bluuebunny/crossref_metadata_2025 huggingface dataset:
Complete sequence of RNA 1 and the presence of tRNA-like structures in all RNAs of Potato mop-top virus, genus Pomovirus
Authors: Savenkov Eugene I. , Sandgren Maria , Valkonen Jari P. T.
Published: August 2015

The complete nucleotide sequence (6043 nt) of RNA 1 from Potato mop-top virus (PMTV-Sw), the type member of the genus Pomovirus, was determined. The first (5′-terminal) open reading frame (ORF 1) encodes a predicted protein of 148 kDa. ORF 2 extends through the opal stop codon of ORF 1 producing a predicted readthrough protein of 206 kDa which resembles the RNA-dependent RNA polymerases (RdRp) of other fungal-transmitted viruses. It includes a methyltransferase, a helicase and a GDD RdRp motif, respectively. Phylogenetic analyses of RdRps indicated that PMTV is most closely related to Beet soil-borne virus (genusPomovirus), Broad bean necrosis virus (genus Pomovirus) and Soil-borne wheat mosaic virus (genus Furovirus), and is more distantly related to the other viruses of the former furovirus group. The 5′ and 3′ termini of RNA 1 in PMTV contained untranslated regions (UTR) of 114 nt and 489 nt, respectively. The 3′-UTR of RNA 1 contained a tRNA-like structure, which has previously been reported in the 3′-UTR of RNA 2 but not RNA 3. However, in this study, the tRNA-like structure was also found in the 3′-UTR of RNA 3, which confirms its presence in the 3′-UTRs of all three RNAs of PMTV.
Resistance of Capsicum annuum ‘Avelar’ to pepper mottle potyvirus and alleviation of this resistance by co-infection with cucumber mosaic cucumovirus are associated with virus movement
Authors: Guerini Michael N. , Murphy John F.
Published: August 2015

Capsicum annuum cv. Avelar plants resist systemic infection by the Florida isolate of pepper mottle potyvirus (PepMoV-FL). Immuno-tissue blot analysis for detection of PepMoV-FL infection in selected stem segments revealed that virus moved down the stem in external phloem, and, over time, accumulated to detectable levels throughout stem sections (appearing to accumulate in external and internal phloem) taken from below the inoculated leaf. At 21 days post-inoculation, PepMoV-FL was detected in stem segments one or two internodes above the inoculated leaf; however, no virus was observed in internal phloem in stem segments beyond these internodes. In contrast to these observations, PepMoV-FL was detected in the internal phloem of all internodes of the stem located above the inoculated leaf, with subsequent movement into non-inoculated leaves, in Avelar plants co-infected with PepMoV-FL and cucumber mosaic cucumovirus (CMV-KM). No apparent enhancement of PepMoV-FL accumulation occurred in protoplasts inoculated with PepMoV-FL alone versus a mixed inoculum of PepMoV-FL and CMV-KM. These findings confirm earlier observations that potyvirus movement up the stem of Capsicum species occurs via internal phloem. It is also shown that PepMoV-FL does not accumulate to detectable levels in internal phloem in the stem of Avelar plants, thereby limiting its movement to within the inoculated leaf and lower portions of the stem; however, co-infection of Avelar plants with CMV-KM alleviates this restricted movement, allowing PepMoV-FL to invade young tissues systemically.
Tomato chlorotic dwarf viroid: an evolutionary link in the origin of pospiviroids
Authors: Singh Rudra P. , Nie Xianzhou , Singh Mathuresh
Published: August 2015

Over 40 isolates of potato spindle tuber viroid (PSTVd) have been reported from potato, other Solanum species and greenhouse tomato. These isolates have sequence similarities in the range 95–99%. A viroid which caused chlorotic leaves and severe dwarfing of plants in greenhouse tomato crops was detected. The viroid was found to hybridize readily with PSTVd probes. It migrated faster than PSTVd in return-polyacrylamide gel electrophoresis and was not amplified in RT–PCR by a primer pair based on the lower strand of the central conserved region of PSTVd. Nucleotide sequencing of the viroid indicated that it is a circular RNA of 360 nt, with less than 90% sequence similarities with PSTVd isolates. The Variable domain (V) has less than 60% and the Terminal Right domain less than 90% sequence similarity, while the remainder of the molecule has greater than 97% similarity with PSTVd. Because of its less-than 90% sequence similarities, unique V domain, lack of seed-transmission and lack of cross-protection by PSTVd, the viroid from tomato is proposed to be a distinct viroid species (tomato chlorotic dwarf viroid; TCDVd) which also differs from two viroids infecting tomato in nature. TCDVd may be an evolutionary link in the development of crop viroids, with Mexican papita viroid as the ancestral viroid.
Assembly of the Epstein–Barr virus BBLF4, BSLF1 and BBLF2/3 proteins and their interactive properties
Authors: Yokoyama Naoaki , Fujii Ken , Hirata Mineo , Tamai Katsuyuki , Kiyono Tohru , Kuzushima Kiyotaka , Nishiyama Yukihiro , Fujita Masatoshi , Tsurumi Tatsuya
Published: August 2015

