bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of the complete genome sequence of black queen-cell virus, a picorna-like virus of honey bees
Authors: Leat Neil , Ball Brenda , Govan Vandana , Davison Sean
Published: August 2015

A virus with picorna-like biophysical properties was isolated from South African honey bees. On the basis of serology, it was identified as an isolate of black queen-cell virus (BQCV). Nucleotide sequence analysis revealed an 8550 nt polyadenylated genome containing two large ORFs. The 5′-proximal ORF (ORF 1) represented 4968 nt while the 3′-proximal ORF (ORF 2) represented 2562 nt. The ORFs were separated by a 208 nt intergenic region and were flanked by a 657 nt 5′-untranslated region and a 155 nt 3′-untranslated region. Deduced amino acid sequences for ORF 1 and ORF 2 were most similar to the non-structural and structural proteins, respectively, of Drosophila C virus (DCV), Rhopalosiphum padi virus (RhPV), Himetobi P virus (HiPV) and Plautia stali intestine virus (PSIV). It is proposed that BQCV belongs to the group of picorna-like, insect-infecting RNA viruses constituted by DCV, RhPV, HiPV and PSIV.
Spread and pathogenic characteristics of a G-deficient rabies virus recombinant: an in vitro and in vivo study
Authors: Etessami Réza , Conzelmann Karl-Klaus , Fadai-Ghotbi Babak , Natelson Benjamin , Tsiang Henri , Ceccaldi Pierre-Emmanuel
Published: August 2015

Rabies virus (RV), a highly neurotropic enveloped virus, is known to spread within the CNS by means of axonal transport. Although the envelope spike glycoprotein (G) of cell-free virions is required for attachment to neuronal receptors and for virus entry, its necessity for transsynaptic spread remains controversial. In this work, a G gene-deficient recombinant RV (SAD ΔG) complemented phenotypically with RV G protein (SAD ΔG+G) has been used to demonstrate the absolute requirement for G in virus transfer from one neuron to another, bothin vitro, in neuronal cell cultures (cell line and primary cultures), andin vivo, in murine animal models. By using a model of stereotaxic inoculation into the rat striatum, infection is shown to be restricted to initially infected cells and not transferred to secondary neurons. In mouse as in rat models of infection, the limited infection did not cause any detectable symptoms, suggesting that G-deficient RV recombinants might be valuable as non-pathogenic, single-round vectors for expression of foreign genes.
Characterization of the equine infectious anaemia virus S2 protein
Authors: Yoon Soonsang , Kingsman Susan M. , Kingsman Alan J. , Wilson Stuart A. , Mitrophanous Kyriacos A.
Published: August 2015

S2 is an accessory protein of equine infectious anaemia virus (EIAV), the function of which is unknown. In order to gain insight into the function of S2, the intracellular localization of the protein, its interaction with viral proteins and its incorporation into viral particles have been investigated. Immunolocalization of S2 revealed punctate staining in the cytoplasm and the S2 protein co-precipitated with the EIAV Gag precursor. Despite overexpression of S2 through the use of a codon-optimized sequence, there was no preferential association of S2 with EIAV particles. These data suggest that S2 may function to organize the Gag protein during particle assembly in the cytoplasm but that it is unlikely to be involved in the early stages of the virus life-cycle.
Activation of cellular interferon-responsive genes after infection of human cells with herpes simplex virus type 1
Authors: Nicholl Mary Jane , Robinson Laurence H. , Preston Chris M.
Published: August 2015

Previous studies have shown that infection of human fibroblasts with human cytomegalovirus (HCMV) results in activation of cellular interferon-responsive gene expression. We demonstrate here that infection of human fibroblasts with herpes simplex virus type 1 (HSV-1) in the absence of de novo protein synthesis also induces the expression of interferon-responsive genes. Five genes tested (encoding ISG54, IFI56, ISG15, 9-27 and MxA) were activated by infection with HSV-1, although the degree of response varied between the individual genes. HSV-1 was a less efficient inducer than HCMV. The effect was a consequence of binding of the virus particle to the cell surface or of the presence of virion components within the infected cell. Induction was mediated by a pathway other than the mechanism through which interferon-α mediates its effects on cellular gene expression.
Marek’s disease virus (MDV) homologues of herpes simplex virus type 1 UL49 (VP22) and UL48 (VP16) genes: high-level expression and characterization of MDV-1 VP22 and VP16
Authors: Dorange Fabien , El Mehdaoui Slimane , Pichon Chantal , Coursaget Pierre , Vautherot Jean-François
Published: August 2015

Genes UL49 and UL48 of Marek’s disease virus 1 (MDV-1) strain RB1B, encoding the respective homologues of herpes simplex virus type 1 (HSV-1) genes VP22 and VP16, were cloned into a baculovirus vector. Seven anti-VP22 MAbs and one anti-VP16 MAb were generated and used to identify the tegument proteins in cells infected lytically with MDV-1. The two genes are known to be transcribed as a single bicistronic transcript, and the detection of only one of the two proteins (VP22) in MSB-1 lymphoma and in chicken embryo skin cells infected with MDV-1 prompted the study of the transcription/translation of the UL49–48 sequence in anin vivoandin vitroexpression system. VP16 was expressedin vitroat detectable levels, whereas it could only be detected at a lower level in a more controlled environment. It was demonstrated that VP22 is phosphorylated in insect cells and possesses the remarkable property of being imported into all cells in a monolayer. VP22 localized rapidly and efficiently to nuclei, like its HSV-1 counterpart. The DNA-binding property of VP22 is also reported and a part of the region responsible for this activity was identified between aa 16 and 37 in the N-terminal region of the protein.
Epstein–Barr virus latent membrane protein 2A has no growth-altering effects when expressed in differentiating epithelia
Authors: Longan Luz , Longnecker Richard
Published: August 2015

