , Fabiani Concetta , Puzelli Simona , Fioretti Alessandro , Foni Emanuela , De Marco Alessandra , Krauss Scott , Webster Robert G. , Donatelli Isabella
Published: August 2015

In Italy, multiple H3N2 influenza viruses were isolated from chickens with mild respiratory disease and were shown to replicate in the respiratory tracts of experimentally infected chickens; this finding is the first to show that H3N2 influenza viruses can replicate and cause disease in chickens. H3N2 influenza viruses in pigs on nearby farms seemed a likely source of the virus; however, antigenic and molecular analyses revealed that the gene segments of the viruses in chickens were mainly of Eurasian avian origin and were distinguishable from those isolated from pigs and wild aquatic birds in Italy. Thus, several different H3 influenza viruses were circulating in Italy, but we failed to identify the source of the chicken H3N2 influenza viruses that have disappeared subsequently from Italian poultry. Until recently, the transmission of influenza viruses (other than the H5 and H7 subtypes) from their reservoir in aquatic birds to chickens was rarely detected and highly pathogenic and non-pathogenic viruses were considered to be restricted to poultry species. However, the recent reports of the transmission of H9N2 and H5N1 influenza viruses to chickens in Hong Kong and, subsequently, to humans and our findings of the transmission of H3N2 influenza viruses to domestic chickens in Italy suggest an increased role for chickens as an intermediate host in the ecology of influenza.
Distribution and accumulation of PrP in gut-associated and peripheral lymphoid tissue of scrapie-affected Suffolk sheep
Authors: Heggebø Ragna , Press Charles McL. , Gunnes Gjermund , González Lorenzo , Jeffrey Martin
Published: August 2015

The distribution of disease-associated prion protein (PrP) was investigated in eight animals (20–24 months of age) from a flock of Suffolk sheep that had experienced frequent cases of natural scrapie over a period of several years. Tissue from the central nervous system (CNS), alimentary tract, peripheral nervous system and lymphoreticular system was examined by histopathology and immunohistochemistry. The lymphoid tissues were subjected further to histoblot and immunofluorescence examination. The four clinically affected PrPARQ/ARQsheep had widespread accumulations of disease-associated PrP in the CNS, lymphoreticular system and peripheral ganglia. In the two PrPARQ/ARQsheep that did not show clinical signs of scrapie, only limited vacuolation and PrP accumulation were detected in the brain, but the results from the lymphoreticular system and peripheral nervous system were comparable with the clinically affected animals. The remaining PrPARR/ARRand PrPARR/ARQsheep did not show proteinase K-resistant PrP accumulations in the lymphoid tissues examined and immunohistochemistry did not reveal the presence of disease-associated PrP. In lymphoid tissues of the PrPARQ/ARQsheep, the dominant localization of disease-associated PrP was in lymphoid nodules and double immunofluorescence labelling for PrP and CD21 provided further support for the role of follicular dendritic cells in scrapie in sheep. A striking finding in the present study was the large accumulations of disease-associated PrP in the lymphoid nodules of the alimentary tract at the late sub-clinical and clinical stage of the infection. The study also identified disease-associated PrP in extra-nodular sites of lymphoid tissues, such as the marginal zone of the spleen, and these observations were used to argue that cells of the mononuclear phagocyte system of sheep may be involved in the uptake, transport, elimination and shedding of the scrapie agent.
Polymorphism of open reading frame 71 of equine herpesvirus-4 (EHV-4) and EHV-1
Authors: Huang Jin-an , Ficorilli Nino , Hartley Carol A. , Allen George P. , Studdert Michael J.
Published: August 2015

