bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification and ultrastructural characterization of a novel virus from fish
Authors: Granzow Harald , Weiland Frank , Fichtner Dieter , Schütze Heike , Karger Axel , Mundt Egbert , Dresenkamp Barbara , Martin Petra , Mettenleiter Thomas C.
Published: August 2015

During routine investigations on fish, a virus (isolate DF 24/00) with novel morphological features and hitherto undescribed morphogenesis was isolated from a white bream (Blicca bjoerkna L.; Teleostei, order Cypriniformes). Cell-free virions consist of a rod-shaped nucleocapsid (120–150×19–22 nm) similar to that seen in baculoviruses. The virion has a bacilliform shape (170–200×75–88 nm) reminiscent of rhabdoviruses with an envelope containing coronavirus-like spikes (20–25 nm). DF 24/00 replicated well in various fish cell lines. Inhibitor studies with 5-iodo-2′-deoxyuridine indicated that the viral genome consists of RNA and chloroform sensitivity correlated with ultrastructural demonstration of enveloped virions. The buoyant density of the virus determined in sucrose was 1·17–1·19 g/ml. Preliminary biochemical characterization revealed the presence of six antigenic glycoproteins, three of which contain sugars with concanavalin-A specificity. Ultrastructurally, morphogenesis of virus progeny was detected only in the cytoplasm. Nucleocapsids were observed to bud through membranes of the endoplasmic reticulum and/or Golgi apparatus into dilated vesicles. Egress of mature virions occurs primarily by exocytosis and, only very rarely, by budding directly at the plasma membrane. Morphologically similar viruses had previously been isolated from grass carp (Ctenopharyngodon idella), blue crab (Callinectis sapidus), European shore crab (Carcinus maenas) and shrimp (Penaeus monodon). To date, none of them has been classified. In summary, the first characterization of a new virus that might represent a member of a novel virus family that has morphological features resembling those found in rhabdo-, corona- and baculoviruses is presented.
Peroral vaccination with a temperature-sensitive mutant of parainfluenza virus type 1 protects mice against respiratory challenge infection
Authors: Liu Beixing , Mori Isamu , Hossain Md. Jaber , Dong Li , Kimura Yoshinobu
Published: August 2015

Peroral vaccination for preventing respiratory infectious diseases was investigated in a murine model using a temperature-sensitive (ts) mutant of parainfluenza virus type 1. Thetsmutant virus administered perorally in drinking water neither multiplied nor caused lesions in the respiratory tract or the central nervous system. However,tsvirus antigen-positive cells appeared in oropharyngeal lymphoid tissues. This type of antigenic stimulation was capable of inducing both humoral and cellular immune responses, together with an augmentation of interferon production and natural killer cell activity, making it possible to protect the mice against challenge infection with a virulent wild-type virus. These results suggest that the oral cavity, a constituent member of the common mucosal immune system, is a candidate organ applicable as a vaccine route against virus respiratory diseases.
The effect of mosquito passage on the La Crosse virus genotype
Authors: Borucki Monica K. , Kempf Brian J. , Blair Carol D. , Beaty Barry J.
Published: August 2015

The genetic consequences of passing three different strains of La Crosse (LAC) virus orally and transovarially in Aedes triseriatus mosquitoes were examined. Two of the LAC strains (WT LAC and LAC ORI) had been passaged numerous times in cell culture; the third strain (SM1-78) had been passaged only once in suckling mice. Genetic changes were monitored in three regions of the LAC genome after oral infection and dissemination in the mosquito, and transovarial transmission (TOT) of the virus to progeny. Sequence analyses were used to characterize the genetic changes occurring in regions of G1, G2 and N open reading frames (ORFs) during passage. Only one mutation was detected in the G1 ORF of SM1-78 virus after mosquito passage; however, numerous nucleotide and amino acid substitutions were detected in the G1 ORF of WT LAC and LAC ORI (cell culture-adapted viruses). In contrast to G1, the N and G2 ORF sequences examined were stable. Mutations introduced into viral genomes during replication in parental mosquitoes were expressed in progeny mosquitoes following TOT. Genetic diversity of virus populations from a single mosquito was examined by single-strand conformation polymorphisms analysis of the variable region of glycoprotein G1. LAC virus RNA genotype diversity was greatest in virus that infected and replicated in the midgut, and declined as virus disseminated from the midgut and infected ovaries and salivary glands.
Genetic analysis of hepatitis A virus strains recovered from the environment and from patients with acute hepatitis
Authors: Pina Sonia , Buti Maria , Jardí Rosend , Clemente-Casares Pilar , Jofre Joan , Girones Rosina
Published: August 2015

The molecular epidemiology of hepatitis A virus (HAV) was studied by analysing HAV strains recovered from environmental water samples over a 7 year period and strains recovered from patients with acute hepatitis over a 5 year period. A total of 54 samples of raw domestic sewage and 66 samples of river water were collected. HAV particles were concentrated and detected by nested RT–PCR. HAV infection in patients with acute hepatitis was serologically diagnosed in 26 of 74 serum samples, which were also analysed by nested RT–PCR. HAV RNA was detected in 57·4% of sewage samples, 39·2% of Llobregat river water samples, 20% of Ter river water samples and 61·6% of serum samples. The HAV genomes detected were characterized further by directly sequencing a region of the 5′ non-translated region, the VP1/2A junction region and, in some samples, the 2B region. Results showed a 95% prevalence of genotype I, with nearly 50% being either subgenotype IA or subgenotype IB. Various strains were found simultaneously in both environmental and clinical samples. These strains were closely related to those described in distant geographical areas. Genotype IIIA was also found in 5% of sewage samples and in 12·5% of serum samples. Strains belonging to a common endemic genotype were not identified. The abundance of HAV in the environment produces a situation of sanitary risk, especially considering the low prevalence of antibodies in the young population.
Molecular epidemiology of simian T-lymphotropic virus (STLV) in wild-caught monkeys and apes from Cameroon: a new STLV-1, related to human T-lymphotropic virus subtype F, in a Cercocebus agilis
Authors: Nerrienet Eric , Meertens Laurent , Kfutwah Anfumbom , Foupouapouognigni Yacouba , Gessain Antoine
Published: August 2015