Previous studies using transgenic mice with B-cell expression of LMP2A demonstrated that LMP2A drives B-cell development and survival signal in the absence of normal B-cell receptor (BCR) signal transduction. To determine if LMP2A may have similar effects in epithelial differentiation, six transgenic murine lines were constructed and analysed with LMP2A expression directed to the epidermis by a keratin 14 (K14) promoter cassette. LMP2A protein expression was verified by immunofluorescence and immunoprecipitation of skin samples using LMP2A-specific antibodies. To evaluate the effects of LMP2A expression on epidermal differentiation, immunofluorescence and histochemistry were performed on tongue and tail samples of transgenic mice and their wild-type littermate controls using differentially expressed keratins. The analysis indicated that LMP2A does not alter the normal epithelial differentiation program in the epithelia of K14–LMP2A transgenic mice.
Open reading frame 2 of porcine circovirus type 2 encodes a major capsid protein
Authors: Nawagitgul Porntippa , Morozov Igor , Bolin Steven R. , Harms Perry A. , Sorden Steven D. , Paul Prem S.
Published: August 2015

Porcine circovirus 2 (PCV2), a single-stranded DNA virus associated with post-weaning multisystemic wasting syndrome of swine, has two potential open reading frames, ORF1 and ORF2, greater than 600 nucleotides in length. ORF1 is predicted to encode a replication-associated protein (Rep) essential for replication of viral DNA, while ORF2 contains a conserved basic amino acid sequence at the N terminus resembling that of the major structural protein of chicken anaemia virus. Thus far, the structural protein(s) of PCV2 have not been identified. In this study, a viral structural protein of 30 kDa was identified in purified PCV2 particles. ORF2 of PCV2 was cloned into a baculovirus expression vector and the gene product was expressed in insect cells. The expressed ORF2 gene product had a molecular mass of 30 kDa, similar to that detected in purified virus particles. The recombinant ORF2 protein self-assembled to form capsid-like particles when viewed by electron microscopy. Antibodies against the ORF2 protein were detected in samples of sera obtained from pigs as early as 3 weeks after experimental infection with PCV2. These results show that the major structural protein of PCV2 is encoded by ORF2 and has a molecular mass of 30 kDa.
Native electrophoresis and Western blot analysis (NEWeB): a method for characterization of different forms of potyvirus particles and similar nucleoprotein complexes in extracts of infected plant tissues
Authors: Manoussopoulos I. N. , Maiss E. , Tsagris M.
Published: August 2015

A combination of native electrophoresis and immunodetection (Western blot) was used for the characterization of nucleoprotein particles of the potyvirus Plum pox virus (PPV). Virus particles were electrophoresed directly from plant extracts in agarose or mixed acrylamide–agarose gels under native conditions, blotted on nitrocellulose membranes, and characterized with the aid of a coat protein-specific antibody. Using this combined methodology, called NEWeB (native electrophoresis and Western blotting), we could show that a population of particles that differ in their electrophoretic mobility can be detected in extracts of Nicotiana benthamiana, that two different strains of PPV can be distinguished in double infections of the same plant and that virus particles from leaves contain detectable levels of helper component proteinase molecules. The potential of the NEWeB method for the study of structure and function of virus particles and similar nucleoprotein complexes in single and mixed infections is discussed.
Distribution of prion protein in the ileal Peyer’s patch of scrapie-free lambs and lambs naturally and experimentally exposed to the scrapie agent
Authors: Heggebø Ragna , Press Charles McL. , Gunnes Gjermund , Inge Lie Kai , Tranulis Michael A. , Ulvund Martha , Groschup Martin H. , Landsverk Thor
Published: August 2015

A sensitive immunohistochemical procedure was used to investigate the presence of prion protein (PrP) in the ileal Peyer’s patch of PrP-genotyped lambs, including scrapie-free lambs and lambs naturally and experimentally exposed to the scrapie agent. The tyramide signal amplification system was used to enhance the sensitivity of conventional immunohistochemical procedures to show that PrP was widely distributed in the enteric nervous plexus supplying the gut wall. In scrapie-free lambs, PrP was also detected in scattered cells in the lamina propria and in the dome and interfollicular areas of the Peyer’s patch. In the follicles, staining for PrP was mainly confined to the capsule and cells associated with vascular structures in the light central zone. In lambs naturally exposed to the scrapie agent, staining was prominent in the dome and neck region of the follicles and was also found to be associated with the follicle-associated epithelium. Similar observations were made in lambs that had received a single oral dose of scrapie-infected brain material from sheep with a homologous and heterologous PrP genotype 1 and 5 weeks previously. These studies show that the ileal Peyer’s patch in young sheep may be an important site of uptake of the scrapie agent and that the biology of this major gut-associated lymphoid tissue may influence the susceptibility to oral infection in sheep. Furthermore, these studies suggest that homology or heterology between PrP genotypes or the presence of PrP genotypes seldom associated with disease does not impede uptake of PrP.
Nucleotide sequence, genome organization and phylogenetic analysis of pineapple mealybug wilt-associated virus-2
Authors: Melzer M. J. , Karasev A. V. , Sether D. M. , Hu J. S.
Published: August 2015