Epstein–Barr virus (EBV) encodes putative helicase–primase proteins BBLF4, BSLF1 and BBLF2/3, which are essential for the lytic phase of viral DNA replication. The BSLF1, BBLF4 and BBLF2/3 proteins were expressed in B95-8 cells after induction of a virus productive cycle, possessing apparent molecular masses of 89 kDa, 90 kDa and 80 kDa, respectively. The anti-BSLF1 or anti-BBLF2/3 protein-specific antibody, which recognizes its target protein in both Western blotting and immunoprecipitation analyses, immunoprecipitated all of the BSLF1, BBLF4 and BBLF2/3 proteins from the extract of the cells with a virus productive cycle, indicating that these viral proteins are assembled togetherin vivo. To characterize their protein–protein interactions in detail, recombinant baculoviruses capable of expressing each of these viral gene products in insect cells were constructed. The assembly of the three virus replication proteins was reproduced in insect cells co- infected with the three recombinant baculoviruses, indicating that complex formation does not require other EBV replication proteins. Furthermore, experiments performed by using the extracts from insect cells co-infected with each pair of the recombinant viruses demonstrated that the BSLF1 protein could interact separately with both the BBLF4 and BBLF2/3 proteins and that the BBLF2/3 protein also interacted with the BBLF4 protein. These observations strongly suggest that within the BBLF4–BSLF1–BBLF2/3 complex each component interacts directly with the other two, resulting in helicase–primase enzyme activity.
Serological evidence for protection by human papillomavirus (HPV) type 6 infection against HPV type 16 cervical carcinogenesis
Authors: Silins Ilvars , Wang Zhaohui , Åvall- Lundqvist Elisabeth , Frankendal Bo , Vikmanis Uldis , Sapp Martin , Schiller John T. , Dillner Joakim
Published: August 2015

Human papillomavirus (HPV) exists as more than 100 genotypes. It is not well-established whether the different HPV types interfere with infection or pathogenesis by each other. Possible interactions in cervical carcinogenesis between infection with the most common HPV types (6, 11, 16, 18 and 33) were studied in a seroepidemiological case- control study of 218 women with primary untreated cervical cancer and 219 healthy age-matched control women. As previously shown, HPV-16 seropositivity was associated with cervical cancer risk [odds ratio (OR), 2·39], but HPV-16 was not associated with cervical cancer risk among HPV-6 seropositive women (OR, 1·0). The relative excess risk due to interaction between HPV-6 and -16 was −2·35 (95% confidence interval, −0·04 to −4·65), indicating significant antagonism. The results suggest that infection with HPV-6 may interfere with HPV-16-associated cervical carcinogenesis.
Generation of recombinant lentogenic Newcastle disease virus from cDNA
Authors: Römer-Oberdörfer Angela , Mundt Egbert , Mebatsion Teshome , Buchholz Ursula J. , Mettenleiter Thomas C.
Published: August 2015

Recombinant lentogenic Newcastle disease virus (NDV) of the vaccine strain Clone-30 was reproducibly generated after simultaneous expression of antigenome-sense NDV RNA and NDV nucleoprotein, phosphoprotein and RNA-dependent RNA polymerase from plasmids transfected into cells stably expressing T7 RNA polymerase. For this purpose, the genome of Clone-30, comprising 15186 nt, was cloned and sequenced prior to assembly into a full-length cDNA clone under control of a T7 RNA polymerase promoter. Recombinant virus was amplified by inoculation of transfection supernatant into the allantoic cavity of embryonated specific-pathogen-free (SPF) chicken eggs. Two marker restriction sites comprising a total of five nucleotide changes artificially introduced into noncoding regions were present in the progeny virus. The recombinant NDV was indistinguishable from the parental wild-type virus with respect to its growth characteristics in cell culture and in embryonated eggs. Moreover, an intracerebral pathogenicity index of 0·29 was obtained for both viruses as determined by intracerebral inoculation of day-old SPF chickens, proving that the recombinant NDV is a faithful copy of the parental vaccine strain of NDV.
Comparative studies of human rotavirus serotype G8 strains recovered in South Africa and the United Kingdom
Authors: Steele A. D. , Parker S. P. , Peenze I. , Pager C. T. , Taylor M. B. , Cubitt W. D.
Published: August 2015

Epidemiological studies on the VP7 serotype prevalence of human rotaviruses in South Africa and the United Kingdom identified several strains which could not be serotyped as G1–G4 by monoclonal antibodies. Further analysis of these strains with a G8-specific monoclonal antibody and with probes for human rotaviruses confirmed them as G8 rotaviruses. These G8 strains exhibited a high degree of sequence identity when compared with each other and with other rotavirus G8 strains. Five South African strains were further characterized as VP6 subgroup I, but with a long RNA electropherotype, which is similar to the G8 strains previously isolated in Finland. In the UK strains, one was VP6 subgroup II with a long RNA electropherotype (similar to the Italian G8 strain). The other two were subgroup I with a short RNA electropherotype. None of these strains exhibited the super-short RNA electropherotype described in the prototype G8 strains recovered from Indonesia (69M).
Human hepatic glyceraldehyde-3-phosphate dehydrogenase binds to the poly(U) tract of the 3′ non-coding region of hepatitis C virus genomic RNA
Authors: Petrik Juraj , Parker Hayley , Alexander Graeme J. M.
Published: August 2015

The unique poly(U/UC) tract, the middle part of the tripartite 3′ non-coding region (3′NCR) of hepatitis C virus (HCV) genomic RNA, may represent a recognition signal for the HCV replicase complex. In this study, several proteins binding specifically to immobilized ribooligonucleotide r(U)25 mimicking this structure were identified using cytosolic extracts from HCV-negative or -positive liver explants, and a prominent 36 kDa protein was studied further. Competition experiments including homoribopolymers revealed binding affinities in the order: oligo/poly(U)≫(A)≫(C)≫(G). The protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a multifunctional protein known to bind RNA. GAPDH bound efficiently to the full-length HCV RNA and binding to various 3′NCR constructs revealed critical dependence upon the presence of the middle part of the 3′NCR. Polypyrimidine tract-binding protein, described previously to bind the 3′NCR, did not bind efficiently to the middle part of 3′NCR and was captured from liver extracts in considerably smaller quantities.
Transmission of Eurasian avian H2 influenza virus to shorebirds in North America
Authors: Makarova N. V. , Kaverin N. V. , Krauss S. , Senne D. , Webster R. G.
Published: August 2015