A serological survey for human T-lymphotropic virus (HTLV)/simian T-lymphotropic virus (STLV) antibodies was performed in 102 wild-caught monkeys and apes from 15 (sub)species originating from Cameroon. Two animals (aMandrillus sphinxand aCercocebus agilis) exhibited a complete HTLV-1 seroreactivity pattern while two others lacked either the p24 (aMandrillus sphinx) or the MTA-1/gp46 bands (aPan troglodytes). Sequence comparison and phylogenetic analyses, using a 522 bpenvgene fragment and the complete LTR, indicated that the two mandrill STLV strains belonged to the HTLV/STLV subtype D clade while the chimpanzee strain clustered in the HTLV/STLV subtype B clade. TheCercocebus agilisSTLV strain, the first one found in this species, was closely related to the two HTLV/STLV subtype F strains. Such data indicate that the African biodiversity of STLV-1 in the wild is far from being known and reinforces the hypothesis of interspecies transmission of STLV-1 from monkeys and apes to humans leading to the present day distribution of HTLV-1 in African inhabitants.
A novel virus capture assay reveals a differential acquisition of host HLA-DR by clinical isolates of human immunodeficiency virus type 1 expanded in primary human cells depending on the nature of producing cells and the donor source
Authors: Cantin Réjean , Martin Geneviève , Tremblay Michel J.
Published: August 2015

Previous findings indicated that HLA-DR is probably one of the most abundant cellular constituents incorporated within the human immunodeficiency virus type 1 (HIV-1) envelope. Given that the life-cycle of HIV-1 has been reported to be modulated by virion-bound host HLA-DR, an improved version of a virus capture technique was developed to assess the degree of HLA-DR incorporation in several clinical isolates of HIV-1 derived from primary human peripheral blood mononuclear cells (PBMCs) and monocyte-derived macrophages (MDM). Analysis of virus stocks purified from PBMCs and MDM indicated that primary isolates of HIV-1 bearing distinct tropism (i.e. T-, macrophage-, and dual-tropic) all incorporate host cell membrane HLA-DR protein. The amount of incorporated HLA-DR varies among the primary HIV-1 isolates tested. Propagation of some clinical HIV-1 isolates in either autologous PBMCs or MDM resulted in differential incorporation of virion-bound cellular HLA-DR depending on the nature of the virus producer cells. Differences in the degree of HLA-DR incorporation were also noticed when macrophage-tropic isolates of HIV-1 were produced in MDM from different donors. Altogether these data show that the efficiency of HLA-DR incorporation into the envelope of primary isolates of HIV-1 is a multifactorial phenomenon since it is affected by the virus isolate itself, the nature of host cells (i.e. PBMCs or MDM) and the donor source.
Cis-acting sequences may contribute to size variation in the surface glycoprotein of bovine immunodeficiency virus
Authors: Li Yuxing , Carpenter Susan
Published: August 2015

Genetic recombination is an important mechanism of retrovirus variation and diversity. Size variation in the surface (SU) glycoprotein, characterized by duplication and insertion, has been observed duringin vivoinfection with several lentiviruses, including bovine immunodeficiency virus (BIV), equine infectious anaemia virus (EIAV) and human immunodeficiency virus type 1. These duplication/insertion events are thought to occur through a mechanism of template switching/strand transfer during reverse transcription. Studies of RNA recombination in a number of virus systems indicate thatcis-acting sequences can modulate the frequency of template switching/strand transfer. The size variable region of EIAV and BIV SU glycoproteins was examined and an AU-rich region and regions of nucleotide sequence identity that may facilitate template switching/strand transfer were identified. Anin vitrostrand transfer assay using donor and acceptor templates derived from the size variable region in BIVenvdetected both precise and imprecise strand transfer products, in addition to full-length products. Sequence analysis of clones obtained from imprecise strand transfer products showed that 87·5% had crossover sites within 10 nt of the crossover site observedin vivo. Mutations in the donor template which altered either the AU-rich region or nucleotide sequence identity dramatically decreased the frequency of imprecise strand transfer. Together, these results suggest thatcis-acting elements can modulate non-homologous recombination events during reverse transcription and may contribute to the genetic and biological diversity of lentivirusesin vivo.
Circulating tumour necrosis factor-α and interferon-γ are detectable during acute and convalescent parvovirus B19 infection and are associated with prolonged and chronic fatigue
Authors: Kerr Jonathan R. , Barah Faraj , Mattey Derek L. , Laing Ian , Hopkins Stephen J. , Hutchinson Ian V. , Tyrrell David A. J.
Published: August 2015

