bluuebunny/crossref_metadata_2025 huggingface dataset:
Leucine at position 278 of the AIK-C measles virus vaccine strain fusion protein is responsible for reduced syncytium formation
Authors: Nakayama Tetsuo , Komase Katsuhiro , Uzuka Rina , Hoshi Akiyoshi , Okafuji Takao
Published: August 2015

The live measles virus (MV) vaccine strain AIK-C was attenuated from the wild-type strain Edmonston by plaque purification at 33 °C. Strain AIK-C grew well at 33 °C with a mixture of small-and medium-sized plaques in Vero cells, but did not grow well at 40 °C. To investigate fusion inducibility, expression plasmids for the fusion (F) and haemagglutinin (H) protein regions of MV strains AIK-C (pAIK-F01 and pAIK-H) and Edmonston (pEdm-F and pEdm-H) were constructed. pEdm-F induced extensive cell fusion in B95a and Vero cells under the control of T7 RNA polymerase, whereas a sharp reduction in syncytium formation was observed when pAIK-F01 was used. Six amino acid differences were determined between pAIK-F01 and pEdm-F. Direct sequencing showed that the seed strain AIK-C contained either Leu or Phe at position 278 of the F protein. Experiments using recombinant F protein plasmids demonstrated that those with Leu at position 278 induced poor syncytium formation, while those with Phe at position 278 (Edmonston type) induced extensive cell fusion. Replacement of Phe with Leu at position 278 of pEdm-F reduced fusion-inducing capability. A full-length infectious clone of AIK-C with Leu at position 278 of the F protein was constructed. The rescued virus produced small plaques in Vero cells. However, the same rescued virus with Phe at position 278 produced large plaques. It was concluded that Leu at position 278 of the F protein of the MV vaccine strain AIK-C is responsible for the formation of small plaques.
Complete nucleotide sequence of avian paramyxovirus type 6 isolated from ducks
Authors: Chang P.-C. , Hsieh M.-L. , Shien J.-H. , Graham D. A. , Lee M.-S. , Shieh H. K.
Published: August 2015

There are nine serotypes of avian paramyxovirus (APMV). Only the genome of APMV type 1 (APMV-1), also called Newcastle disease virus (NDV), has been completely sequenced. In this study, the complete nucleotide sequence of an APMV-6 serotype isolated from ducks is reported. The 16236 nt genome encodes eight proteins, nucleocapsid protein (NP), phosphoprotein (P), V protein, matrix protein (M), fusion protein (F), small hydrophobic (SH) protein, haemagglutinin–neuraminidase (HN) protein and large (L) protein, which are flanked by a 55 nt leader sequence and a 54 nt trailer sequence. Sequence comparison reveals that the protein sequences of APMV-6 are most closely related to those of APMV-1 (NDV) and -2, with sequence identities ranging from 22 to 44%. However, APMV-6 contains a gene that might encode the SH protein, which is absent in APMV-1, but present in the rubulaviruses simian virus type 5 and mumps virus. The presence of an SH gene in APMV-6 might provide a link between the evolution of APMV and rubulaviruses. Phylogenetic analysis demonstrates that APMV-6, -1, -2 (only the F and HN sequences were available for analysis) and -4 (only the HN sequences were available for analysis) all cluster into a single lineage that is distinct from other paramyxoviruses. This result suggests that APMV should constitute a new genus within the subfamily Paramyxovirinae.
Characterization of hepatitis D virus genotype III among Yucpa Indians in Venezuela
Authors: Nakano Tatsunori , Shapiro Craig N. , Hadler Stephan C. , Casey John L. , Mizokami Masashi , Orito Etsuro , Robertson Betty H.
Published: August 2015

The complete genome sequences of hepatitis D virus (HDV) strains isolated from three Yucpa Amerindians in Venezuela were determined and found to be genotype III. Comparison of these three genotype III sequences demonstrated the presence of a hypervariable region containing numerous substitutions, insertions/deletions and a highly conserved region containing the self-cleavage domains, which have been reported previously for genotypes I and II. Amino acid changes within the first 90 amino acids of the hepatitis D antigen (HDAg) were found in the genotype III sequences, while the remainder of the HDAg-coding sequence was conserved. The secondary structure for the RNA-editing site differed between genotypes I and III. It was concluded that the serious delta hepatitis outbreaks characterized epidemiologically in the Yucpa Amerindians were caused by HDV genotype III isolates that were related to HDV genotype III isolates from other regions of South America.
Immunogenicity of an E1-deleted recombinant human adenovirus against rabies by different routes of administration
Authors: Vos Ad , Neubert Andreas , Pommerening Elke , Müller Thomas , Döhner Leopold , Neubert Larissa , Hughes Kenneth
Published: August 2015

The immunogenic properties of an E1-deleted, human adenovirus type 5 (Ad5) vaccine virus with activity against rabies were examined in mice, foxes and dogs using different routes of administration. NMRI mice received 105·8, 105·3, 104·3, 103·3and 102·3TCID50by peroral or intramuscular (i.m.) administration. Furthermore, six mice received 105·8TCID50intracerebrally (i.c.). The construct elicited marked seroconversion in mice after oral administration. Immunoreactivity in mice was even more pronounced i.m. and i.c. After direct oral administration (108·0TCID50) in foxes, six of eight animals developed rabies virus-neutralizing antibodies (VNA). All foxes immunized by direct injection (107·7TCID50) in the membrane of the jejunum were shown to seroconvert. Pre-existing immunity against canine adenovirus did not hinder the development of rabies VNA after oral application of the construct (108·0TCID50). Fox cubs (24–29 days old) born from rabies-immune vixens were shown to develop very high levels of rabies VNA after i.m. administration (108·0TCID50), indicating that the immunogenicity of the construct could surpass maternally transferred immunity. In dogs, the construct (108·0TCID50) induced a very strong immune response after i.m. administration. However, no immune response was detectable in dogs after direct oral administration (108·3TCID50) or after endoscopic deposition in the smaller intestine (108·0TCID50). Hence, it must be concluded that the construct is not suitable for oral vaccination of dogs against rabies.
A PCR primer system for detecting oncoretroviruses based on conserved DNA sequence motifs of animal retroviruses and its application to human leukaemias and lymphomas
Authors: Burmeister Thomas , Schwartz Stefan , Thiel Eckhard
Published: August 2015