Open reading frame (ORF) 71 genes of both equine herpesvirus-1 (EHV-1) and EHV-4 encode a unique glycoprotein, which has been described to vary in molecular mass from 200 to 450 kDa. Using PCR and nucleotide sequence analysis, it was shown that the ORF 71 genes of EHV-1 and EHV-4 are polymorphic due to a variable number of reiterated sequences in two regions, designated regions A and B. Region A was threonine-rich and was located near the N terminus. Region B comprised a 38 amino acid repeat near the C terminus that expanded following cell culture adaptation. Western blot analysis of viruses showed that EHV-4 gp2 was modified by glycosylation and that variation in region A resulted in the marked differences in the molecular mass of EHV-4 gp2.
In vitro and in vivo expression of foreign genes by transmissible gastroenteritis coronavirus-derived minigenomes
Authors: Alonso Sara , Sola Isabel , Teifke Jens P. , Reimann Ilona , Izeta Ander , Balasch Mónica , Plana-Durán Juan , Moormann Rob J. M. , Enjuanes Luis
Published: August 2015

A helper-dependent expression system based on transmissible gastroenteritis coronavirus (TGEV) has been developed using a minigenome of 3·9 kb (M39). Expression of the reporter gene β-glucuronidase (GUS) (2–8 μg per 106cells) and the porcine respiratory and reproductive syndrome virus (PRRSV) ORF5 (1–2 μg per 106cells) has been shown using a TGEV-derived minigenome. GUS expression levels increased about eightfold with the m.o.i. and were maintained for more than eight passages in cell culture. Nevertheless, instability of the GUS and ORF5 subgenomic mRNAs was observed from passages five and four, respectively. About a quarter of the cells in culture expressing the helper virus also produced the reporter gene as determined by studying GUS mRNA production byin situhybridization or immunodetection to visualize the protein synthesized. Expression of GUS was detected in the lungs, but not in the gut, of swine immunized with the virus vector. Around a quarter of lung cells showing replication of the helper virus were also positive for the reporter gene. Interestingly, strong humoral immune responses to both GUS and PRRSV ORF5 were induced in swine with this virus vector. The large cloning capacity and the tissue specificity of the TGEV-derived minigenomes suggest that these virus vectors are very promising for vaccine development.
Antigenic and genetic diversity among swine influenza A H1N1 and H1N2 viruses in Europe
Authors: Marozin S. , Gregory V. , Cameron K. , Bennett M. , Valette M. , Aymard M. , Foni E. , Barigazzi G. , Lin Y. , Hay A.
Published: August 2015

Three subtypes of influenza A viruses, H1N1, H1N2 and H3N2, co-evolve in pigs in Europe. H1N2 viruses isolated from pigs in France and Italy since 1997 were closely related to the H1N2 viruses which emerged in the UK in 1994. In particular, the close relationship of the neuraminidases (NAs) of these viruses to the NA of a previous UK H3N2 swine virus indicated that they had not acquired the NA from H3N2 swine viruses circulating in continental Europe. Moreover, antigenic and genetic heterogeneity among the H1N2 viruses appeared to be due in part to multiple introductions of viruses from the UK. On the other hand, comparisons of internal gene sequences indicated genetic exchange between the H1N2 viruses and co-circulating H1N1 and/or H3N2 subtypes. Most genes of the earlier (1997–1998) H1N2 isolates were more closely related to those of a contemporary French H1N1 isolate, whereas the genes of later (1999–2000) isolates, including the HAs of some H1N2 viruses, were closely related to those of a distinct H1N1 antigenic variant which emerged in France in 1999. In contrast, an H3N2 virus isolated in France in 1999 was closely related antigenically and genetically to contemporary human A/Sydney/5/97-like viruses. These studies reveal interesting parallels between genetic and antigenic drift of H1N1 viruses in pig and human populations, and provide further examples of the contribution of genetic reassortment to the antigenic and genetic diversity of swine influenza viruses and the importance of the complement of internal genes in the evolution of epizootic strains.
Identification of a cell surface 30 kDa protein as a candidate receptor for Hantaan virus
Authors: Kim Tae-Yeon , Choi Yun , Cheong Hong-Seok , Choe Joonho
Published: August 2015