To investigate whether cytokine responses may have a bearing on the symptoms and outcome of parvovirus B19 infection, circulating cytokines were measured during acute infection (n=51), follow-up of acute infection (n=39) and in normal healthy controls (n=50). At acute B19 virus infection (serum anti-B19 IgM-positive), patients ranged in age from 4 to 54 years, with a mean age of 28·2 years. The male:female ratio was 1:4·1 and symptoms were rash (n=15), arthralgia (n=31), fatigue (n=8), lymphadenopathy (n=4), foetal hydrops (n=3), transient aplastic crisis (n=2), neutropenia (n=2), myelodysplasia (n=1), thrombocytopenia (n=1) and pancytopenia (n=1). Of these patients, 39 were contacted after a follow-up period of 2–37 months (mean of 22·5 months). In comparison with normal controls, detectable IL-6 was associated with acute B19 virus infection (26%;P=0·0003), but not with follow-up (6%;P=0·16). Detection of interferon (IFN)-γ was associated with acute B19 virus infection (67%;P<0·0001) and follow-up (67%;P<0·0001). Detection of tumour necrosis factor (TNF)-α was associated with acute B19 virus infection (49%;P<0·0001) and follow-up (56%;P<0·0001). IL-1β was detected in acute infection (20%), but not at follow-up. At acute B19 virus infection, detection of serum/plasma IL-6 was associated with rheumatoid factor (P=0·038) and IFN-γ (⩾7 pg/ml) was associated with fatigue in those patients of ⩾15 years of age (P=0·022). At follow-up, fatigue was associated with IFN-γ (⩾7 pg/ml) and/or TNF-α (⩾40 pg/ml) (P=0·0275). Prolonged upregulation of serum IFN-γ and TNF-α appears to represent a consistent host response to symptomatic B19 virus infection.
Genetic polymorphism of human herpesvirus-7 among human populations
Authors: Franti Michael , Gessain Antoine , Darlu Pierre , Gautheret-Dejean Agnès , Kosuge Haruhiko , Mauclère Philippe , Aubin Jean-Thierry , Gurtsevitch Vladimir , Yamanishi Koichi , Agut Henri
Published: August 2015

The analysis of three human herpesvirus-7 (HHV-7) genes encoding phosphoprotein p100, glycoprotein B and major capsid protein respectively had previously shown the existence of distinct gene alleles, leading to the concept of HHV-7 variants. We have analysed the distribution of HHV-7 variants among 297 distinct subjects who belonged to different human populations from Africa, Asia, Europe and America. Two variants, designated Co1 and Co2, were found in 52% and 20% of studied subjects. Ten other variants, designated Co3–Co12, were less frequent and classified into two groups related to Co1 and Co2 respectively. While the former group was ubiquitous and the most frequent in Africa and Asia, the latter one was predominantly found in European and Mongol populations. Despite the high stability of the HHV-7 genome, a few nucleotide substitutions at precise positions define distinct variants which, to some extent, behave as markers of human populations.
Effects of inactivation of the coat protein and movement genes of Tomato bushy stunt virus on early accumulation of genomic and subgenomic RNAs
Authors: Qiu Wenping , Scholthof Herman B.
Published: August 2015

Accumulation of RNA of Tomato bushy stunt virus (TBSV) was examined within the first few hours after infection of Nicotiana benthamiana protoplasts to determine the influence of the coat protein (CP), the movement-associated proteins P22 and P19 and RNA sequences at very early stages of replication. The results showed that P19 had no effect on early RNA replication, whereas the absence of CP and/or P22 expression delayed RNA accumulation only marginally. Removal of CP-coding sequences had no added negative effects, but when the deletion extended into the downstream p22 gene, it not only eliminated synthesis of subgenomic RNA2 but also delayed accumulation of genomic RNA by 10 h. At times beyond 20 h post-transfection, RNA accumulated to normal high levels for all mutants. This illustrates that TBSV RNA sequences that have negligible impact on overall RNA levels observed late in infection can actually have pronounced effects at very early stages.
Determination of the substrate specificity of turnip mosaic virus NIa protease using a genetic method
Authors: Kang Hara , Lee Yong Jae , Goo Jae Hwan , Park Woo Jin
Published: August 2015

The RNA genome of turnip mosaic potyvirus (TuMV) encodes a large polyprotein that is processed to mature proteins by virus-encoded proteases. The TuMV NIa protease is responsible for the cleavage of the polyprotein at seven different locations. These cleavage sites are defined by a conserved sequence motif Val-Xaa-His-Gln↓, with the scissile bond located after Gln. To determine the substrate specificity of the NIa protease, amino acid sequences cleaved by the NIa protease were obtained from randomized sequence libraries using a screening method referred to as GASP (geneticassay forsite-specificproteolysis). Based on statistical analysis of the obtained sequences, a consensus substrate sequence was deduced: Yaa-Val-Arg-His-Gln↓Ser, with Yaa being an aliphatic amino acid and the scissile bond being located between Gln and Ser. This result is consistent with the conserved cleavage sequence motif, and should provide insight into the molecular activity of the NIa protease.
P34.8 (GP37) is not essential for baculovirus replication
Authors: Cheng Xiao-Wen , Krell Peter J. , Arif Basil M.
Published: August 2015