Many C- and D-type retroviruses are known to cause a broad spectrum of malignant diseases in animals. Certain genome regions of these animal retroviruses are highly conserved between different animal species. It should be possible to detect new members of the retrovirus family with consensus PCR primers derived from these conserved sequence motifs. The consensus PCR primers developed in this study are generic enough to detect nearly all known oncogenic mammalian and avian exogenous C- and D-type retroviruses but do not amplify human endogenous retroviral sequences. In contrast to previous investigations, the present study involved highly stringent PCR conditions and truly generic PCR primers. Forty-four samples from patients with various immunophenotyped malignant diseases (acute and chronic T-/B-cell lymphocytic leukaemias, acute myeloid leukaemias, T-/B-cell lymphomas, chronic myeloproliferative disorders) and three cell lines (Hodgkin’s lymphoma, Burkitt’s lymphoma) have thus far been investigated using these PCR primers. The fact that no retroviruses have been found argues against an involvement of known animal oncoretroviruses or related hitherto undetected human retroviruses in the aetiopathogenesis of these diseases. The retrovirus detection system developed here may be used to confirm suspected retroviral involvement in other (malignant or nonmalignant) human diseases as well as to identify new animal retroviruses.
A study of the Autographa californica multiple nucleopolyhedrovirus ODV envelope protein p74 using a GFP tag
Authors: Slack Jeffrey M. , Dougherty Edward M. , Lawrence Susan D.
Published: August 2015

The Autographa
californica multiple nucleopolyhedrovirus (AcMNPV) protein p74 is associated with the occlusion-derived virus (ODV) envelope. p74 is essential for oral infectivity of ODV and has been proposed to play a role in midgut attachment and/or fusion. In this study, p74 protein was expressed in-frame with green fluorescent protein (GFP) to create a p74–GFP chimera. The C-terminal GFP portion of the chimera facilitated visualization of the trafficking of p74 in baculovirus-infected Spodoptera frugiperda (Sf-9) cells. p74–GFP chimeric proteins localized in the intranuclear ring zone of the nucleus and were found to co-precipitate with the microvesicle fraction of cell lysates. A series of truncations of p74 was expressed as p74–GFP chimeras in recombinant baculoviruses. When C-terminal region S580–F645 was deleted from p74, p74–GFP chimera localization became non-specific and chimeras became soluble. p74 region S580–F645 directed GFP to the intranuclear ring zone in a similar pattern to full-length p74. The hydrophobic C terminus of p74 plays a role in protein localization and possibly in transmembrane anchoring and insertion.
The human herpesvirus-8 (Kaposi’s sarcoma-associated herpesvirus) ORF 40/41 region encodes two distinct transcripts
Authors: AuCoin David P. , Pari Gregory S.
Published: August 2015

The human herpesvirus-8 (HHV-8) locus encoding ORFs 40/41 is a candidate homologue for the Epstein–Barr virus BBLF 2/3 gene, which encodes the putative primase-associated factor. Northern blot data revealed that two transcripts originated from the HHV-8 ORF 40/41 region. The sizes of these transcripts (2.2 and 0.7 kb) suggested that one transcript was the result of a spliced form of ORFs 40 and 41 and the second transcript originated from a region within ORF 41. cDNA sequence and 5′ RACE analysis revealed the removal of an intron between ORFs 40 and 41 and a transcriptional start site 82 nt upstream of ORF 40. The start of transcription for the smaller transcript was mapped to within ORF 41. Regions upstream of the transcriptional start sites were subcloned into a luciferase reporter vector, and transient luciferase assays indicated that distinct promoters drive the expression of each transcript.
The vaccinia virus F12L protein is associated with intracellular enveloped virus particles and is required for their egress to the cell surface
Authors: van Eijl Henriette , Hollinshead Michael , Rodger Gaener , Zhang Wei-Hong , Smith Geoffrey L.
Published: August 2015

The vaccinia virus (VV) F12L gene encodes a 65 kDa protein that is expressed late during infection and is important for plaque formation, EEV production and virulence. Here we have used a recombinant virus (vF12LHA) in which the F12L protein is tagged at the C terminus with an epitope recognized by a monoclonal antibody to determine the location of F12L in infected cells and whether it associates with virions. Using confocal and electron microscopy we show that the F12L protein is located on intracellular enveloped virus (IEV) particles, but is absent from immature virions (IV), intracellular mature virus (IMV) and cell-associated enveloped virus (CEV). In addition, F12L shows co-localization with endosomal compartments and microtubules. F12L did not co-localize with virions attached to actin tails, providing further evidence that actin tails are associated with CEV but not IEV particles. In vΔF12L-infected cells, virus morphogenesis was arrested after the formation of IEV particles, so that the movement of these virions to the cell surface was inhibited and CEV particles were not found. Previously, virus mutants lacking IEV- or EEV-specific proteins were either unable to make IEV particles (vΔF13L and vΔB5R), or were unable to form actin tails after formation of CEV particles (vΔA36R, vΔA33R, vΔA34R). The F12L deletion mutant therefore defines a new stage in the morphogenic pathway and the F12L protein is implicated as necessary for microtubule-mediated egress of IEV particles to the cell surface.
Solution structures of potato virus X and narcissus mosaic virus from Raman optical activity
Authors: Blanch Ewan W. , Robinson David J. , Hecht Lutz , Syme Christopher D. , Nielsen Kurt , Barron Laurence D.
Published: August 2015