The genome of pineapple mealybug wilt-associated closterovirus-2 (PMWaV-2) was cloned from double-stranded RNA isolated from diseased pineapple and its sequence determined. The 3′-terminal 14861 nt of the single-stranded RNA genome contains ten open reading frames (ORFs) which, from 5′ to 3′, potentially encode a >204 kDa polyprotein containing papain-like protease, methyltransferase and helicase domains (ORF1a), a 65 kDa RNA-dependent RNA polymerase (ORF1b), a 5 kDa hydrophobic protein (ORF2), a 59 kDa heat shock protein 70 homologue (ORF3), a 46 kDa protein (ORF4), a 34 kDa coat protein (ORF5), a 56 kDa diverged coat protein (ORF6), a 20 kDa protein (ORF7), a 22 kDa protein (ORF8) and a 6 kDa protein (ORF9). A 132 nt untranslated region was present at the 3′ terminus of the genome. This genome organization is typical of the monopartite closteroviruses, including the putative +1 ribosomal frameshift allowing expression of ORF1b. Phylogenetic analysis revealed that within the family Closteroviridae the mealybug-transmitted PMWaV-2 is more closely related to other mealybug-transmitted members than to those which are transmitted by aphids or whiteflies. Within this group, PMWaV-2 shares the greatest sequence identity with grapevine leafroll-associated virus-3, another mealybug-transmitted closterovirus.
Genetic variability among group A and B respiratory syncytial viruses in Mozambique: identification of a new cluster of group B isolates
Authors: Roca Anna , Loscertales Mari-Paz , Quintó Llorenç , Pérez-Breña Pilar , Vaz Neide , Alonso Pedro-Luis , Saiz Juan-Carlos
Published: August 2015

Respiratory syncytial virus (RSV) is the major cause of acute lower respiratory tract infection in children and vulnerable adults, but little is known regarding RSV infection in Africa. In this report, a recent RSV outbreak in Mozambique was studied and results showed that 275 of 3192 (8·6%) nasopharyngeal aspirates tested were RSV-positive by ELISA. RSV presents two antigenic groups (A and B) with a high genetic and antigenic variability between and within them. Analysis by a new RFLP assay of RT–PCR amplified N protein gene products showed a higher prevalence of group B RSV than that of group A (85% versus 15%). However, genetic variability of the G protein gene was higher among group A RSV strains. The frequency and pattern of glycosylation sites were also quite different between both groups. In addition, two different phylogenetic clusters of Mozambican viruses were found within each group, but only sequences from cluster B-I were relatively distinct from previously described isolates. The implications of such differences in the antigenic and immunogenic characteristics of each group are discussed.
Variation of African horsesickness virus nonstructural protein NS3 in southern Africa
Authors: van Niekerk M. , van Staden V. , van Dijk A. A. , Huismans H.
Published: August 2015

NS3 protein sequences of recent African horsesickness virus (AHSV) field isolates, reference strains and current vaccine strains in southern Africa were determined and compared. The variation of AHSV NS3 was found to be as much as 36·3% across serotypes and 27·6% within serotypes. NS3 proteins of vaccine and field isolates of a specific serotype were found to differ between 2·3% and 9·7%. NS3 of field isolates within a serotype differed up to 11·1%. Our data indicate that AHSV NS3 is the second most variable AHSV protein, the most variable being the major outer capsid protein, VP2. The inferred phylogeny of AHSV NS3 corresponded well with the described NS3 phylogenetic clusters. The only exception was AHSV-8 NS3, which clustered into different groups than previously described. No obvious sequence markers could be correlated with virulence. Our results suggest that NS3 sequence variation data could be used to distinguish between field isolates and live attenuated vaccine strains of the same serotype.
Construction and characterization of recombinant porcine adenovirus serotype 5 expressing the transmissible gastroenteritis virus spike gene
Authors: Tuboly Tamás , Nagy Éva
Published: August 2015

Five recombinant porcine adenoviruses of serotype 5 (PAdV-5) carrying the full-length or the 5′ 2·2 kb half of the transmissible gastroenteritis virus (TGEV) spike (S) gene were generated by homologous recombination in E. coli strain BJ5183 cells and subsequent transfection of swine testicle cells. The foreign genes were inserted into the E3 region of PAdV-5. One recombinant virus had no deletion in the E3 region, whereas a 1·2 kb fragment was removed from the E3 region in the remainder of the recombinant viruses. One stable construct with a 4·4 kb insertion had a genome size of 109·6% of the wild-type genome, the largest reported for any recombinant adenovirus. Only those viruses that carried the S gene in the left to right orientation expressed the S gene. Three recombinant viruses were tested by oral immunization of pigs and both antibody response and virus shedding were monitored. None of the pigs showed clinical signs and the virus was recovered from rectal swabs until 6–7 days post-infection. Viruses expressing the S gene induced TGEV- and PAdV-5-specific virus-neutralizing antibodies. Moreover, TGEV-specific secretory IgA was detected in the small intestine and in the lungs of the immunized animals.
The VP1-unique region of parvovirus B19: amino acid variability and antigenic stability
Authors: Dorsch Simone , Kaufmann Bärbel , Schaible Uwe , Prohaska Elke , Wolf Hans , Modrow Susanne
Published: August 2015