Influenza A virus of the H2 subtype caused a serious pandemic in 1957 and may cause similar outbreaks in the future. To assess the evolution and the antigenic relationships of avian influenza H2 viruses, we sequenced the haemagglutinin (HA) genes of H2 isolates from shorebirds, ducks and poultry in North America and derived a phylogenetic tree to establish their interrelationships. This analysis confirmed the divergence of H2 HA into two geographical lineages, American and Eurasian. One group of viruses isolated from shorebirds in North America had HA belonging to the Eurasian lineage, indicating an interregional transmission of the H2 gene. Characterization of HA with a monoclonal antibody panel revealed that the antigenicity of the Delaware strains differed from the other avian strains analysed. The data emphasizes the importance of avian influenza surveillance.
Bunyavirus superinfection and segment reassortment in transovarially infected mosquitoes
Authors: Borucki Monica K. , Chandler Laura J. , Parker Beulah M. , Blair Carol D. , Beaty Barry J.
Published: August 2015

Rapid evolution of bunyaviruses may occur by RNA segment reassortment between closely related viruses. Reassortment between viruses occurs in dually infected mosquitoes when two different viruses are simultaneously ingested or when the second virus is ingested within 2 days of the first virus. By 3 days after oral infection, interference to superinfection occurs, thus limiting the potential for evolution.Aedes triseriatusmosquitoes can also be transovarially infected (TI+) with LaCrosse (LAC) virus. In these studies, the potential for oral superinfection of TI+ mosquitoes was assessed. Approximately 20% of mosquitoes TI+ with either a temperature-sensitive LAC virus or wild-type (wt) LAC virus became superinfected by ingesting blood meals containing wt LAC or snowshoe hare (SSH) viruses. LAC virus TI+ mosquitoes superinfected with SSH virus were detected by blot hybridization or RT–PCR. Viruses from these mosquitoes were plaque purified and genotyped using RT–PCR. Reassortant genomes were detected in 2·3% of the viruses genotyped, and 4·0% of the genomes tested were diploid for one genome segment.
Characterization of the transcriptional repressor RBP in Epstein–Barr virus-transformed B cells
Authors: Krauer Kenia G. , Buck Marion , Sculley Tom
Published: August 2015

RBP, a transcriptional repressor, is intricately involved in Epstein–Barr virus (EBV) transformation of human B cells. The EBV nuclear proteins EBNA-2, -3, -4 and -6 all utilize RBP to regulate the transcription of both cellular and viral genes. This study investigates the isoforms of the RBP protein in Burkitt’s lymphoma (BL) cells and in EBV-transformed lymphoblastoid cell lines (LCLs). Two-dimensional gel electrophoresis showed the presence of two different cellular isoforms of RBP; the molecular masses and isoelectric points of these two isoforms corresponded to RBP-Jκ and RBP-2N. Fractionation studies and green fluorescent protein (GFP)-tagged expression studies demonstrated that both RBP isoforms were located predominantly in the cell nucleus. Interestingly, GFP-tagged RBP-Jκ showed diffuse, uniform nuclear staining, whereas GFP-tagged RBP-2N showed a discrete nuclear pattern, demonstrating differences between the two isoforms. Within the nuclear fraction of EBV-negative BL cells, RBP existed both in a free form and bound to chromatin, whereas in LCLs the intranuclear RBP was predominantly chromatin-bound. Expression of the EBV latent proteins was found to lead to the sequestering of RBP from the cytoplasm into the cell nucleus and to an increase in the chromatin-bound forms of RBP.
Dissociation of patching by latent membrane protein-1 of Epstein–Barr virus from its stimulation of NF-κB activity
Authors: Bloss Tim , Kaykas Ajamete , Sugden Bill
Published: August 2015

Alterations were made in the amino terminus and the first two transmembrane-spanning regions of the latent membrane protein-1 (LMP-1) of Epstein–Barr virus. These mutant proteins were tested for their abilities to patch and to stimulate NF-κB activity. A subset of these derivatives retains the wild-type topology of LMP-1 in the plasma membrane, but has lost the ability to patch. Deletion of residues 9–20 of LMP-1, which contain potential SH3-binding motifs, abrogates patching of LMP-1. However, mutation of the prolines within these motifs, which eliminates binding of LMP-1 to SH3 domainsin vitro, does not prevent patching by LMP-1. Deletion of the first two transmembrane regions of LMP-1 does prevent it patching. Some of the derivatives of LMP-1 which do not patch do stimulate NF-κB activity. Patching by LMP-1 appears to be a higher-order assemblage of protein that is compatible with the stimulation of NF-κB activity but is not necessary for this signalling.
‘Primer alignment-and-extension’: a novel mechanism of viral RNA recombination responsible for the rescue of inactivated poliovirus cDNA clones
Authors: Pierangeli Alessandra , Bucci M. , Forzan M. , Pagnotti P. , Equestre M. , Pérez Bercoff R.
Published: August 2015