Potato virus X (PVX) and narcissus mosaic virus (NMV) were studied using vibrational Raman optical activity (ROA) in order to obtain new information on the structures of their coat protein subunits. The ROA spectra of the two intact virions are very similar to each other and similar to that of tobacco mosaic virus (TMV) studied previously, being dominated by signals characteristic of proteins with helix bundle folds. In particular, PVX and NMV show strong positive ROA bands at ∼1340 cm−1 assigned to hydrated α-helix and perhaps originating in surface exposed helical residues, together with less strong positive ROA intensity in the range ∼1297–1312 cm−1 assigned to α-helix in a more hydrophobic environment and perhaps originating in residues at helix–helix interfaces. The positive ∼1340 cm−1 ROA band of TMV is less intense than those of PVX and NMV, suggesting that TMV contains less hydrated α-helix. Small differences in other spectral regions reflect differences in some loop, turn and side-chain compositions and conformations among the three viruses. A pattern recognition program based on principal component analysis of ROA spectra indicates that the coat protein subunit folds of PVX and NMV may be very similar to each other and similar to that of TMV. These results suggest that PVX and NMV may have coat protein subunit structures based on folds similar to the TMV helix bundle and hence that the helical architecture of the PVX and NMV particles may be similar to that of TMV but with different structural parameters.
Characterization of Chelonus inanitus polydnavirus segments: sequences and analysis, excision site and demonstration of clustering
Authors: Wyder Stefan , Tschannen Adrian , Hochuli Anita , Gruber Andreas , Saladin Verena , Zumbach Sonja , Lanzrein Beatrice
Published: August 2015

Polydnaviruses (genera Ichnovirus and Bracovirus) have a segmented genome of circular double-stranded DNA molecules, replicate in the ovary of parasitic wasps and are essential for successful parasitism of the host. Here we show the first detailed analysis of various segments of a bracovirus, the Chelonus inanitus virus (CiV). Four segments were sequenced and two of them, CiV12 and CiV14, were found to be closely related while CiV14.5 and CiV16.8 were unrelated. CiV12, CiV14.5 and CiV16.8 are unique while CiV14 occurs also nested in another larger segment. All four segments are predicted to contain genes and predictions could be substantiated in most cases. Comparison with databases revealed no significant similarities at either the nucleotide or amino acid level. Inverted repeats with identities between 77% and 92% and lengths between 26 bp and 100 bp were found on all segments outside of predicted genes. Hybridization experiments indicate that CiV12 and CiV14 are both flanked by other virus segments, suggesting that proviral CiV segments are clustered in the genome of the wasp. The integration/excision site of CiV14 was analysed and compared to that of CiV12. On both termini of proviral CiV12 and CiV14 as well as in the excised circular molecule and the rejoined DNA a very similar repeat of 14 bp was found. A model to illustrate where the terminal repeats might recombine to yield the circular molecule is presented. Excision of CiV12 and CiV14 is restricted to the female and sets in at a very specific time-point in pupal–adult development.
Characterization of neutralization sites on the circulating variant of swine vesicular disease virus (SVDV): a new site is shared by SVDV and the related coxsackie B5 virus
Authors: Borrego Belén , Carra Elena , García-Ranea Juan Antonio , Brocchi Emiliana
Published: August 2015

Using a panel of new monoclonal antibodies (mAbs), five neutralizing, conformation-dependent sites have been identified on the antigenic variant of swine vesicular disease virus (SVDV) circulating currently. In studies on the antigenic conservation of these sites, the four antigenic/genetic groups of SVDV described showed distinguishable patterns, confirming this classification. By sequencing mAb-resistant mutants, the five sites have been mapped precisely and localized on a three-dimensional model of the SVDV capsid. All were found to be orientated, to a different extent, towards the external surface of the capsid. Three of the five sites, located in VP1, VP2 and VP3, correspond to epitopes identified previously in historic isolates as sites 1, 2a and 3b, respectively. Another site, site IV, which maps to position 258 of VP1, corresponds to an epitope reported recently and is described in this study to be specific for isolates of the most recent antigenic group of SVDV. A fifth site is described for the first time and corresponds to the unique neutralizing site that is common to both SVDV and coxsackie B5 virus; it maps to positions 95 and 98 of VP1, but may also include positions nearby that belong to site 1 on the BC-loop of VP1, suggesting the classification of site Ia. These results may have useful diagnostic and epidemiological applications, since mAbs to the new conserved site Ia provide universal reagents for SVDV detection systems, while the specificity of mAbs to site IV make them unique markers for the most recent strains of SVDV.
Genetic characterization of wild-type genotype VII hepatitis A virus
Authors: Ching Karen Z. , Nakano Tatsunori , Chapman Louisa E. , Demby Austin , Robertson Betty H.
Published: August 2015