The unique region of structural protein VP1 of parvovirus B19 (erythrovirus B19) is important for eliciting neutralizing antibodies that are responsible for eliminating the virus from the peripheral blood and for inducing lifelong immunity. Neutralizing human MAbs bind a conformationally defined epitope spanning VP1 residues 30–42. The DNA sequence encoding the VP1-unique region was determined in parvovirus B19 isolated from peripheral blood and amniotic fluid of nine acutely infected pregnant women, five arthritis patients and two chronically infected children. The amino acid sequences of the VP1-unique region exhibited higher variability in comparison with other B19-specific proteins. To analyse the influence of amino acid variations on antibody binding and protein conformation, two variants of the VP1-unique region were selected and expressed in E. coli as intein-fusion proteins. The selected variants displayed a number of amino acid exchanges in the VP1-unique region and had mutations in the determined epitope and adjacent regions. After purification via affinity chromatography, the dissociation constants K
D of VP1-specific human MAbs interacting with the variant antigens and a viral prototype of the VP1-unique region were determined with a quartz crystal microbalance biosensor. A value of 5·4×10−8 M was determined for the prototype isolate pJB; the affinity constants for the variant VP1-unique regions were similar. Comparable values were obtained for interaction of antibodies with non-infectious VP1/VP2 capsids produced by recombinant baculovirus and with B19 virions from amniotic fluid. It is concluded that the conformation of the epitope is unaffected by mutations or the environment of the VP1-unique region in virus capsids.
Analysis of the capsid processing strategy of Thosea asigna virus using baculovirus expression of virus-like particles
Authors: Pringle Fiona M. , Kalmakoff James , Ward Vernon K.
Published: August 2015

Thosea asignavirus (TaV), a putative member of the genusBetatetravirusof the familyTetraviridae, is predicted to have a novel capsid expression strategy compared with other characterized tetraviruses. The capsid precursor protein is cleaved twice to generate three proteins. Two of the proteins, L (58·3 kDa) and S (6·8 kDa), are incorporated into the TaV virion. The third, non-structural protein, produced from the N terminus of the precursor protein, is up to 17 kDa in size and is of unknown function. The TaV capsid precursor protein sequence without the 17 kDa N-terminal region was modelled against the solved structure fromNudaureliaω virus (NωV) using SwissModel. The TaV model was very similar to the solved structure determined for subunit A of NωV and had features that are conserved between tetraviruses and nodaviruses, including the positioning of the cleavage site between the L and S capsid proteins. The production of virus-like particles (VLPs) using the baculovirus expression system was used to analyse the capsid processing strategy employed by TaV. VLPs were formed in both the presence and absence of the 17 kDa N-terminal region of the capsid precursor. VLPs were not formed when the L and S regions were expressed from separate promoters, indicating that cleavage between the L and S capsid proteins was an essential part of TaV capsid assembly. Expression of the TaV 17 kDa protein in bacteria did not produce intracellular tubules similar to those formed by bacterial expression of the p17 protein fromHelicoverpa armigerastunt virus.
Partial dissociation of PrPSc deposition and vacuolation in the brains of scrapie and BSE experimentally affected goats
Authors: Foster James , Goldmann Wilfred , Parnham David , Chong Angie , Hunter Nora
Published: August 2015

The diagnosis of transmissible spongiform encephalopathies (TSEs) depends on the detection of vacuolation in brain sections taken from affected individuals and/or the identification of the disease-associated isoform of the PrP (prion) protein (PrPSc). During the course of an investigation, goats clinically affected following experimental infection with three different sources of TSE (SSBP/1, CH1641 and BSE) developed widespread vacuolar degeneration in the brain. With BSE, PrPSc was clearly recognized in affected goat brain by immunocytochemistry (icc) and Western blotting, but in contrast the experimental scrapie sources SSBP/1 and CH1641 showed almost no or very little PrPSc by icc. Western blot analysis of PrPSc from BSE-affected and SSBP/1-affected goat brain showed that the protein was present in brain affected by both TSE sources, but could not be used to determine how much protein was present. It became clear that PrPSc and vacuolation could be partially dissociated following challenge with two of the three TSE sources. Subtle differences in glycosylation patterns between BSE- and SSBP/1-associated PrP protein isoforms could also be recognized, although these experimentally generated results should not be regarded as a BSE/scrapie differential test. However, our study warns that the reliance on PrPSc determination by icc alone as a means by which to diagnose TSE infection may generate false negative results.
The distinct disease phenotypes of the common and yellow vein strains of Tomato golden mosaic virus are determined by nucleotide differences in the 3′-terminal region of the gene encoding the movement protein
Authors: Saunders Keith , Wege Christina , Veluthambi Karuppannan , Jeske Holger , Stanley John
Published: August 2015

In Nicotiana benthamiana, the common strain of the bipartite geminivirus Tomato golden mosaic virus (csTGMV) induces extensive chlorosis whereas the yellow vein strain (yvTGMV) produces veinal chlorosis on systemically infected leaves. In Datura stramonium, csTGMV produces leaf distortion and a severe chlorotic mosaic whereas yvTGMV produces only small chlorotic lesions on systemically infected leaves. Genetic recombination and site-directed mutagenesis studies using infectious clones of csTGMV and yvTGMV have identified a role in symptom production for the gene encoding the movement protein (MP). The MP amino acid at position 272, either valine (csTGMV) or isoleucine (yvTGMV), influenced symptoms in both hosts by inducing an intermediate phenotype when exchanged between the two strains. Exchange of an additional strain-specific MP amino acid at position 288, either glutamine (csTGMV) or lysine (yvTGMV), resulted in the change of symptom phenotype to that of the other strain. In situ hybridization analysis in N. benthamiana demonstrated that there was no qualitative difference in the tissue distribution of the two strains although csTGMV accumulated in higher amounts, suggesting that the efficiency of virus movement rather than distinct differences in tissue specificity of the strains is responsible for the symptom phenotypes.
Complete nucleotide sequence and host range of South African cassava mosaic virus: further evidence for recombination amongst begomoviruses
Authors: Berrie L. C. , Rybicki E. P. , Rey M. E. C.
Published: August 2015