Previous reports have indicated that p34.8 (gp37) may be essential for the replication of Autographa californica nucleopolyhedrovirus (AcMNPV) because no virus with inactivated p34.8 was isolated. We have ascertained the requirement for this gene by attempting to inactivate it with a large insertion [the gene encoding GFP (green fluorescent protein)] or by deleting all the conserved domains from the open reading frame (ORF). The gene encoding GFP was inserted into the NotI site of the p34.8 ORF and a viral plaque containing the insertion was propagated in SF-21 cells. Similarly, 531 bp (NotI–XbaI) containing all conserved domains were deleted from the ORF. All mutants were authenticated by PCR amplification, restriction endonuclease analysis, DNA sequencing, and Southern and Northern blot analysis. It was found that inactivation of p34.8 of AcUW1-LacZ (AcMNPV containing a lacZ gene in the p10 locus) had no effect on the biological property of virus, such as virulence and kinetics. These two independent methods showed that p34.8 is not essential for replication and that this locus could provide another site for the engineering of baculoviruses.
The tegument protein ppUL25 of human cytomegalovirus (CMV) is a major target antigen for the anti-CMV antibody response
Authors: Lazzarotto Tiziana , Varani Stefania , Gabrielli Liliana , Pignatelli Sara , Landini Maria Paola
Published: August 2015

A viral protein of approximately 82 kDa is the only structural protein of human cytomegalovirus (CMV) that is strongly immunogenic during natural infection and the corresponding gene of which is still unknown. In this work, strong evidence is presented that this protein is the product of UL25.
Characterization of hepatitis B virus genotypes among Yucpa Indians in Venezuela
Authors: Nakano Tatsunori , Lu Ling , Hu Xiaolei , Mizokami Masashi , Orito Etsuro , Shapiro Craig N. , Hadler Stephan C. , Robertson Betty H.
Published: August 2015

The complete genome sequences of hepatitis B virus (HBV) from 12 HBV-infected Yucpa Indians of Venezuela, a group with highly endemic HBV, were amplified and sequenced. The 12 isolates were closely related to each other, with 98·6–100% nucleotide identity. A phylogenetic tree based on the complete genome indicated clearly that they were genotype F. Three individuals had evidence of infection with two different HBV deletion mutants. In two individuals, a three amino acid deletion was identified just prior to the ‘a’ determinant loop of the S region. A third individual was infected with virus that contained a complete core reading frame and a population that contained a deletion in the middle of the core region. These results indicate that genotype F HBV is present in the Venezuelan Yucpa Amerindians and the complete genome sequence allowed the identification of two unique deletion mutants in a limited set of samples.
Genetic analysis of full-length genomes and subgenomic sequences of TT virus-like mini virus human isolates
Authors: Biagini Philippe , Gallian Pierre , Attoui Houssam , Touinssi Mhammed , Cantaloube Jean-François , de Micco Philippe , de Lamballerie Xavier
Published: August 2015

The phylogenetic relationship between the complete genomic sequences of ten Japanese and one French isolate of TT virus-like mini virus (TLMV) was investigated. Analysis of the variability of the nucleotide sequences and the detection of signature patterns for overlapping genes suggested that ORFs 1 and 2 are probably functional. However, this was not the case for a putative third ORF, ORF3. Throughout the viral genome, several nucleotide or amino acid motifs that are conserved in circoviruses such as TT virus (TTV) and chicken anaemia virus were identified. Phylogenetic analysis distinguished three main groups of TLMV and allowed the identification of putative recombination breakpoints in the untranslated region. TLMV genomes were detected by PCR in the plasma of 38/50 French blood donors tested and were also identified in peripheral blood mononuclear cells, faeces and saliva. A phylogenetic study of 37 TLMV strains originating from France, Japan and Brazil showed that groupings were not related to geographical origin.
Human immunodeficiency virus type 2 lentiviral vectors: packaging signal and splice donor in expression and encapsidation
Authors: D’Costa Jenice , Brown Heidi M. , Kundra Priya , Davis-Warren Alberta , Arya Suresh K.
Published: August 2015

Retroviral vectors provide the means for gene transfer with long-term expression. The lentivirus subgroup of retroviruses, such as human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2), possesses a number of regulatory and accessory genes and other special elements. These features can be exploited to design vectors for transducing non-dividing as well as dividing cells with the potential for regulated transgene expression. Encapsidation of the transgene RNA in lentiviral vectors is determined by the leader sequence-based multipartite packaging signal. Embedded in the packaging signal is a major splice donor site that, this study shows, is not by itself essential for transgene expression or encapsidation. We designed HIV-2 vectors that contained all the sequence elements thought to be necessary and sufficient for vector RNA encapsidation. Unexpectedly, despite abundant expression, only a small fraction of the transgene RNA was encapsidated and the titre of the vector was low. Redesign of the vector with a mutant splice donor resulted in increased vector RNA encapsidation and yielded vectors with high titre. Inefficient encapsidation by the conventionally designed vector was not due to suboptimal Rev responsive element (RRE)–Rev function. Varying the length of RRE in the vector did not change vector RNA encapsidation, nor did the introduction of a synthetic intron into the mutant vector. The vector RNA with the intact splice donor may have been excessively spliced, decreasing the amount of packageable RNA. A titre of 105 transducing units (TU)/ml was readily obtained for vectors with the neo or GFP transgene, and the vector could be concentrated to a titre of 1–5×107  TU/ml.
The rapamycin sensitivity of human T-cell leukaemia virus type I-induced T-cell proliferation is mediated independently of the polypyrimidine motifs in the 5′ long terminal repeat
Authors: Rose Nicola J. , Lever Andrew M. L.
Published: August 2015