In the course of experiments designed to assess the potential role of alternative open reading frames (ORF) present in the 5′-terminal untranslated region (5′-UTR) of poliovirus type 1 (Mahoney strain) genomic RNA, we came across a double mutation that completely abrogated the infectivity of full-length cDNA clones. The infectivity was rescued in trans by cotransfecting COS-1 cells with short RNA transcripts of the wild-type 5′-UTR of poliovirus type 2 Lansing, provided a free 3′-OH was available. Direct sequencing of the viral RNA revealed that the infectious viruses recovered were recombinants Lansing/Mahoney, with variable points of ‘crossing-over’. A novel mechanism of RNA–RNA recombination, which we propose to call ‘primer alignment-and-extension’, is described that would explain the high rate of recombination of RNA viruses observed in natural conditions.
Antigenic properties and population stability of a foot-and-mouth disease virus with an altered Arg-Gly-Asp receptor-recognition motif
Authors: Ruiz-Jarabo Carmen M. , Sevilla Noemí , Dávila Mercedes , Gómez-Mariano Gema , Baranowski Eric , Domingo Esteban
Published: August 2015

The antigenic properties and genetic stability of a multiply passaged foot-and-mouth disease virus (FMDV) clone C-S8c1 with an Arg-Gly-Gly triplet (RGG) instead of the Arg-Gly-Asp (RGD) integrin-recognition motif at positions 141 to143 of capsid protein VP1 are described. Clear antigenic differences between FMDV RGG and clone C-S8c1 have been documented in ELISA, enzyme-linked immunoelectrotransfer (Western) blot and neutralization assays using site A-specific monoclonal antibodies and anti-FMDV polyclonal antibodies from swine and guinea pigs. The results validate with a live virus the role of the RGD (in particular Asp-143) in recognition of (and neutralization by) antibodies, a role previously suggested by immunochemical and structural studies with synthetic peptides. The FMDV RGG was genetically stable in a large proportion of serial infections of BHK-21 cells. However, a revertant virus with RGD was generated in one out of six passage series. Interestingly, this revertant FMDV did not reach dominance but established an equilibrium with its parental FMDV RGG, accompanied by an increase of quasispecies complexity at the sequences around the RGG triplet. FMDV RGG exhibited a selective disadvantage relative to other RGD-containing clones isolated from the same parental FMDV population. The results suggest that large antigenic variations can be prompted by replacements at critical capsid sites, including those involved in receptor recognition. These critical replacements may yield viruses whose stability allows them to replicate efficiently and to expand the sequence repertoire of an antigenic site.
Sequence analysis of a porcine enterovirus serotype 1 isolate: relationships with other picornaviruses
Authors: Doherty Michelle , Todd Daniel , McFerran Neil , Hoey Elizabeth M.
Published: August 2015

The majority of the genomic sequence of a porcine enterovirus serotype 1 (PEV-1) isolate was determined. The genome was found to contain a large open reading frame which encoded a leader protein prior to the capsid protein region. This showed no sequence identity to other picornavirus leader regions and the sequence data suggested that it does not possess proteolytic activity. The 2A protease was small and showed considerable sequence identity to the aphthoviruses and to equine rhinovirus serotype 2. The 2A/2B junction possessed the typical cleavage site (NPG/P) exhibited by these viruses. The other proteins shared less than 40% sequence identity with equivalent proteins from other picornavirus genera. Phylogenetic analyses of the P1 and 3D sequences indicated that this virus forms a distinct branch of the family Picornaviridae. On the basis of results presented in this paper PEV-1 has been assigned to a new picornavirus genus. The phylogeny of the virus in relation to other picornaviruses is discussed.
A novel internal open reading frame product expressed from a polycistronic mRNA of porcine epidemic diarrhoea virus may not contribute to virus attenuation
Authors: Singh Mahender
Published: August 2015

Cell-culture-adapted (ca) porcine epidemic diarrhoea virus (PEDV) contains three internal open reading frames (I ORF) within the nucleocapsid protein gene and lacks the downstream counterpart of porcine transmissible gastroenteritis virus ORF7 or feline infectious peritonitis virus ORF6a. To confirm whether such features also exist in wild-type (wt) PEDV, the 3′ 1800 nucleotides of its genome were sequenced and were found to be identical to those of ca virus. The coding potential of I-1 ORF was ascertained by transient expression in Vero cells followed by immunofluorescence using antipeptide sera. The I-1 protein was synthesized as a 12 kDa non-phosphorylated PEDV-specific protein that was not present in detectable amounts in virions. However, a low copy number of I-1 in the virion would suggest it is a structural component. Nevertheless, identical nucleotide sequences and gene expression strategies of attenuated ca virus and its virulent parent, wt PEDV, demonstrate that the 3′ 1800 nucleotides or the genes and gene products encoded therein may not contribute to virus attenuation.
Properties of human foamy virus relevant to its development as a vector for gene therapy
Authors: Hill Claire L. , Bieniasz Paul D. , McClure Myra O.
Published: August 2015

The Spumaviridae (foamy viruses) are increasingly being considered as potential vectors for gene therapy, yet little has been documented of their basic cell biology. This study demonstrates that human foamy virus (HFV) has a broad tropism and that the receptor for HFV is expressed not only on many mammalian, but on avian and reptilian cells. Receptor interference assays using an envelope-expressing cell line and a vesicular stomatitis virus/HFV pseudotype virus demonstrate that the cellular receptor is common to all primate members of the genus. The majority of foamy virus particles assemble and remain sequestered intracellularly. A rapid and quantitative method of assaying foamy virus infectivity by reverse transcriptase activity facilitates the use of classical protocols to increase infectious virus titres in vitro to ⩾106 TCID/ml.
Measles virus-induced immunosuppression in cotton rats is associated with cell cycle retardation in uninfected lymphocytes
Authors: Niewiesk Stefan , Ohnimus Hartmut , Schnorr Jens-Jörg , Götzelmann Michaela , Schneider-Schaulies Sibylle , Jassoy Christian , ter Meulen Volker
Published: August 2015