The complete genome sequence of the only identified genotype VII hepatitis A virus (HAV), strain SLF88, was obtained from PCR amplicons generated by a modified long PCR approach. There was 90% nucleotide identity in the 5′ untranslated region compared to other known HAV sequences. In the remainder of the genome containing the long open reading frame, there was about 85% nucleotide identity to human HAV genotypes IA and IB and 80% identity to simian HAV genotype V. Compared to HAV strain HM-175, the capsid amino acids were highly conserved, with only four homologous amino acid changes, while an increasing number of amino acid differences was seen in the P2 and P3 genome regions. While nucleotide variability within the three functional coding regions did not differ, the P3D region was found to have the largest number of amino acid changes compared to HM-175.
Multiple glycosylated forms of the respiratory syncytial virus fusion protein are expressed in virus-infected cells
Authors: Rixon Helen W. McL. , Brown Craig , Brown Gaie , Sugrue Richard J.
Published: August 2015

Analysis of the respiratory syncytial virus (RSV) fusion (F) protein in RSV-infected Vero cells showed the presence of a single F1 subunit and at least two different forms of the F2 subunit, designated F2a (21 kDa) and F2b (16 kDa), which were collectively referred to as [F2]a/b. Enzymatic deglycosylation of [F2]a/b produced a single 10 kDa product suggesting that [F2]a/b arises from differences in the glycosylation pattern of F2a and F2b. The detection of [F2]a/b was dependent upon the post-translational cleavage of the F protein by furin, since its appearance was prevented in RSV-infected Vero cells treated with the furin inhibitor dec-RVKR-cmk. Analysis by protein cross-linking revealed that the F1 subunit interacted with [F2]a/b, via disulphide bonding, to produce equivalent F protein trimers, which were expressed on the surface of infected cells. Collectively, these data show that multiple F protein species are expressed in RSV-infected cells.
A DNA vaccine containing an infectious Marek’s disease virus genome can confer protection against tumorigenic Marek’s disease in chickens
Authors: Tischer B. Karsten , Schumacher Daniel , Beer Martin , Beyer Jörg , Teifke Jens Peter , Osterrieder Kerstin , Wink Kerstin , Zelnik Vladimir , Fehler Frank , Osterrieder Nikolaus
Published: August 2015

A DNA vaccine containing the infectious BAC20 clone of serotype 1 Marek’s disease virus (MDV) was tested for its potential to protect against Marek’s disease (MD). Chickens were immunized at 1 day old with BAC20 DNA suspended either in PBS, as calcium phosphate precipitates, incorporated into chitosan nanoparticles, in Escherichia coli DH10B cells, or bound to gold particles for gene-gun delivery. Challenge infection with MDV strain EU1 was performed at 12 days old, and four out of seven birds immunized with BAC20 DNA in saline by the intramuscular route remained free of MD until day 77 after challenge infection. A delay in the development of the disease could be observed in some animals vaccinated with other BAC20 DNA formulations, but clinical MD and tumour formation were evident in all but one bird. Five out of seven animals immunized with the vaccine virus CVI988 were protected against MD, but none out of seven birds survived EU1 challenge infection after injection of negative-control plasmid DNA. In a second animal experiment, five out of 12 chickens immunized with BAC20 DNA and six out of eight birds immunized with virus reconstituted from BAC20 DNA remained free of MD after challenge infection. In contrast, none out of 12 chickens survived challenge infection after immunization with BAC20 DNA lacking the essential gE gene or with gE-negative BAC20 virus. The results suggested that an MDV BAC DNA vaccine has potential to protect chickens against MD, but that in vivo reconstitution of vaccine virus is a prerequisite for protection.
Emergence of dengue virus type 4 genotype IIA in Malaysia
Authors: AbuBakar Sazaly , Wong Pooi-Fong , Chan Yoke-Fun
Published: August 2015

Phylogenetic analyses of the envelope (E) gene sequence of five recently isolated dengue virus type 4 (DENV-4) suggested the emergence of a distinct geographical and temporal DENV-4 subgenotype IIA in Malaysia. Four of the isolates had direct ancestral lineage with DENV-4 Indonesia 1973 and showed evidence of intra-serotypic recombination with the other recently isolated DENV-4, MY01-22713. The E gene of isolate MY01-22713 had strong evidence of an earlier recombination involving DENV-4 genotype II Indonesia 1976 and genotype I Malaysia 1969. These results suggest that intra-serotypic recombination amongst DENV-4 from independent ancestral lineages may have contributed to the emergence of DENV-4 subgenotype IIA in Malaysia.
Genome sequence analysis of Tamana bat virus and its relationship with the genus Flavivirus
Authors: de Lamballerie X. , Crochu S. , Billoir F. , Neyts J. , de Micco P. , Holmes E. C. , Gould E. A.
Published: August 2015