The immunosuppressant rapamycin can regulate the translation of a subset of messenger RNAs, a phenotype which has been linked to the presence of a polypyrimidine motif [C(N)4–14] downstream of the mRNA cap structure. T-cell clones naturally infected with transcriptionally active human T-cell leukaemia virus, type I (HTLV-I) undergo autologous proliferation; this phenotype is inhibited by rapamycin but not FK506, which reverses the rapamycin effect. Within the R region of the HTLV-I 5′ long terminal repeat (LTR) there are seven polypyrimidine motifs. We sought to determine if these were involved in the sensitivity of proliferation to the presence of rapamycin. Here we illustrate the generation of anin vitromodel of this rapamycin-sensitivity and the analysis of LTR mutants which were created to determine the importance of the polypyrimidine motifs. Reporter gene assays suggest the effect is independent of the polypyrimidine motifs in the virus leader sequence.
Genetic and ultrastructural characterization of a European isolate of the fatal endotheliotropic elephant herpesvirus
Authors: Ehlers B. , Burkhardt S. , Goltz M. , Bergmann V. , Ochs A. , Weiler H. , Hentschke J.
Published: August 2015

A male Asian elephant (Elephas maximus) died at the Berlin zoological gardens in August 1998 of systemic infection with the novel endotheliotropic elephant herpesvirus (ElHV-1). This virus causes a fatal haemorrhagic disease in Asian elephants, the so-called endothelial inclusion body disease, as reported from North American zoological gardens. In the present work, ElHV-1 was visualized ultrastructurally in affected organ material. Furthermore, a gene block comprising the complete glycoprotein B (gB) and DNA polymerase (DPOL) genes as well as two partial genes was amplified by PCR-based genome walking and sequenced. The gene content and arrangement were similar to those of members of the Betaherpesvirinae. However, phylogenetic analysis with gB and DPOL consistently revealed a very distant relationship to the betaherpesviruses. Therefore, ElHV-1 may be a member of a new genus or even a new herpesvirus subfamily. The sequence information generated was used to set up a nested-PCR assay for diagnosis of suspected cases of endothelial inclusion body disease. Furthermore, it will aid in the development of antibody-based detection methods and of vaccination strategies against this fatal herpesvirus infection in the endangered Asian elephant.
The zinc ring finger in the bICP0 protein encoded by bovine herpesvirus-1 mediates toxicity and activates productive infection
Authors: Inman Melissa , Zhang Yange , Geiser Vicki , Jones Clinton
Published: August 2015

The bICP0 protein encoded by bovine herpesvirus 1 (BHV-1) is believed to activate transcription and consequently productive infection. Expression of full-length bICP0 protein is toxic in transiently transfected mouse neuroblastoma cells (neuro-2A) in the absence of other viral genes. However, bICP0 does not appear to directly induce apoptosis. Although bICP0 is believed to be functionally similar to the herpes simplex virus type 1-encoded ICP0, the only protein domain that is well conserved is a C3HC4zinc ring finger located near the N terminus of both proteins. Site-specific mutagenesis of the zinc ring finger of bICP0 demonstrated that it was important for inducing aggregated chromatin structures in transfected cells and toxicity. The zinc ring finger was also required for stimulating productive infection in bovine cells and fortrans-activating the thymidine kinase (TK) promoter of herpes simplex virus type 1. Deletion of amino acids spanning 356–677 of bICP0 altered subcellular localization of bICP0 and preventedtrans-activation of the TK promoter. However, this deletion did not preventtrans-activation of the viral genome. Taken together, these studies indicated that bICP0 has several functional domains, including the zinc ring finger, which stimulate productive infection and influence cell survival.
The complete nucleotide sequence of porcine adenovirus serotype 5
Authors: Nagy Miklós , Nagy Éva , Tuboly Tamás
Published: August 2015

The complete nucleotide sequence of porcine adenovirus serotype 5 (PAdV-5) has been determined and the putative genomic map was constructed. The size of the genome was found to be 32621 nucleotides. Twenty-eight putative ORFs were identified by their homology to other adenovirus or other virus and eukaryotic genes. Several special protein sequence motifs were identified by their homology to similar protein motifs. The putative promoter regions, polyadenylation and splice sites were predicted and the early and late transcription units were determined. Based on sequence analysis and RNA secondary structure prediction, sequences for virus-associated RNA could not be recognized. Phylogenetic analysis showed that PAdV-5 was more closely related to certain bovine adenoviruses than to other porcine adenoviruses.
Site-specific mutation of the hepatitis B virus enhancer II B1 element: effect on virus transcription and replication
Authors: Xie Youhua , Li Mei , Wang Yuan , Hofschneider Peter Hans , Weiss Ludwig
Published: August 2015

The hepatitis B virus (HBV) enhancer II (EII) is highly liver-specific and plays an important role in regulating the transcription of all HBV genes. In this report, mutational analysis on the B1F-binding site in the major functional unit of HBV EII is described. The activity of HBV EII in EII–CAT reporter plasmids was significantly decreased when the sequence of the B1F-binding site in EII was mutated. Furthermore, a single point mutation in the B1 element that aborted the binding of B1F caused a dramatic decrease in viral gene transcription initiated from the HBV core promoter, which resulted in a reduction of the production of the HBV e antigen and pregenomic RNA, the template for viral DNA replication. In conclusion, the interaction of B1F with its target binding sequence in the EII region is crucial for liver-specific transcription and DNA replication of the virus.
Divergent replication kinetics of two phenotypically different parvoviruses of rats
Authors: Ball-Goodrich Lisa J. , Johnson Elizabeth , Jacoby Robert
Published: August 2015