Cellular receptors play an important role in virus pathogenesis. As a first step in virus infection, viruses attach to specific receptors on the surface of cells; Hantaan virus infects susceptible cells by attaching to a receptor located on the cell surface. To date, the identity of the Hantaan virus host cell receptor remains unknown. To determine the protein on the cell surface to which Hantaan virus binds, a virus overlay protein-binding assay was performed with radiolabelled virus. A 30 kDa (30K) protein was identified as a putative receptor for Hantaan virus. The specificity of virus interactions with this protein was demonstrated with a competition assay using unlabelled Hantaan virus and poliovirus. Unlabelled Hantaan virus inhibited the binding of radiolabelled Hantaan virus to this 30 kDa protein, whereas poliovirus did not. A polyclonal antibody against the 30K protein blocked binding of Hantaan virus to Vero-E6 cells and, consequently, virus infection. Blocking with the anti-30K antibody reduced virus infection of cells by 70%. These data strongly suggest that the 30 kDa surface protein is a putative receptor for Hantaan virus.
Antigenic sites of foot-and-mouth disease virus (FMDV): an analysis of the specificities of anti-FMDV antibodies after vaccination of naturally susceptible host species
Authors: Aggarwal N. , Barnett P. V.
Published: August 2015

Of the known neutralizing antigenic sites of foot-and-mouth disease virus (FMDV), site 1 or A, formed in part by the G–H loop of VP1, has historically been considered immunodominant because of evidence implicating its importance in the induction of a protective immune response. However, no systematic study has been done to determine the relative importance of the various specificities of antibodies against the known neutralizing antigenic sites of FMDV in the polyclonal immune response of a natural host after vaccination. In this report, we have adopted a monoclonal antibody-based competition ELISA and used antibodies specific to sites 1, 2 and 3 to provide some insight into this issue. Following vaccination of the three main target species, cattle, pigs and sheep, with an O1serotype strain, results indicate that none of these three antigenic sites can be considered immunodominant in a polyclonal serum. Interestingly, pigs did not respond to epitopes on the carboxy terminus end of VP1 as efficiently as the ruminant species. In addition to the known sites, other as yet undefined sites might also be important in the induction of a protective immune response. Possible implications for the design of new vaccine strategies for foot-and-mouth disease are discussed.
Multimerization reactions of coxsackievirus proteins 2B, 2C and 2BC: a mammalian two-hybrid analysis
Authors: de Jong Arjan S. , Schrama Ina W. J. , Willems Peter H. G. M. , Galama Jochem M. D. , Melchers Willem J. G. , van Kuppeveld Frank J. M.
Published: August 2015

Recently, homomultimerization and heteromultimerization reactions of the poliovirus P2 region proteins were investigated using a yeast two-hybrid approach (Cuconatiet al.,Journal of Virology72, 1297–1307, 1998). In this study, we investigated multimerization reactions of the 2B, 2C and 2BC proteins of the closely related coxsackie B3 virus (CBV3) using a mammalian two-hybrid system. This system allows the characterization of protein:protein interactions within a cellular environment that more closely mimics the native protein environment. Homomultimerization reactions were observed with the 2BC protein and, albeit weakly, with the 2B protein, but not with the 2C protein. To identify the determinants involved in the 2BC and 2B homomultimerization reactions, several mutants containing deletions or point mutations in the 2B region were tested. Disruption of the hydrophobic character of either the cationic amphipathic α-helix or the second hydrophobic domain of the 2B protein disturbed both the 2BC:2BC and the 2B:2B homomultimerization reactions. Disruption of either the cationic or the amphipathic character of the α-helix or deletion of the N-terminal 30 amino acids of the 2B protein, however, had no effect on the 2BC and 2B homomultimerization reactions. Heteromultimerization reactions were observed between proteins 2BC and 2B, and also between proteins 2BC and 2C, but not between the 2B and 2C proteins. The 2BC:2B and 2BC:2C heteromultimerization reactions were also mediated by hydrophobic determinants located in the amphipathic α-helix and the second hydrophobic domain. The nature of the interactions and their implications for the virus life-cycle are discussed.