Tamana bat virus (TABV, isolated from the batPteronotus parnellii) is currently classified as a tentative species in the genusFlavivirus. We report here the determination and analysis of its complete coding sequence. Low but significant similarity scores between TABV and member-viruses of the genusFlaviviruswere identified in the amino acid sequences of the structural, NS3 and NS5 genes. A series of cysteines located in the envelope protein and the most important enzymatic domains of the virus helicase/NTPase, methyltransferase and RNA-dependent RNA polymerase were found to be highly conserved. In the serine-protease domain, the catalytic sites were conserved, but variations in sequence were found in the putative substrate-binding sites, implying possible differences in the protease specificity. In accordance with this finding, the putative cleavage sites of the TABV polyprotein by the virus protease are substantially different from those of flaviviruses. The phylogenetic position of TABV could not be determined precisely, probably due to the extremely significant genetic divergence from other member-viruses of the familyFlaviviridae. However, analysis based on both genetic distances and maximum-likelihood confirmed that TABV is more closely related to the flaviviruses than to the other genera. These findings have implications for the evolutionary history and taxonomic classification of the family as a whole: (i) the possibility that flaviviruses were derived from viruses infecting mammals rather than from mosquito viruses cannot be excluded; (ii) using the current criteria for the definition of genera in the familyFlaviviridae, TABV should be assigned to a new genus.
Lymphoid activation: a confounding factor in AIDS vaccine development?
Authors: Richardson Jennifer , Broche Sophie , Baud Sandrine , Leste-Lasserre Thierry , Féménia Françoise , Levy Daniel , Moraillon Anne , Pancino Gianfranco , Sonigo Pierre
Published: August 2015

In a previous vaccination trial, inoculation of env gene DNA failed to elicit a detectable antibody response, yet accelerated virus dissemination in most immunized cats following challenge with feline immunodeficiency virus. This result raised the possibility that cell-mediated immune responses had given rise to immune-mediated enhancement of infection. Since high-level replication of immunodeficiency viruses in lymphocytes requires cellular activation, antigen-specific responses or non-specific polyclonal activation may have increased the frequency of optimal target cells. In the present DNA vaccination trial, although designed so as to minimize non-specific polyclonal activation, immune-mediated enhancement was nonetheless observed in certain immunized cats. Moreover, rapid virus dissemination in vivo was associated with the presence of T-helper responses prior to challenge, and was linked to increased susceptibility of lymphocytes to ex vivo infection. Immune activation may thus be a confounding factor in vaccination against lentivirus infection, diminishing vaccine efficacy and giving rise to immune-mediated enhancement.
Characterization of a human immunodeficiency virus type 1 pre-integration complex in which the majority of the cDNA is resistant to DNase I digestion
Authors: Khiytani Dheeraj K. , Dimmock Nigel J.
Published: August 2015

The human immunodeficiency virus type 1 (HIV-1) pre-integration complex (PIC) is a cytoplasmic nucleoprotein structure derived from the core of the virion and is responsible for reverse transcription of viral RNA to cDNA, transport to the nucleus and integration of the cDNA into the genome of the infected target cell. Others have shown by Mu phage-mediated PCR footprinting that only the LTRs of the cDNA of PICs isolated early in infection are protected by bound protein, while the rest of the genome is susceptible to nuclease attack. Here, using DNase I footprinting, we confirmed that the majority of the cDNA of PICs isolated at 8·5 h after infection with cell-free virus was sensitive to digestion with DNase I and that only part of the LTRs (approximately 6% of the total cDNA) was protected. However, PICs isolated 90 min later (at 10 h post-infection) were very different in that the majority (approximately 90%) of cDNA was protected from nuclease degradation. These late PICs were integration active in vitro. We conclude that HIV-1 has at least two types of PIC, an early PIC characterized by protein bound only at the LTRs, and a late, and possibly more mature form, in which protein is bound along the length of the cDNA.
On the evolution and molecular epidemiology of the potyvirus Papaya ringspot virus
Authors: Bateson Marion F. , Lines Rosemarie E. , Revill Peter , Chaleeprom Worawan , Ha Cuong V. , Gibbs Adrian J. , Dale James L.
Published: August 2015

The potyvirus Papaya ringspot virus (PRSV) is found throughout the tropics and subtropics. Its P biotype is a devastating pathogen of papaya crops and its W biotype of cucurbits. PRSV-P is thought to arise by mutation from PRSV-W. However, the relative impact of mutation and movement on the structure of PRSV populations is not well characterized. To investigate this, we have determined the coat protein sequences of isolates of both biotypes of PRSV from Vietnam (50), Thailand (13), India (1) and the Philippines (1), and analysed them together with 28 PRSV sequences already published, so that we can better understand the molecular epidemiology and evolution of PRSV. In Thailand, variation was greater among PRSV-W isolates (mean nucleotide divergence 7·6%) than PRSV-P isolates (mean 2·6%), but in Vietnamese populations the P and W biotypes were more but similarly diverse. Phylogenetic analyses of PRSV also involving its closest known relative, Moroccan watermelon mosaic virus, indicate that PRSV may have originated in Asia, particularly in the Indian subcontinent, as PRSV populations there are most diverse and hence have probably been present longest. Our analyses show that mutation, together with local and long-distance movement, contributes to population variation, and also confirms an earlier conclusion that populations of the PRSV-P biotype have evolved on several occasions from PRSV-W populations.
Preclinical diagnosis of chronic wasting disease in captive mule deer (Odocoileus hemionus) and white-tailed deer (Odocoileus virginianus) using tonsillar biopsy
Authors: Wild Margaret A. , Spraker Terry R. , Sigurdson Christina J. , O’Rourke Katherine I. , Miller Michael W.
Published: August 2015