Measles virus (MV)-induced immune suppression during acute measles often leads to secondary viral, bacterial and parasitic infections which severely complicate the course of disease. Previously, we have shown that cotton rats are a good animal model to study MV-induced immune suppression, where proliferation inhibition afterex vivostimulation of cotton rat spleen cells is induced by the viral glycoproteins (fusion and haemagglutinin proteins). We have now tested a variety of putative mechanisms of MV-induced immune suppression in this animal model. Proliferation inhibition is not due to fusion mediated by the MV glycoproteins and subsequent lysis of cells. Other putative mechanisms like classical anergy (unresponsiveness towards IL-2) or apoptosis do not seem to play a role in MV-induced immune suppression. In contrast, it was shown that spleen cells from infected animals preferentially accumulate in the G0/G1phase and progress more slowly through the cell cycle after mitogen stimulation in comparison to cells from non-infected animals. These data indicate a retardation of the cell cycle which is correlated with proliferation inhibition and might have severe consequences in mounting an effective immune response.
Dynamics of rabies virus quasispecies during serial passages in heterologous hosts
Authors: Kissi B. , Badrane H. , Audry L. , Lavenu A. , Tordo N. , Brahimi M. , Bourhy H.
Published: August 2015

To understand the mutations and genetic rearrangements that allow rabies virus infections of new hosts and adaptation in nature, the quasispecies structure of the nucleoprotein and glycoprotein genes as well as two noncoding sequences of a rabies virus genome were determined. Gene sequences were obtained from the brain and from the salivary glands of the original host, a naturally infected European fox, and after serial passages in mice, dogs, cats and cell culture. A relative genetic stasis of the consensus sequences confirmed previous results about the stability of rabies virus. At the quasispecies level, the mutation frequency varies, in the following order: glycoprotein region (21·9×10−4 mutations per bp), noncoding sequence nucleoprotein–phosphoprotein region (7·2–7·9×10−4 mutations per bp) and nucleoprotein gene region (2·9–3·7×10−4 mutations per bp). These frequencies varied according to the number, type of heterologous passages and the genomic region considered. The shape of the quasispecies structure was dramatically modified by passages in mice, in which the mutation frequencies increased by 12–31×10−4 mutations per bp, depending on the region considered. Nonsynonymous mutations were preponderant particularly in the glycoprotein gene, stressing the importance of positive selection in the maintenance and fixation of substitutions. Two mechanisms of genomic evolution of the rabies virus quasispecies, while adapting to environmental changes, have been identified: a limited accumulation of mutations with no replacement of the original master sequence and a less frequent but rapid selective overgrowth of favoured variants.
Characteristics of a new birnavirus associated with a warm-water fish cell line
Authors: Riji John K. , Richards R. H.
Published: August 2015

A warm-water fish cell line developed from blotched snakehead caudal peduncle (BSN) was found to have persistent birnavirus infection. Purified virus particles were of icosahedral shape and had 57±1·6 nm diameter. The BSN virus was resistant to 5-iodo-2′-deoxyuridine and induced yellowish-green cytoplasmic inclusions when stained with acridine orange. The virus was resistant to chloroform, acid and alkaline pH and heat treatment at 56 °C for 2 h. Purified virions had a buoyant density of 1·33 g/ml in CsCl and contained two genomic segments with molecular masses of 2·56×106 and 2·00×106 Da and four structural polypeptides of 112 (polyprotein, PP), 91 (VP1), 44 (VP2) and 37 (VP3) kDa. Reciprocal β cross-neutralization tests incorporating four classical strains of infectious pancreatic necrosis virus (IPNV) (WB, Sp, Ab and TV-1) and the BSN virus established the complete serological distinctness of the virus from IPNV. Considering the uniqueness of the virus, the name blotched snakehead virus is proposed for this agent.
Sequence analysis and in vitro expression of genes 6 and 11 of an ovine group B rotavirus isolate, KB63: evidence for a non-defective, C-terminally truncated NSP1 and a phosphorylated NSP5
Authors: Shen S. , McKee T. A. , Wang Z. D. , Desselberger U. , Liu D. X.
Published: August 2015

An ovine group B rotavirus (GBR) isolate, KB63, was isolated from faeces of a young goat with diarrhoea in Xinjiang, People’s Republic of China. Sequence determination and comparison of genes 6 and 11 with the corresponding sequences of GBR strains ADRV and IDIR showed that they were the cognate genes encoding NSP1 and NSP5, respectively. While the overall identities of nucleotide sequences between these two genes and the corresponding genes of strains ADRV and IDIR were in the range 52·6–57·2%, the identities of deduced amino acid sequences were only 34·9–46·3%. These results demonstrate that the substantial diversity of NSP1 observed among group A rotaviruses (GAR) also exists within GBRs and that a high degree of diversity also exists among NSP5 of GBRs, in contrast to GAR NSP5. The NSP1 gene of KB63 contains three ORFs, whereas the NSP1 genes of other GBR strains contain only two. ORFs 2 and 3 of the KB63 gene may be derived from a single ORF corresponding to ORF2 of other GBR strains by the usage of a stop codon created by an upstream single base deletion and single point mutations. In vitro expression studies showed that ORFs 1 and 2, but not 3, of gene 6 can be translated, suggesting that ORF2 may encode a C-terminally truncated, potentially functional product. It may play a role, together with the product of ORF1, in virus replication, as the virus can be passaged further in kids. Similarly, gene 11 can be translated in vitro. Like its counterpart in GARs, the protein encoded by gene 11 was shown to be phosphorylated in vitro.
Improved detection systems for TT virus reveal high prevalence in humans, non-human primates and farm animals
Authors: Leary Thomas P. , Erker James C. , Chalmers Michelle L. , Desai Suresh M. , Mushahwar Isa K.
Published: August 2015