Complete nucleotide sequences of the DNA-A (2800 nt) and DNA-B (2760 nt) components of a novel cassava-infecting begomovirus, South African cassava mosaic virus (SACMV), were determined and compared with various New World and Old World begomoviruses. SACMV is most closely related to East African cassava mosaic virus (EACMV) in both its DNA-A (85% with EACMV-MH and -MK) and -B (90% with EACMV-UG2-Mld and EACMV-UG3-Svr) components; however, percentage sequence similarities of less than 90% in the DNA-A component allowed SACMV to be considered a distinct virus. One significant recombination event spanning the entire AC4 open reading frame was identified; however, there was no evidence of recombination in the DNA-B component. Infectivity of the cloned SACMV genome was demonstrated by successful agroinoculation of cassava and three other plant species (Phaseolus vulgaris, Malva parviflora and Nicotiana benthamiana). This is the first description of successful infection of cassava with a geminivirus using Agrobacterium tumefaciens.
Molecular strategy for ‘serotyping’ of human enteroviruses
Authors: Caro Valérie , Guillot Sophie , Delpeyroux Francis , Crainic Radu
Published: August 2015

To explore further the phylogenetic relationships between human enteroviruses and to develop new diagnostic approaches, we designed a pair of generic primers in order to study a 1452 bp genomic fragment (relative to the poliovirus Mahoney genome), including the 3′ end of the VP1-coding region, the 2A- and 2B-coding regions, and the 5′ moiety of the 2C-coding region. Fifty-nine of the 64 prototype strains and 45 field isolates of various origins, involving 21 serotypes and 6 strains untypeable by standard immunological techniques, were successfully amplified with these primers. By determining the nucleotide sequence of the genomic fragment encoding the C-terminal third of the VP1 capsid protein we developed a molecular typing method based on RT–PCR and sequencing. If field isolate sequences were compared to human enterovirus VP1 sequences available in databases, nucleotide identity score was, in each case, highest with the homotypic prototype (74.8 to 89.4%). Phylogenetic trees were generated from alignments of partial VP1 sequences with several phylogeny algorithms. In all cases, the new classification of enteroviruses into five identified species was confirmed and strains of the same serotype were always monophyletic. Analysis of the results confirmed that the 3′ third of the VP1-coding sequence contains serotype-specific information and can be used as the basis of an effective and rapid molecular typing method. Furthermore, the amplification of such a long genomic fragment, including non-structural regions, is straightforward and could be used to investigate genome variability and to identify recombination breakpoints or specific attributes of pathogenicity.
Distribution of the prion protein in sheep terminally affected with BSE following experimental oral transmission
Authors: Foster J. D. , Parnham D. W. , Hunter N. , Bruce M.
Published: August 2015

This study has examined the distribution of PrPSc in sheep by immunocytochemistry of tissues recovered from terminally affected animals following their experimental infection by the oral route with BSE. Despite a wide range of incubation period lengths, affected sheep showed a similar distribution of high levels of PrPSc throughout the central nervous system. PrPSc was also found in the lymphoid system, including parts of the digestive tract, and some components of the peripheral nervous system. These abundant PrPSc deposits in sheep in regions outside the central nervous system are in direct contrast with cattle infected with BSE, which show barely detectable levels of PrPSc in peripheral tissues. A number of genetically susceptible, challenged animals appear to have survived.
PrPCWD in the myenteric plexus, vagosympathetic trunk and endocrine glands of deer with chronic wasting disease
Authors: Sigurdson Christina J. , Spraker Terry R. , Miller Michael W. , Oesch Bruno , Hoover Edward A.
Published: August 2015

Accumulated evidence in experimental and natural prion disease systems supports a neural route of infectious prion spread from peripheral sites of entry to the central nervous system. However, little is known about prion trafficking routes in cervids with a naturally occurring prion disease known as chronic wasting disease (CWD). In the brain, the pathogenic isoform of the prion protein (PrPCWD) accumulates initially in the dorsal motor nucleus of the vagus nerve. To assess whether alimentary-associated neural pathways may play a role in prion trafficking, neural and endocrine tissues from mule deer naturally infected with CWD (n=6) were examined by immunohistochemistry. PrPCWD was detected in the myenteric plexus, vagosympathetic trunk, nodose ganglion, pituitary, adrenal medulla and pancreatic islets. No to scant PrPCWD staining was detected in other nerves or ganglia (brachial plexus, sciatic nerve, gasserian ganglion, coeliac ganglion, cranial cervical ganglion, spinal nerve roots) of CWD-positive deer and no PrPCWD was detected in nerves or endocrine tissues from 11 control deer. These findings suggest that: (i) transit of PrPCWD in nerves, either centrifugally or centripetally, is one route of prion trafficking and organ invasion and (ii) endocrine organs may also be targets for cervid pathogenic prion accumulation.
E5 transforming proteins of papillomaviruses do not disturb the activity of the vacuolar H+-ATPase
Authors: Ashby Andrew D. M. , Meagher Liam , Campo M. Saveria , Finbow Malcolm E.
Published: August 2015