The usefulness of tonsillar biopsy on live deer for preclinical diagnosis of the transmissible spongiform encephalopathy chronic wasting disease (CWD) was evaluated. Disease was tracked in a CWD-endemic herd using serial tonsillar biopsies collected at 6 to 9 month intervals from 34 captive mule deer (Odocoileus hemionus) and five white-tailed deer (O. virginianus). Tonsillar biopsies were examined for accumulation of PrPCWD, the protein marker for infection, using immunohistochemical (IHC) staining. 26/34 (76%) mule deer and 4/5 (80%) white-tailed deer had PrPCWD accumulation in tonsillar biopsies; CWD was subsequently confirmed by post-mortem examination in all 30 of these tonsillar-positive deer. Six mule deer with IHC-negative tonsillar biopsies had positive brain and tonsillar IHC staining upon death 12 to 40 months following the last biopsy. PrPCWD accumulation in tonsillar biopsy was observed 2 to 20 months before CWD-related death and up to 14 months before onset of clinical signs of CWD. Tonsillar biopsies from 3-month-old mule deer (n=6) were IHC negative, but PrPCWD accumulation was detected in tonsillar biopsies from 7/10 mule deer by 19 months of age. Tonsillar biopsy evaluated with IHC staining is a useful technique for the preclinical diagnosis of CWD in live mule deer and white-tailed deer when intensive management approaches are possible.
Canine vaccine recipients recognize an immunodominant region of the rabies virus glycoprotein
Authors: Johnson N. , Mansfield K. L. , Fooks A. R.
Published: August 2015

To investigate the immune response to anti-rabies vaccination in the principal recipient (the domestic dog), four truncated fragments of the rabies virus glycoprotein were expressed as glutathione S-transferase fusion proteins. Immune sera from vaccinated rabbits and dogs were then used to probe for reactivity with these expressed proteins. In two rabbits and four dogs tested, the dominant antibody response to non-conformational antigenic sites appeared to be directed to a region of the glycoprotein between amino acids 222 and 332. The N-terminal fragment of the glycoprotein was also significantly antigenic. Further studies to assess whether the antibody response to the internal domain could neutralize the rabies Challenge Virus Standard (CVS) strain, using antibody depletion, suggested that this fraction did contribute to the ability of post-vaccination sera to neutralize and therefore protect against infection.
Salmonid viral haemorrhagic septicaemia virus: fusion-related enhancement of virus infectivity by peptides derived from viral glycoprotein G or a combinatorial library
Authors: Mas V. , Pérez L. , Encinar J. A. , Pastor M. T. , Rocha A. , Perez-Paya E. , Ferrer-Montiel A. , Gonzalez Ros J. M. , Estepa A. , Coll J. M.
Published: August 2015

To search for enhancers and/or inhibitors of viral haemorrhagic septicaemia virus (VHSV, a salmonid rhabdovirus) infectivity, a total of 51 peptides from a pepscan of viral envelope protein G, a recombinant peptide from protein G (frg11) and 80 peptide mixtures from an α-helix-favoured combinatorial library were screened. However, contrary to what occurs in many other enveloped viruses, only peptides enhancing rather than inhibiting VHSV infectivity were found. Because some of the enhancer pepscan G peptides and frg11 were derived from phospholipid-binding or fusion-related regions identified previously, it was suggested that enhancement of virus infectivity might be related to virus–cell fusion. Furthermore, enhancement was significant only when the viral peptides were pre-incubated with VHSV at the optimal low pH of fusion, before being adjusted to physiological pH and assayed for infectivity. Enhancement of VHSV infectivity caused by the pre-incubation of VHSV with peptide p5 (SAAEASAKATAEATAKG), one of the individual enhancer peptides defined from the screening of the combinatorial library, was independent of the pre-incubation pH. However, it was also related to fusion because the binding of p5 to protein G induced VHSV to bypass the endosome pathway of infection and reduced the low-pH threshold of fusion, thus suggesting an alternative virus entry pathway for p5–VHSV complexes. Further investigations into VHSV enhancer peptides might shed some light on the mechanisms of VHSV fusion.
A release-competent influenza A virus mutant lacking the coding capacity for the neuraminidase active site
Authors: Gubareva Larisa V. , Nedyalkova Marina S. , Novikov Dmitri V. , Murti K. Gopal , Hoffmann Erich , Hayden Frederick G.
Published: August 2015

Both influenza A virus surface glycoproteins, the haemagglutinin (HA) and neuraminidase (NA), interact with neuraminic acid-containing receptors. The influenza virus A/Charlottesville/31/95 (H1N1) has shown a substantially reduced sensitivity to NA inhibitor compared with the A/WSN/33 (H1N1) isolate by plaque-reduction assays in Madin–Darby canine kidney (MDCK) cells. However, there was no difference in drug sensitivity in an NA inhibition assay. The replacement of the HA gene of A/WSN/33 with the HA gene of A/Charlottesville/31/95 led to a drastic reduction in sensitivity of A/WSN/33 to NA inhibitor in MDCK cells. Passage of A/Charlottesville/31/95 in cell culture in the presence of an NA inhibitor resulted in the emergence of mutant viruses (delNA) whose genomes lacked the coding capacity for the NA active site. The delNA mutants were plaque-to-plaque purified and further characterized. The delNA-31 mutant produced appreciable yields (∼106 p.f.u./ml) in MDCK cell culture supernatants in the absence of viral or bacterial NA activity. Sequence analysis of the delNA mutant genome revealed no compensatory substitutions in the HA or other genes compared with the wild-type. Our data indicate that sialylation of the oligosaccharide chains in the vicinity of the HA receptor-binding site of A/Charlottesville/31/95 virus reduces the HA binding efficiency and thus serves as a compensatory mechanism for the loss of NA activity. Hyperglycosylation of HA is common in influenza A viruses circulating in humans and has the potential to reduce virus sensitivity to NA inhibitors.
Natural killer cell activation after infection with lactate dehydrogenase-elevating virus
Authors: Markine-Goriaynoff Dominique , Hulhoven Xavier , Cambiaso César L. , Monteyne Philippe , Briet Thérèse , Gonzalez Maria-Dolores , Coulie Pierre , Coutelier Jean-Paul
Published: August 2015