Rat virus (RV) is an important infectious agent of laboratory rats because of its high prevalence and capacity to disrupt research. Additionally, RV infection serves as a model for characterizing virus–host interactions during acute, persistent and prenatal infection. Our research has examined the pathogenesis of two RV strains, RV-UMass and RV-Y. RV-UMass is more pathogenic, causes a higher level of persistent infection and transmits to the foetus after oronasal inoculation of the pregnant dam. To determine in vitro distinctions between the strains that may account for these differences and to provide a benchmark for characterizing virus replication in vivo, synchronized in vitro replication of both RV strains was defined and compared. The results demonstrated that RV replication has replicative intermediates, virus transcripts and proteins similar to those reported for the prototype parvovirus, minute virus of mice. However, the replicative cycle of RV-UMass was 12 h compared with 24 h for RV-Y, and RV-UMass and RV-Y differed in kinetics of virus DNA replication, transcription and protein accumulation. Additionally, in situ analysis correlated well with kinetics data as determined by Southern and Northern blot analysis. Sequence comparisons between the strains also determined coding differences that may contribute to phenotypic differences.
Upstream stimulating factor affects human immunodeficiency virus type 1 (HIV-1) long terminal repeat-directed transcription in a cell-specific manner, independently of the HIV-1 subtype and the core-negative regulatory element
Authors: Naghavi Mojgan H. , Estable Mario C. , Schwartz Stefan , Roeder Robert G. , Vahlne Anders
Published: August 2015

Human immunodeficiency virus type 1 (HIV-1) is classified into subtypes on the basis of phylogenetic analysis of sequence differences. Inter- and intra-subtype polymorphism extends throughout the genome, including the long terminal repeat (LTR). In this study, the importance of the upstream stimulating factor (USF)-binding site (E-box) in the core-negative regulatory element (NRE) of the LTR of HIV-1 subtypes A, B, C, D, E and G was investigated.In vivo, USF was found to repress transcription directed from representative HIV-1 LTR sequences of all the subtypes tested in an epithelial cell line, yet activate the same transcription in a T-cell line. Mutation of the core-NRE USF site of the representative subtype B LTR did not affect the cell-specific, subtype-independent, dual role of USF.In vitrobinding assays showed that recombinant USF43interacts with the core-NRE from subtypes B and C, but not A, D, E or G. Thus, USF affects LTR-directed transcription in a cell-specific manner, independently of both the HIV-1 subtype from which the LTR was derived and the core-NRE USF site sequences.
Adaptation of primate cell-adapted hepatitis A virus strain HM175 to growth in guinea pig cells is independent of mutations in the 5′ nontranslated region
Authors: Frings Werner , Dotzauer Andreas
Published: August 2015

Previous studies of hepatitis A virus (HAV) genotypes after adaptation of wild-type virus to growth in cell cultures of primate origin identified determinants for growth in cell culture in the viral 2B and 2C protein-coding regions of the genome and demonstrated that an increased growth efficiency in a particular cell line was achieved by subsequent mutations in the 5′ nontranslated region (5′NTR). The results reported in this study demonstrate that the passage of HAV adapted to primate BS-C-1 cells in guinea pig cells resulted in increased growth efficiency in the rodent cells and decreased growth efficiency in BS-C-1 cells. This adaptation occurred without mutation in the 5′NTR, but the viral 2B and 2C proteins seem to play a role during adaptation to the new environment, as one mutation occurred in each protein. Although the data presented here do not clearly identify which region of the viral genome underwent mutations to improve the interaction of the viruses with guinea pig proteins, they do confirm that the 5′NTR is not the only region responsible for providing host cell-specific information.
Foot-and-mouth disease type O viruses exhibit genetically and geographically distinct evolutionary lineages (topotypes)
Authors: Samuel A. R. , Knowles N. J.
Published: August 2015

Serotype O is the most prevalent of the seven serotypes of foot-and-mouth disease (FMD) virus and occurs in many parts of the world. The UPGMA method was used to construct a phylogenetic tree based on nucleotide sequences at the 3′ end of the VP1 gene from 105 FMD type O viruses obtained from samples submitted to the OIE/FAO World Reference Laboratory for FMD. This analysis identified eight major genotypes when a value of 15% nucleotide difference was used as a cut-off. The validity of these groupings was tested on the complete VP1 gene sequences of 23 of these viruses by bootstrap resampling and construction of a neighbour-joining tree. These eight genetic lineages fell within geographical boundaries and we have used the term topotype to describe them. Using a large sequence database, the distribution of viruses belonging to each of the eight topotypes has been determined. These phylogenetically based epidemiological studies have also been used to identify viruses that have transgressed their normal ecological niches. Despite the high rate of mutation during replication of the FMD virus genome, the topotypes appear to represent evolutionary cul-de-sacs.
Determination of the complete nucleotide sequences of RNA1 and RNA2 from greasy grouper (Epinephelus tauvina) nervous necrosis virus, Singapore strain
Authors: Tan Corrina , Huang Bin , Chang Siow Foong , Ngoh Gim Hue , Munday Barry , Chen Shih Chu , Kwang Jimmy
Published: August 2015

The complete nucleotide sequences of RNA1 and RNA2 from greasy grouper (Epinephelus tauvina) nervous necrosis virus (GGNNV), Singapore strain, were determined. 5′RACE and RNA ligation were used to obtain the complete nucleotide sequences of the 5′ and 3′ non-coding regions (NCRs). GGNNV RNA1 was determined to be 3103 nt long, containing an ORF of 982 aa, while GGNNV RNA2 was determined to be 1433 nt long, containing an ORF of 338 aa. Both GGNNV RNAs are longer than those of other published betanodavirus sequences and the additional nucleotides were located within the NCRs. Analysis of GGNNV RNA2 revealed that it is closely related to redspotted grouper nervous necrosis virus and that both grouper viruses share the same neutralization epitope. Predicted domains for six RNA-dependent RNA polymerase motifs and two putative ORFs (proteins B1 and B2) were confirmed by sequence analysis of GGNNV RNA1.
Functional equivalence of late gene promoters in bean golden mosaic virus with those in tomato golden mosaic virus
Authors: Hung Hsiu-Cheng , Petty Ian T. D.
Published: August 2015