TT virus is a newly described agent infecting humans. Initially isolated from a patient (initials T.T.) with unexplained hepatitis, the virus has since been found in both normal and diseased individuals. In the present study, we utilized genomic-length sequences from distinct genotypes of TT virus to design PCR-based assays using conserved oligonucleotide primers from three independent regions of the virus genome. Each of the three assays was found to be superior to the PCR-based assays previously published. The most sensitive of the new assays was utilized to demonstrate the prevalence of TT virus to be at least 34·1% in volunteer blood donors, 39·6% in commercial blood donors, 59·6% in non-A–GB hepatitis cases, 81·7% in injectable drug users and 95·9% in haemophiliacs. In an attempt to identify a possible source of human infection, we found TT virus sequences to be present in 19% of chickens, 20% of pigs, 25% of cows and 30% of sheep. Sequence determination and phylogenetic analyses demonstrated that isolates from farm animals were not genetically distinct from those found in humans. This study clearly demonstrates that previously reported PCR assays dramatically underestimate the true prevalence of TT virus within the human population. Due to the high rate of infection in both blood donors and those with non-A–GB hepatitis, these results question the causal role of TT virus in cases of unexplained hepatitis. Further, it is possible that domesticated farm animals serve as a source of human infection.
Antigenic domain 1 of human cytomegalovirus glycoprotein B induces a multitude of different antibodies which, when combined, results in incomplete virus neutralization
Authors: Speckner Andrea , Glykofrydes Diana , Ohlin Mats , Mach Michael
Published: August 2015

Glycoprotein B (gB, gpUL55) is the major antigen for the induction of neutralizing antibodies against human cytomegalovirus (HCMV), making it an attractive molecule for active and passive immunoprophylaxis. The region between aa 552 and 635 of HCMV gB (termed AD-1) has been identified as the immunodominant target for the humoral immune response following natural infection. AD-1 represents a complex domain which requires a minimal continuous sequence of more than 70 aa for antibody binding. Neutralizing as well as non-neutralizing antibodies can bind to AD-1 in a competitive fashion. The fine specificity of AD-1-binding monoclonal antibodies (MAbs) and affinity-purified human polyclonal antibodies was analysed by using recombinant proteins containing single amino acid substitutions spanning the entire AD-1 domain. Our results revealed that all MAbs had individual patterns of binding to the mutant proteins indicating the presence of a considerable number of distinct antibody-binding sites on AD-1. The neutralization capacity of antibodies could not be predicted from their binding pattern to AD-1 mutant proteins. Polyclonal human antibodies purified from different convalescent sera showed identical binding patterns to the mutant proteins suggesting that the combined antibody specificities present in human sera are comparable between individuals. Neutralization capacities of polyclonal human AD-1 antibodies did not exceed 50% indicating that, during natural infection, a considerable proportion of non-neutralizing antibodies are induced and thus might provide an effective mechanism to evade complete virus neutralization.
A naturally occurring deleted form of RNA 2 of Potato mop-top virus
Authors: Torrance Lesley , Cowan Graham H. , Sokmen Miray Arli , Reavy Brian
Published: August 2015

A spontaneous deletion in RNA 2 of Potato mop-top virus (PMTV) was identified by RT–PCR. The deletion occurred reproducibly during manual passage of two isolates of PMTV and during fungal inoculation of plants with viruliferous soil. The borders of the deletion were conserved in all instances and sequence analyses showed that a contiguous segment of 2113 nucleotides was deleted internally from the genomic RNA 2, leaving the 5′- and 3′-terminal sequences. RT–PCR experiments also showed that the deletion was present in preparations of PMTV particles.
Role of the 3′ untranslated region of baculovirus p10 mRNA in high-level expression of foreign genes
Authors: van Oers Monique M. , Vlak Just M. , Voorma Harry O. , Thomas Adri A. M.
Published: August 2015

The p10 gene of Autographa californica nucleopolyhedrovirus has two putative AATAAA polyadenylation signals. The downstream signal is used predominantly, as was determined by analysing 3′ cDNA ends. This downstream motif is followed by a GT-rich sequence, known to be important for efficient polyadenylation in mammalian systems. To analyse the importance of polyadenylation for p10 gene expression, recombinant viruses with altered 3′ untranslated regions (UTRs) were tested using chloramphenicol acetyltransferase (CAT) as a reporter. Surprisingly, after inactivation of the downstream AATAAA motif, CAT expression remained at the same high level as observed with a wild-type 3′ UTR. Polyadenylation occurred 24–28 nucleotides further downstream, probably due to an ATTAAA sequence motif. Replacing the p10 3′ UTR with the SV40 early terminator sequence as part of an hsp70–lacZ–SV40 gene cassette, which is commonly used in baculovirus expression vectors, resulted in a reduction in reporter gene expression. Polyadenylation occurred far more efficiently in the original p10 3′ UTR than in the SV40 terminator sequence, as was shown by testing the SV40 terminator separately. These results indicate that in order to obtain high levels of foreign gene expression, vectors that provide a wild-type p10 3′ UTR are to be preferred over those containing the hsp70–lacZ–SV40 gene cassette. Comparison of the p10 genes of various baculoviruses showed the presence of at least one AATAAA or ATTAAA motif in combination with a GT-rich sequence in the 3′ UTR, suggesting an evolutionary conservation of these two elements, thereby maintaining the high level of p10 gene expression.
Behavioural changes in the rat following infection with varicella-zoster virus
Authors: Fleetwood-Walker S. M. , Quinn J. P. , Wallace C. , Blackburn-Munro G. , Kelly B. G. , Fiskerstrand C. E. , Nash A. A. , Dalziel R. G.
Published: August 2015