Early after infection, lactate dehydrogenase-elevating virus (LDV) alters the immune system by polyclonally activating B lymphocytes, which leads to IgG2a-restricted hypergammaglobulinaemia, and by suppressing the secretion of Th2 cytokines. Considering that these alterations may involve cells of the innate immune system and cytokines such as interferon-gamma (IFN-γ), we analysed the effect of LDV on natural killer (NK) cells. Within a few days of infection, a strong and transient NK cell activation, characterized by enhancedIFN-γ message expression and cytolysis, was observed. LDV triggered a large increase in serum IFN-γ levels. Because NK cells and IFN-γ may participate in the defence against virus infection, we analysed their possible role in the control of LDV titres with a new agglutination assay. Our results indicate that neither the activation of NK cells nor the IFN-γ secretion affect the early and rapid virus replication that follows LDV inoculation.
The jaagsiekte sheep retrovirus envelope gene induces transformation of the avian fibroblast cell line DF-1 but does not require a conserved SH2 binding domain
Authors: Allen Thomas E. , Sherrill Kate J. , Crispell Sara M. , Perrott Matthew R. , Carlson Jonathan O. , DeMartini James C.
Published: August 2015

Ovine pulmonary adenocarcinoma, caused by jaagsiekte sheep retrovirus (JSRV), is a naturally occurring retrovirus-induced pulmonary neoplasm of sheep. We report here that expression of the JSRV env gene is sufficient to transform an avian embryo fibroblast cell line, DF-1. DF-1 cells transfected with an avian sarcoma–leukaemia retroviral expression vector containing the JSRV env gene [pRCASBP(A)-J:env] exhibited changes consistent with transformation, including contraction and rounding of cells with formation of dense foci. Transfection with a reporter construct expressing the green fluorescent protein did not induce morphological changes in DF-1 cells, eliminating the possibility that the vector, the transfection protocol or culturing techniques were responsible for the transformed phenotype. When pRCASBP(A)-J:env-transfected cells were inoculated into nude mice, tumours formed, verifying that the DF-1 cells were tumorigenic. Analysis of the JSRV env gene revealed a conserved tyrosine (597) and methionine (600) residue in the cytoplasmic tail within the transmembrane domain of the envelope, which creates a known binding site of SH2 domains in the p85 subunit of phosphatidylinositol 3-kinase. However, when this tyrosine residue was mutated to serine or alanine, transformation was not affected. Furthermore, mutation of the methionine residue to valine or leucine also failed to eliminate JSRV env-mediated transformation. These results are in contrast to mutational analysis performed in JSRV env-transformed murine NIH-3T3 cells in which both the tyrosine and methionine residues are necessary for transformation. These findings suggest that more than one mechanism may be involved in JSRV env-mediated transformation.
The dominant hepatitis B virus genotype identified in Tibet is a C/D hybrid
Authors: Cui Chaoyin , Shi Jinxiu , Hui Lijian , Xi Huifeng , Zhuoma  , Quni  , Tsedan  , Hu Gengxi
Published: August 2015

There are no reports on DNA sequences of hepatitis B virus (HBV) strains from Tibet, although this highland area has a high HBsAg-positive population. We characterized HBV isolates from sera of 26 HBsAg-positive Tibetans. To determine the HBV genotypes and their phylogenetic relationships, we sequenced two genomic regions, one including the pre-S1/pre-S2/S region and the other including the pre-C/C region. The sequences were classified into two different genotypes based on different regions of the genome, except for one isolate. To clarify this finding, two complete HBV genomes that represented the two groups of isolates were sequenced. From the sequencing results, we concluded that HBV strains in Tibet may be classified as genotype C, and there are at least two subgroups. The dominant subgroup is a C/D hybrid with serotype ayw2, and the other is genotype C with serotype adw. This is the first report of complete nucleotide sequences of HBV from Tibet. These results contribute to the investigation of recombinant HBV strains throughout the world and should encourage further study of genotypes and recombination in HBV from this particular region.
Excretion of herpes simplex virus type 2 glycoprotein D into the culture medium
Authors: Murata Takayuki , Goshima Fumi , Takakuwa Hiroki , Nishiyama Yukihiro
Published: August 2015

Glycoprotein D (gD) of herpes simplex virus type 2 (HSV-2) was excreted from infected cells into the medium. Peptide mapping analysis and lectin binding assays suggested that the gD in the medium is secreted after full glycosylation and cleavage at its C terminus. Release of HSV-2 gD was inhibited by addition of either tunicamycin or brefeldin A, suggesting that the gD in the medium was secreted through the endoplasmic reticulum and Golgi apparatus.
Human cytomegalovirus infection inhibits epidermal growth factor (EGF) signalling by targeting EGF receptors
Authors: Fairley J. A. , Baillie J. , Bain M. , Sinclair J. H.
Published: August 2015