Papillomaviruses contain a gene, E5, that encodes a short hydrophobic polypeptide that has transforming activity. E5 proteins bind to the 16 kDa subunit c (proteolipid) of the eukaryotic vacuolar H+-ATPase (V-ATPase) and this binding is thought to disturb the V-ATPase and to be part of transformation. This link has been examined in the yeastSaccharomyces cerevisiae. The E5 proteins from human papillomavirus (HPV) type 16, bovine papillomavirus (BPV) type 1, BPV-4 E5 and various mutants of E5 and the p12′ polypeptide from human T-lymphotropic virus (HTLV) type I all bound to theS. cerevisiaesubunit c (Vma3p) and could be found in vacuolar membranes. However, none affected the activity of the V-ATPase. In contrast, a dominant-negative mutant of Vma3p (E137G) inactivated the enzyme and gave the characteristic VMA phenotype. A hybrid V-ATPase containing a subunit c from Norway lobster also showed no disruption. Sedimentation showed that HPV-16 E5 was not part of the active V-ATPase. It is concluded that the binding of E5 and E5-related proteins to subunit c does not affect V-ATPase activity or function and it is proposed that the binding may be due to a chaperone function of subunit c.
Effect of the pseudorabies virus US3 protein on nuclear membrane localization of the UL34 protein and virus egress from the nucleus
Authors: Klupp Barbara G. , Granzow Harald , Mettenleiter Thomas C.
Published: August 2015

The alphaherpesvirus UL34 protein is necessary for the primary envelopment of intranuclear capsids at the inner leaflet of the nuclear membrane. In herpes simplex virus type 1, the UL34 protein is exclusively phosphorylated by the protein kinase encoded by the non-essential US3 gene. To investigate the effect of the pseudorabies virus (PrV) US3 product on the intracellular localization of the UL34 protein and on virus morphogenesis, PrV US3 deletion mutants were isolated and characterized. Immunofluorescence analyses demonstrated that in the absence of the US3 protein, the localization of the UL34 polypeptide to the nuclear membrane was not as pronounced as that seen with US3, although immunoelectron microscopy indicated the presence of the UL34 protein in both leaflets of the nuclear membrane. Ultrastructurally, an accumulation of enveloped virions in the perinuclear space in large invaginations of the inner nuclear membrane was observed, which were shown by immunoelectron microscopy to contain the UL34 protein, but not glycoproteins gB or gC. Thus, the US3 protein appears to be involved in the de-envelopment of perinuclear virions by fusion with the outer leaflet of the nuclear membrane. Surprisingly, no difference in the phosphorylation of the PrV UL34 protein was observed in the presence or absence of the US3 kinase. Therefore, the observed effects of the PrV US3 protein on the intracellular localization of the UL34 protein and on virus morphogenesis are probably not due to the phosphorylation of the UL34 protein by the US3 kinase.
Recombination in human herpesvirus-8 strains from Uganda and evolution of the K15 gene
Authors: Kakoola Dorothy N. , Sheldon Julie , Byabazaire Naomi , Bowden Rory J. , Katongole-Mbidde Edward , Schulz Thomas F. , Davison Andrew J.
Published: August 2015

Human herpesvirus-8 (HHV-8) is believed to be the aetiological agent of Kaposi’s sarcoma (KS). KS accounts for half the reported cancer cases in Uganda, and occurs in endemic and epidemic [human immunodeficiency virus (HIV)-associated] forms. We confirmed a high prevalence (74%) of HHV-8 antibodies in 114 HIV-negative Ugandan blood donors, and characterized the genomes of HHV-8 strains present in 30 adult Ugandan KS patients. Phylogenetic analysis of the uniquely variable K1 gene indicated that the majority of KS patients were infected by the B subtype of HHV-8, several by the A5 subtype, and one by a variant of the C subtype. Sequence analysis of nine strains at several other genome loci spaced out across the genome indicated that five are recombinants between subtypes when considered independently of previously published definitions of parental (unrecombined) genotypes. When previously published parental genotypes were taken into account, seven of the nine strains appeared to be recombinants. Analysis of the K15 gene, which exists in HHV-8 in two highly diverged alleles, indicated that the P allele predominates, with only a single strain bearing the M allele. Divergence between the M allele in the latter strain and that in the previously sequenced BC1 strain is at least as great as that between representatives of the P allele. This indicates that introduction of the M allele into extant HHV-8 subtypes did not occur by a single, relatively recent recombination event as was concluded from a previous study in which very limited variation in the M allele was reported.
Evaluation of a prototype sub-unit vaccine against equine arteritis virus comprising the entire ectodomain of the virus large envelope glycoprotein (GL): induction of virus-neutralizing antibody and assessment of protection in ponies
Authors: Castillo-Olivares J. , de Vries A. A. F. , Raamsman M. J. B. , Rottier P. J. M. , Lakhani K. , Westcott D. , Tearle J. P. , Wood J. L. N. , Mumford J. A. , Hannant D. , Davis-Poynter N. J.
Published: August 2015

An Escherichia coli-expressed recombinant protein (6hisGLecto) comprising the entire ectodomain (aa 18–122) of equine arteritis virus (EAV) glycoprotein GL, the immunodominant viral antigen, induced higher neutralizing antibody titres than other GL-derived polypeptides when compared in an immunization study in ponies. The potential of the recombinant GL ectodomain to act as a sub-unit vaccine against EAV was evaluated further in three groups of four ponies vaccinated with doses of 35, 70 or 140 μg of protein. All vaccinated animals developed a virus-neutralizing antibody (VNAb) response with peak titres 1–2 weeks after the administration of a booster on week 5 (VNAb titres of 1·8–3·1), 13 (VNAb titres of 1·4–2·9) or 53 (VNAb titres of 1·2–2·3). Vaccinated and unvaccinated control ponies were infected with EAV at different times post-vaccination to obtain information about the degree of protection relative to the levels of pre-challenge VNAb. Vaccination conferred varying levels of protection, as indicated by reduced or absent pyrexia, viraemia and virus excretion from the nasopharynx. The degree of protection correlated well with the levels of pre-challenge VNAb and, in particular, with levels of virus excretion. These results provide the first evidence that a sub-unit vaccine protects horses against EAV. The use of the sub-unit vaccine in combination with a differential diagnostic test based on other EAV antigens would enable serological discrimination between naturally infected and vaccinated equines.
Different evolutionary patterns are found within human immunodeficiency virus type 1-infected patients
Authors: Casado Concepción , García Soledad , Rodríguez Carmen , del Romero Jorge , Bello Gonzalo , López-Galíndez Cecilio
Published: August 2015