In the bipartite geminivirus tomato golden mosaic virus (TGMV), the activity of late gene promoters is up-regulated by the multifunctional viral protein AL2. Cis-acting sequences required for AL2-mediated promoter responses have not been well characterized. However, nucleotide sequence analysis has implicated a motif termed the conserved late element (CLE). The CLE is present in TGMV and many other begomoviruses, although it is not ubiquitous. Here we analysed the regulation of late gene expression in bean golden mosaic virus (BGMV), one of the begomoviruses which lacks the CLE. Transient reporter gene assays showed that BGMV late gene promoters were trans-activated in Nicotiana benthamiana protoplasts, both by the homologous BGMV AL2 protein and by the heterologous TGMV AL2 protein. The BGMV AL2 protein also trans-activated TGMV late gene promoters. Consistent with these results, we found that hybrid viruses with the late gene promoters exchanged between BGMV and TGMV were viable in planta.
The DNA form of a retroviroid-like element characterized in cultivated carnation species
Authors: Hegedűs Krisztina , Palkovics László , Tóth Endre Kristóf , Dallmann Géza , Balázs Ervin
Published: August 2015

Carnation small viroid-like RNA (CarSV RNA) is a small (275 nt), circular molecule which is unique among plant viroid-like RNAs in having a tandemly repeated homologous DNA. This DNA form was found fused to DNA sequences of carnation etched ring caulimovirus (CERV) in certain Spanish carnation plants. The observation of a growth abnormality consisting of extensive shoot proliferation in cultivated carnations in Hungary prompted the molecular analysis of these plants, in which both CarSV RNA and DNA forms were detected. Several CarSV DNA sequences were characterized in various Dianthus caryophyllus cultivars which were symptomless or showed different symptoms. CarSV DNA forms showing minor sequence heterogeneities and deletions occurred in the same plant. Unit-length CarSV DNA sequences were proven to accumulate in the plant cell nucleus. The plants studied here were not infected by any of the viruses (including CERV) or other cellular pathogens described previously in carnation.
Evolutionarily conserved RNA secondary structures in coding and non-coding sequences at the 3′ end of the hepatitis G virus/GB-virus C genome
Authors: Cuceanu N. M. , Tuplin A. , Simmonds P.
Published: August 2015

Hepatitis G virus (HGV)/GB virus C (GBV-C) causes persistent, non-pathogenic infection in a large proportion of the human population. Epidemiological and genetic evidence indicates a long-term association between HGV/GBV-C and related viruses and a range of primate species, and the co-speciation of these viruses with their hosts during primate evolution. Using a combination of covariance scanning and analysis of variability at synonymous sites, we previously demonstrated that the coding regions of HGV/GBV-C may contain extensive secondary structure of undefined function (Simmonds & Smith, Journal of Virology 73, 5787–5794, 1999). In this study we have carried out a detailed comparison of the structure of the 3′untranslated region (3′UTR) of HGV/GBV-C with that of the upstream NS5B coding sequence. By investigation of free energies on folding, secondary structure predictive algorithms and analysis of covariance between HGV/GBV-C genotypes 1–4 and the more distantly related HGV/GBV-C chimpanzee variant, we obtained evidence for extensive RNA secondary structure formation in both regions. In particular, the NS5B region contained long stem–loop structures of up to 38 internally paired nucleotides which were evolutionarily conserved between human and chimpanzee HGV/GBV-C variants. The prediction of similar structures in the same region of hepatitis C virus may allow the functions of these structures to be determined with a more tractable experimental model.
Interferon-α inhibits hepatitis C virus subgenomic RNA replication by an MxA-independent pathway
Authors: Frese Michael , Pietschmann Thomas , Moradpour Darius , Haller Otto , Bartenschlager Ralf
Published: August 2015

Hepatitis C virus (HCV) persists in the majority of infected individuals and is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Chronic hepatitis C is currently treated with interferon (IFN)-α or with a combination of IFN-α and ribavirin. The availability of an HCV replicon system (Lohmann et al., Science 285, 110–113, 1999) allowed the investigation of the effects of IFN on genuine HCV replication in cultured cells. It is shown here that IFN-α inhibits subgenomic HCV RNA replication in HuH-7 human hepatoma cells. Immunofluorescence, Western blot and Northern blot analysis revealed that levels of both HCV protein and replicon RNA were reduced after treatment with IFN-α in a dose-dependent manner. In further experiments, it was investigated whether MxA plays a role in the inhibition of HCV. The human MxA protein is an IFN-induced GTPase that has antiviral activity against various RNA viruses. However, HCV RNA replication was not affected in transfected HuH-7 cells that transiently overexpressed MxA. Moreover, a dominant-negative mutant of MxA did not interfere with the antiviral activity of IFN-α against HCV RNA replication. Taken together, these results demonstrate that IFN-α inhibits HCV replicons via an MxA-independent pathway.
Activity of Toscana and Rift Valley fever virus transcription complexes on heterologous templates
Authors: Accardi Luisa , Prehaud Christophe , Di Bonito Paola , Mochi Stefania , Bouloy Michèle , Giorgi Colomba
Published: August 2015