Infection with human cytomegalovirus (HCMV) is known to involve complex interactions between viral and cellular factors resulting in perturbation of a number of cellular functions. Specifically, HCMV infection targets control of the cell cycle, cellular transcription and immunoregulation, presumably to optimize the cellular environment for virus persistence and productive infection. Here, we show that HCMV infection also prevents external signalling to the cell by disrupting the function of epidermal growth factor receptor (EGFR). Infection with HCMV resulted in a decrease in cell-surface expression of EGFR. This decrease was correlated with a concomitant decrease in steady-state levels of EGFR protein. Consistent with this, HCMV inhibited EGF-mediated receptor autophosphorylation. Infection with a mutant HCMV deleted of all viral gene products known to be involved in down-regulation of MHC Class I receptors still resulted in this down-regulation, implying that EGFR down-regulation by HCMV is mediated by a novel virus function. We suggest that a primary goal of HCMV is to ‘isolate’ the infected cell from host-mediated signals so that the cell responds solely to an array of virus-specific signals which optimize the cell for virus production.
Analysis of the human herpesvirus-6 immediate-early 1 protein
Authors: Stanton Richard , Fox Julie D. , Caswell Richard , Sherratt Emma , Wilkinson Gavin W. G.
Published: August 2015

Herpesvirus immediate-early (IE) gene products play key roles in establishing productive infections, regulating reactivation from latency and evading immune recognition. Analyses of HHV-6 IE gene expression have revealed that the IE1 gene of the HHV-6A and HHV-6B variants exhibits a higher degree of sequence variation than other regions of the genome and no obvious similarity to its positional analogue in HCMV. We have analysed expression of the HHV-6 U1102 (HHV-6A) and Z29 (HHV-6B) IE1 gene products using transient expression vectors, stable cell lines and in the context of lytic virus infection. The IE1 transcripts from both variants demonstrate a similar pattern of splice usage within their translated regions. The HHV-6 IE1 proteins from both variants traffic to, and form a stable interaction with, PML-bodies (also known as ND10 or PODS). Remarkably, PML-bodies remained structurally intact and associated with the IE1 protein throughout lytic HHV-6 infection. Immunoprecipitation studies demonstrated that HHV-6 IE1 from both variants is covalently modified by conjugation to the small ubiquitin-like protein SUMO-1. Overexpression of SUMO-1 in cell lines resulted in substantially enhanced levels of IE1 expression; thus sumoylation may bestow stability to the protein. These results indicate that the HHV-6 IE1 protein interacts with PML-bodies yet, unlike other herpesviruses, HHV-6 appears to have no requirement or mechanism to induce PML-body dispersal during lytic replication.
RNA-dependent RNA polymerase complex of Brome mosaic virus: analysis of the molecular structure with monoclonal antibodies
Authors: Dohi Koji , Mise Kazuyuki , Furusawa Iwao , Okuno Tetsuro
Published: August 2015

Viral RNA-dependent RNA polymerase (RdRp) plays crucial roles in the genomic replication and subgenomic transcription of Brome mosaic virus (BMV), a positive-stranded RNA plant virus. BMV RdRp is a complex of virus-encoded 1a and 2a proteins and some cellular factors, and associates with the endoplasmic reticulum at an infection-specific structure in the cytoplasm of host cells. In this study, we investigate the gross structure of the active BMV RdRp complex using monoclonal antibodies raised against the 1a and 2a proteins. Immunoprecipitation experiments showed that the intermediate region between the N-terminal methyltransferase-like domain and the C-terminal helicase-like domain of 1a protein, and the N terminus region of 2a protein are exposed on the surface of the solubilized RdRp complex. Inhibition assays for membrane-bound RdRp suggested that the intermediate region between the methyltransferase-like and the helicase-like domains of 1a protein is located at the border of the region buried within a membrane structure or with membrane-associated material.
The formation and function of extracellular enveloped vaccinia virus
Authors: Smith Geoffrey L. , Vanderplasschen Alain , Law Mansun
Published: August 2015

Vaccinia virus produces four different types of virion from each infected cell called intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV) and extracellular enveloped virus (EEV). These virions have different abundance, structure, location and roles in the virus life-cycle. Here, the formation and function of these virions are considered with emphasis on the EEV form and its precursors, IEV and CEV. IMV is the most abundant form of virus and is retained in cells until lysis; it is a robust, stable virion and is well suited to transmit infection between hosts. IEV is formed by wrapping of IMV with intracellular membranes, and is an intermediate between IMV and CEV/EEV that enables efficient virus dissemination to the cell surface on microtubules. CEV induces the formation of actin tails that drive CEV particles away from the cell and is important for cell-to-cell spread. Lastly, EEV mediates the long-range dissemination of virus in cell culture and, probably,in vivo. Seven virus-encoded proteins have been identified that are components of IEV, and five of them are present in CEV or EEV. The roles of these proteins in virus morphogenesis and dissemination, and as targets for neutralizing antibody are reviewed. The production of several different virus particles in the VV replication cycle represents a coordinated strategy to exploit cell biology to promote virus spread and to aid virus evasion of antibody and complement.
Epstein–Barr virus replication in tongue epithelial cells
Authors: Herrmann Kathrin , Frangou Phroso , Middeldorp Jaap , Niedobitek Gerald
Published: August 2015

Epstein–Barr virus (EBV) persistently infects B-cells in humans and can be shed into the saliva. The cellular source of infectious virus is uncertain. Hairy leukoplakia, an AIDS-associated lesion of the tongue, supports EBV replication in epithelial cells. However, the general significance of this observation has remained doubtful. Using immunohistochemistry and in situ hybridization, we demonstrate evidence of EBV replication in tongue epithelial cells in 4 of 168 samples from 84 autopsy cases. Thus, in patients who do not have AIDS, squamous epithelial cells of the tongue rarely support EBV replication. However, all individuals with evidence of EBV replication were either on immunosuppressive therapy or were terminally ill cancer patients, suggesting that an impairment of the immune system may have allowed EBV replication to occur at this site. Thus, our findings are consistent with the idea that EBV replication in oropharyngeal epithelial cells is an infrequent event.