In order to study the evolution in vivo of human immunodeficiency virus type 1 (HIV-1) in patients with normal clinical evolution, six individuals were selected from a group of 46 patients followed for 1 to 4 years. Patients were selected not by clinical progression characteristics but on the basis of virus genetic variability, as analysed by heteroduplex mobility assay and RNase A mismatch cleavage method. Two patients displayed a homogeneous virus population, two showed very heterogeneous quasispecies and two presented two distinct variants within the virus population. Virus quasispecies were studied by nucleotide sequencing of the C2-fusion domain of the env gene. Virus evolution was approached by analysing the distribution of genetic distances, calculation of divergence and heterogeneity as well as the K
a/K
s ratio and by the construction of the phylogenetic trees. Three patients displayed the same tree topology, characterized by the presence of independent clades supported by high bootstrap values, whereas this pattern was not present in the other three patients. In the three patients displaying independent clades, a recombination analysis was carried out between distinct subpopulations and recombinant variants were identified. In one patient of this group, different selective pressures were detected in distinct virus clades, measured by their corresponding K
a/K
s ratios, revealing that different evolutionary forces are occurring at the same time within the same patient. These results show that multiple evolutionary patterns can be found in typical HIV-1-infected patients.
Measurement of membrane fusion activity from viral membrane fusion proteins based on a fusion-dependent promoter induction system in insect cells
Authors: Slack J. M. , Blissard G. W.
Published: August 2015

A number of viral membrane fusion proteins can be expressed alone on the surface of host cells, and then triggered to induce cell-to-cell fusion or syncytium formation. Although rapid and easily observed, syncytium formation is not easily quantified and differences in fusion activity are not easily distinguished or measured. To address this problem, we developed a rapid and quantitative cell-to-cell fusion system that is useful for comparative analysis and may be suitable for high throughput screening. In this system, expression of a reporter protein, enhanced green fluorescent protein (EGFP), is dependent on cell-to-cell fusion.Spodoptera frugiperda(Sf9) insect cells expressing a chimeric Lac repressor-IE1 protein were fused toSf9 cells containing anEGFPreporter construct under the control of a responsivelacoperator-containing promoter. Membrane fusion efficiency was measured from the resulting EGFP fluorescence activity.Sf9 cells expressing theOrgyia pseudotsugatamulticapsid nucleopolyhedrovirus (OpMNPV) GP64 envelope fusion protein were used as a model to test this fusion assay. Subtle changes in fusion activities of GP64 proteins containing single amino acid substitutions in a putative membrane fusion domain were distinguished, and decreases in EGFP fluorescence corresponded to decreases in the hydrophobicity in the small putative membrane fusion domain.
The complete sequence of the Cydia pomonella granulovirus genome
Authors: Luque Teresa , Finch Ruth , Crook Norman , O’Reilly David R. , Winstanley Doreen
Published: August 2015

The nucleotide sequence of the DNA genome of Cydia pomonella granulovirus (CpGV) was determined and analysed. The genome is composed of 123500 bp and has a G+C content of 45·2%. It contains 143 ORFs of 150 nucleotides or more that show minimal overlap. One-hundred-and-eighteen (82·5%) of these putative genes are homologous to genes previously identified in other baculoviruses. Among them, 73 are homologous to genes of Autographa californica nucleopolyhedrovirus (AcMNPV), whereas 108 and 98 are homologous to genes of Xestia c-nigrum GV (XcGV) and Plutella xylostella GV (PxGV), respectively. These homologues show on average 37·4% overall amino acid sequence identity to those from AcMNPV and 45% to those from XcGV and PxGV. The CpGV gene content was compared to that of other baculoviruses. Several genes reported to have major roles in baculovirus biology were not found in the CpGV genome, such as gp64, the major budded virus glycoprotein gene in some nucleopolyhedroviruses, and lef-7, involved in DNA replication. However, the CpGV genome encodes the large and small subunits of ribonucleotide reductase, three inhibitor of apoptosis (iap) homologues and two protein tyrosine phosphatases. The CpGV, PxGV and XcGV genomes present a noticeably high level of conservation of gene order and orientation. A striking feature of the CpGV genome is the absence of typical homologous repeat sequences. However, it contains one major repeat region and 13 copies of a single 73–77 bp imperfect palindrome.
Cloning and sequence analysis of an infectious clone of Citrus yellow mosaic virus that can infect sweet orange via Agrobacterium-mediated inoculation
Authors: Huang Qi , Hartung John S.
Published: August 2015