A transcription system for Toscana virus (TOSV) (a member of the family Bunyaviridae, genus Phlebovirus) was constructed. For in vivo expression, the TOSV transcription system uses the viral N and L proteins and an S-like RNA genome containing the chloramphenicol acetyltransferase reporter gene in the antisense orientation flanked by the viral genomic 5′- and 3′-terminal S sequences. It was found that the N and L proteins represent the minimal protein requirement for an active transcription complex. To investigate the possibility of reassortment between TOSV and Rift Valley fever virus (RVFV), the activity of their polymerase complexes was tested on their heterologous S-like RNA genomes and this showed that both virus complexes were active. Moreover, hybrid transcriptase complexes with protein components originating from the two viruses were tested on both virus templates and only the combination RVFV L + TOSV N on RVFV S-like RNA was found to be active in this assay. These results suggest that virus reassortants might be generated whenever the two viruses infect the same host.
Infection kinetics, prostacyclin release and cytokine-mediated modulation of the mechanism of cell death during bluetongue virus infection of cultured ovine and bovine pulmonary artery and lung microvascular endothelial cells
Authors: DeMaula Christopher D. , Jutila Mark A. , Wilson Dennis W. , MacLachlan N. James
Published: August 2015

Bluetongue virus (BTV) infection causes a haemorrhagic disease in sheep, whereas BTV infection typically is asymptomatic in cattle. Injury to the endothelium of small blood vessels is responsible for the manifestations of disease in BTV-infected sheep. The lungs are central to the pathogenesis of BTV infection of ruminants; thus endothelial cells (ECs) cultured from the pulmonary artery and lung microvasculature of sheep and cattle were used to investigate the basis for the disparate expression of bluetongue disease in the two species. Ovine and bovine microvascular ECs infected at low multiplicity with partially purified BTV were equally susceptible to BTV-induced cell death, yet ovine microvascular ECs had a lower incidence of infection and produced significantly less virus than did bovine microvascular ECs. Importantly, the relative proportions of apoptotic and necrotic cells were significantly different in BTV-infected EC cultures depending on the species of EC origin and the presence of inflammatory mediators in the virus inoculum. Furthermore, BTV-infected ovine lung microvascular ECs released markedly less prostacyclin than the other types of ECs. Results of thesein vitrostudies are consistent with the marked pulmonary oedema and microvascular thrombosis that characterize bluetongue disease of sheep but which rarely, if ever, occur in BTV-infected cattle.
Differential roles of B cells and IFN-γ-secreting CD4+ T cells in innate and adaptive immune control of genital herpes simplex virus type 2 infection in mice
Authors: Harandi Ali M. , Svennerholm Bo , Holmgren Jan , Eriksson Kristina
Published: August 2015

The role of B, CD4+T and CD8+T cells in both primary genital infection with attenuated herpes simplex virus type 2 (HSV-2) and development of protective immunity to a later challenge with virulent HSV-2 using lymphocyte-deficient mice has been elucidated. Following primary inoculation with attenuated thymidine kinase-deficient (TK−) HSV-2, B cell-deficient (μMT) mice developed a local viraemia and transient genital inflammation, suggesting a role for B cells in the innate control of local infection and inflammation. Natural antibodies are implicated in this process, as passive transfer of normal serum into μMT mice significantly reduced HSV-2 TK−shedding in the vaginal lumen, although it did not affect subsequent inflammation. Protection against lethal HSV-2 challenge was noted in HSV-2-vaccinated wild-type, CD8+T cell-deficient and μMT mice and was characterized by strong virus-specific IFN-γ responsesin vitroand delayed type hypersensitivity (DTH) responsesin vivo. In contrast, CD4+T cell-deficient (CD4−/−) mice had impaired HSV-2-specific IFN-γ production and DTH responses and succumbed rapidly to genital HSV-2 challenge. However, protective responses to HSV-2 could be induced in HSV-2-vaccinated CD4−/−mice by treatment with recombinant IFN-γ. Taken together, these results suggest that CD4+T cells secreting IFN-γ are critical for immune protection against lethal genital HSV-2 re-infection, whereas B cells/natural antibodies have anti-viral and -inflammatory effects in the innate control of a primary infection.
Towards a protein interaction map of potyviruses: protein interaction matrixes of two potyviruses based on the yeast two-hybrid system
Authors: Guo Deyin , Rajamäki Minna-Liisa , Saarma Mart , Valkonen Jari P. T.
Published: August 2015

A map for the interactions of the major proteins from Potato virus A (PVA) and Pea seed-borne mosaic virus (PSbMV) (members of the genus Potyvirus, family Potyviridae) was generated using the yeast two-hybrid system (YTHS). Interactions were readily detected with five PVA protein combinations (HC–HC, HC–CI, VPg–VPg, NIa–NIb and CP–CP) and weak but reproducible interactions were detected for seven additional combinations (P1–CI, P3–NIb, NIaPro–NIb, VPg–NIa, VPg–NIaPro, NIaPro–NIa and NIa–NIa). In PSbMV, readily detectable interactions were found in five protein combinations (HC–HC, VPg–VPg, VPg–NIa, NIa–NIa and NIa–NIb) and weaker but reproducible interactions were detected for three additional combinations (P3–NIa, NIa–NIaPro and CP–CP). The self-interactions of HC, VPg, NIa and CP and the interactions of VPg–NIa, NIa–NIaPro and NIa–NIb were, therefore, common for the two potyviruses. The multiple protein interactions revealed in this study shed light on the co-ordinated functions of potyviral proteins involved in virus movement and replication.