Following the establishment of a chronic varicella-zoster virus infection in the rat, behavioural allodynia and hyperalgesia were observed in the injected, but not the contralateral hind limb up to 33 days post-infection. This model may prove useful in investigating mechanisms involved in the establishment of post-herpetic neuralgia.
Transcription-modulatory activities of differentially spliced cDNAs encoding the E2 protein of human papillomavirus type 16
Authors: Alloul Nathalie , Sherman Levana
Published: August 2015

Human papillomavirus (HPV) type 16 expresses a variety of alternatively spliced polycistronic mRNAs encoding the E2 transcription-regulatory protein. These mRNAs initiate at the p97 promoter and contain the 880/2708 (a-type), 880/2581 (a′-type) and 226/2708 (d-type) splice sites upstream from the E2 open reading frame (ORF). Recent studies investigating the translational capacities of partial cDNAs representing three of these mRNAs indicated their abilities to function in E2 protein translation, although at different efficiencies. In the present study, the transcription-regulatory activities of the E2 cDNAs towards the virus long control region (LCR) have been examined. LCR regulation was evaluated in transient transfection assays by using the chloramphenicol acetyltransferase reporter gene linked to the HPV-16 LCR. Transfections were carried out into fibroblast (Cf2Th) and epithelial (C33A) cell lines. It is shown that all three E2 cDNAs transrepressed the virus LCR in a dose-dependent manner. Transrepression was mainly dependent on the function of the E2 ORF and was abolished or markedly reduced by premature termination or truncation of the E2 ORF. Transrepression activities exhibited by the various E2 cDNAs correlated with the previously defined efficiencies of E2 protein translation from the respective templates. The truncated E2 cDNAs exhibited variable low regulatory activities that correlated with the activities of the 5′ ORFs contained in each cDNA. The E6I and E1C ORFs transactivated the virus LCR whereas the E6IV cDNA transrepressed LCR activity. Thus, the 5′ ORFs contribute in different manners to the overall activities of the polycistronic cDNAs.
In vitro infection of cells of the monocytic/macrophage lineage with bovine leukaemia virus
Authors: Doménech Ana , Goyache Joaquín , Llames Louie , Jesús Payá M. , Suárez Guillermo , Gómez-Lucía Esperanza
Published: August 2015

The oncogenic retrovirus bovine leukaemia virus (BLV) primarily infects B cells. Most infected animals remain asymptomatic for long periods of time before an increase in circulating B cells or localized tumours can be observed. This long clinical latency period may be explained by cells of the monocyte/macrophage lineage (M/M) becoming infected and acting as a reservoir for the virus, as shown for other retroviruses (human immunodeficiency virus-1, feline immunodeficiency virus). M/M cells in different stages of differentiation (HL-60, THP-1, U-937, J774, BGM, PM2, primary macrophages of sheep and cows) were cultured with BLV produced by permanently infected donor cells (FLKBLV and BLV-bat2). Donor cells were inhibited from multiplying by either irradiation or treatment with mitomycin C. In other experiments, supernatant from donor cells containing virus was used. In co-culture with the donor cells, the less differentiated monocytic cells showed severe cellular changes such as differentiation, vacuolization, cell lysis and membrane blebbing; apoptosis was a frequent phenomenon. Budding and extracellular viruses were also observed. The more differentiated macrophage cells, although they showed less signs of infection by microscopy, had a complete BLV protein profile, as seen by Western blotting; bands corresponding to p24CA (Gag) and its precursors were clearly seen. In addition, gp51SU was identified by syncytia formation assays. It is concluded that M/M cells may be infected by BLV, the consequences of the infection differing according to the type of cell.
Several protein regions contribute to determine the nuclear and cytoplasmic localization of the influenza A virus nucleoprotein
Authors: Bullido Rosario , Gómez-Puertas Paulino , Albo Carmen , Portela Agustín
Published: August 2015

A systematic analysis was carried out to identify the amino acid signals that regulate the nucleo-cytoplasmic transport of the influenza A virus nucleoprotein (NP). The analysis involved determining the intracellular localization of eight deleted recombinant NP proteins and 14 chimeric proteins containing the green fluorescent protein fused to different NP fragments. In addition, the subcellular distribution of NP derivatives that contained specific substitutions at serine-3, which is the major phosphorylation site of the A/Victoria/3/75 NP, were analysed. From the results obtained, it is concluded that the NP contains three signals involved in nuclear accumulation and two regions that cause cytoplasmic accumulation of the fusion proteins. One of the karyophilic signals was located at the N terminus of the protein, and the data obtained suggest that the functionality of this signal can be modified by phosphorylation at serine-3. These findings are discussed in the context of the transport of influenza virus ribonucleoprotein complexes into and out of the nucleus.
Growth of infectious salmon anaemia virus in CHSE-214 cells and evidence for phenotypic differences between virus strains
Authors: Kibenge Frederick S. B. , Lyaku Japhet R. , Rainnie Don , Hammell K. Larry
Published: August 2015