Citrus yellow mosaic virus(CYMV), a member of the familyCaulimoviridae, genusBadnavirus, causes citrus mosaic disease, a disease that occurs commonly in India. The CYMV genome has been cloned and its complete nucleotide sequence determined. Its DNA genome is 7559 bp in length and contains six putative open reading frames (ORFs), all on the plus-strand of the genome and each capable of encoding proteins with a molecular mass of greater than 10 kDa. ORF 3, the largest ORF, encodes a putative polyprotein for functions involved in virus movement, assembly and replication. The other ORFs encode proteins whose exact functions are not completely understood. The genome also contains a plant tRNAmet-binding site, which may serve as a primer for minus-strand DNA synthesis, in its intergenic region. Phylogenetic analysis of the badnaviruses revealed that CYMV is most closely related toCacao swollen shoot virus. It was demonstrated that a construct containing 1·4 copies of the cloned CYMV genome could infect sweet orange viaAgrobacterium-mediated inoculation.
Interaction of picornavirus 2C polypeptide with the viral negative-strand RNA
Authors: Banerjee Rajeev , Dasgupta Asim
Published: August 2015

The picornavirus membrane-associated polypeptide 2C is believed to be required for viral RNA synthesis. Hepatitis A virus (HAV)- and human rhinovirus (HRV)-encoded recombinant 2C proteins have been expressed, purified and examined for their ability to interact with the terminal sequences of viral positive- and negative-strand RNAs. The results demonstrate that both the HAV- and the HRV-encoded 2C polypeptide specifically interact with the 3′-terminal sequences of the negative-strand RNA, but not with the complementary sequences at the 5′ terminus of the positive-strand RNA. This interaction was detected by both mobility gel shift and UV cross-linking assays. Furthermore, complex formation exhibited dose-dependency and competition assays confirmed specificity. These results are consistent with our previous observation using the poliovirus 2C protein. The implication of the picornavirus 2C protein binding to the 3′-terminal sequence of the negative-strand untranslated region in viral RNA synthesis is discussed.
Equine rhinitis B virus: a new serotype
Authors: Huang Jin-an , Ficorilli Nino , Hartley Carol A. , Wilcox Rebbecca S. , Weiss Marianne , Studdert Michael J.
Published: August 2015

Equine rhinovirus serotype 3 isolate P313/75 was assigned, with an unclassified genus status, to the familyPicornaviridae. The sequence from the 5′ poly(C) tract to the 3′ poly(A) tract of P313/75 was determined. The sequence is 8821 bases in length and contains a potential open reading frame for a polyprotein of 2583 amino acids. Sequence comparison and phylogenic analysis suggest that P313/75 is most closely related to the prototype equine rhinitis B virus (ERBV) strain P1436/71, formerly named equine rhinovirus type 2. A high degree of sequence similarity was found in the P2 and P3 regions of the two genomes. However, the deduced amino acid sequences of the P1 region of P313/75 and ERBV strain P1436/71 contained significant differences, which presumably account for the serological segregation of the two viruses. It is suggested that P313/75 can be classified as a new serotype of the genusErbovirus, tentatively named ERBV2. Seroepidemiological data indicate that ERBV2 infection of horses may be common (24%) in Australia.
Increased replication of respiratory syncytial virus (RSV) in pulmonary infiltrates is associated with enhanced histopathological disease in bonnet monkeys (Macaca radiata) pre-immunized with a formalin-inactivated RSV vaccine
Authors: Ponnuraj Esther M. , Hayward Anthony R. , Raj Anthony , Wilson Harry , Simoes Eric A. F.
Published: August 2015

The pathology of respiratory syncytial virus (RSV) disease in bonnet monkeys parallels findings with human RSV disease. RSV-infected animals pre-immunized with a formalin-inactivated (FI) RSV vaccine develop inflammation in peribronchiolar, perivascular, interstitial and intra-alveolar sites with lung inflammation scores significantly higher than animals with a primary RSV infection and those pre-immunized with an FI-Vero cell control vaccine (P=0·05). Animals previously infected and re-exposed to RSV had significantly lower alveolar, interstitial and total lung inflammation scores than in primary infection (P=0·05). Immunization with two intra-muscular doses of 0·5 ml of the FI-RSV vaccine administered 21 days apart resulted in little serum-neutralizing and ELISA antibody, low levels of secretory IgA and a low lymphocyte proliferative response that was significantly lower than the response observed in animals that were previously infected with live RSV. Higher RSV virus titres were detected in the lungs and lung lavage fluid of monkeys immunized with the FI-RSV vaccine than in those with a primary infection (P=0·001). RSV was detected byin situhybridization in pulmonary inflammatory infiltrates, where the single most abundant infiltrating cellular species was macrophages, so it may be these cells that support the enhanced virus replication that contributes to the enhanced pulmonary pathology of FI-RSV immunization.
Characterization of two decades of temporal co-circulation of four mumps virus genotypes in Denmark: identification of a new genotype
Authors: Tecle Tesfaldet , Böttiger Blenda , Örvell Claes , Johansson Bo
Published: August 2015

Twenty-nine Danish virus isolates and 14 serum samples from patients with mumps were genotyped by nucleotide sequencing of the small hydrophobic (SH) protein gene and the deduced 57 amino acid sequences were aligned with sequences of mumps virus strains published previously. Four neurovirulent genotypes of the SH protein gene, genotypes C, D, H and a new genotype, designated J, were found. There was a dynamic fluctuation of the different genotypes over the two decade period of time. Genotype J was found from 1981 to 1988; genotypes C and H exhibited a similar distribution in time. Genotype D was found between 1979 and 1982, it then disappeared and reappeared again in 1996. From 1996 onwards, genotype D was found to be the predominant genotype, which is in contrast to the situation seen in the neighbouring country of Sweden, where, since 1985, only genotype A has been found.