Infectious salmon anaemia virus (ISAV) is a new orthomyxovirus-like virus. Thirteen isolates of ISAV (11 from Canada, one from Norway and one from Scotland) were studied for their replication in the CHSE-214 cell line compared with that in the SHK-1 cell line. All isolates replicated in SHK-1 cells, producing CPE between 3 and 12 days post-inoculation (p.i.). Six Canadian isolates also replicated in CHSE-214 cells, with production of CPE between 4 and 17 days p.i. Analysis of a one-step growth curve of ISAV in CHSE-214 cells showed that progeny virions remained predominantly cell-associated, accounting for the focalized nature of the CPE in the cell monolayer. One isolate (HKS 36) replicated in CHSE-214 cells, as shown by positive RT–PCR results of blind passages, but was non-cytopathic. All of the isolates were analysed for genetic heterogeneity by RT–PCR and RFLP with EcoRI and XhoI in a fraction of genome segment 2. The Canadian isolates showed a different RFLP profile to those of isolates Glesvaer/2/90 from Norway and 390/98 from Scotland. Structural proteins of four isolates, ‘Back Bay 98’, RPC/NB-877, RPC/NB-049 and Glesvaer/2/90, were examined further by SDS–PAGE. All viruses showed four major polypeptides, designated here as VP1–VP4, in Coomassie blue-stained gels. In isolates Glesvaer/2/90 and RPC/NB-877, these viral proteins had estimated molecular masses of 74, 53, 46 and 26·5 kDa, respectively. Viral proteins in isolates ‘Back Bay 98’ and RPC/NB-049 were of similar sizes, except that VP3 was 43 kDa. Taken together, these results show that there are phenotypic differences among strains of ISAV.
Subcellular distribution analysis of the cucumber mosaic virus 2b protein
Authors: Mayers Carl N. , Palukaitis Peter , Carr John P.
Published: August 2015

The cucumoviral 2b protein is a viral counterdefence factor that interferes with the establishment of virus-induced gene silencing in plants. Synthetic peptides were used to generate an antibody to the 2b protein encoded by the Fny strain of cucumber mosaic virus (Fny-CMV). This polyclonal antibody was able to recognize the Fny-CMV 2b protein in a 10000 g pellet fraction of infected tobacco. No protein of equivalent size was detected in mock-inoculated or tobacco mosaic virus-infected samples. This represents the first demonstration of 2b protein expression by a subgroup I strain of CMV. Subcellular fractionation experiments on CMV-infected tobacco leaf tissue showed that the Fny-CMV 2b protein accumulated within a fraction that sedimented at forces of less than 5000 g and that the 2b protein was solubilized only by treatment with urea or SDS. These results suggested that the 2b protein associates either with the nucleus or cytoskeleton of the host cell. Further analysis showed that the 2b protein was enriched in a fraction that sedimented through a 2·2 M sucrose cushion. This fraction was also enriched in histones, suggesting that the CMV 2b protein associates preferentially with the host cell nucleus.
Virus-like particles assemble in plants and bacteria expressing the coat protein gene of Indian peanut clump virus
Authors: Bragard C. , Duncan G. H. , Wesley S. V. , Naidu R. A. , Mayo M. A.
Published: August 2015

cDNA copies of the coat protein (CP) gene of Indian peanut clump virus (IPCV)-H were introduced into cells of Nicotiana benthamiana or Escherichia coli by transformation with vectors based on pROKII or pET respectively. In both plant and bacterial cells, IPCV CP was expressed and assembled to form virus-like particles (VLP). In plant extracts, the smallest preponderant particle length was about 50 nm. Other abundant lengths were about 85 and about 120 nm. The commonest VLP length in bacterial extracts was about 30 nm. Many of the longer VLP appeared to comprise aggregates of shorter particles. The lengths of the supposed ‘monomer’ VLP corresponded approximately to those expected for encapsidated CP gene transcript RNA. Immunocapture RT–PCR, using primers designed to amplify the CP gene, confirmed that the VLP contained RNA encoding IPCV-H CP. The results show that encapsidation does not require the presence of the 5′-terminal untranslated sequence of the virus RNA and suggest that if there is an ‘origin of assembly’ motif or sequence, it lies within the CP gene. When transgenic plants expressing IPCV-H CP were inoculated with IPCV-L, a strain that is serologically distinct from IPCV-H, the virus particles that accumulated contained both types of CP.
Expression of Epstein–Barr virus lytic gene BRLF1 in nasopharyngeal carcinoma: potential use in diagnosis
Authors: Feng Ping , Ren Ee Chee , Liu Dingxiang , Chan Soh Ha , Hu Huaizhong
Published: August 2015

Tumour cells of undifferentiated nasopharyngeal carcinoma (NPC) consistently harbour Epstein–Barr virus (EBV) genes. Expression of mRNA transcripts associated with EBV latency has been demonstrated in such cells. However, expression of EBV lytic genes has not been well elucidated, although various lines of evidence have suggested that there is EBV replication in NPC tumour cells. We have studied mRNA expression of representative EBV lytic genes by RT–PCR in nasopharynx biopsies obtained from NPC and control individuals. In both NPC and control biopsies, EBV lytic genes BZLF1, BALF2 and BCLF1 were detected readily. However, BRLF1 was detected in NPC biopsies only. The BRLF1 gene was then cloned and expressed in vitro, and the protein product, Rta, was used as an antigen to detect specific antibodies by immunoprecipitation in plasma samples obtained from NPC patients and healthy controls. IgG antibodies directed against Rta were detected in 44 of 53 NPC plasma samples (83·0%), but only in 1 of 53 control samples (1·9%). Furthermore, the antibody binding regions were found in the C-terminal two-thirds of Rta. This serological result confirms indirectly that BRLF1 is specifically expressed in NPC tumour cells. Rta might play an important role in NPC pathogenesis, considering its multiple functions in EBV replication and cell cycles. Moreover, the detection of IgG antibodies directed against Rta could be developed into a diagnostic parameter for NPC.