Putative full-length clones of the genomic DNA segments of subterranean clover stunt virus and identification of the segment coding for the viral coat protein

Subterranean clover stunt disease is an economically important aphid-borne virus disease affecting certain pasture and grain legumes in Australia. The virus associated with the disease, subterranean clover stunt virus (SCSV), was previously found to be representative of a new type of single-stranded DNA virus. Analysis of the virion DNA and restriction mapping of double-stranded cDNA synthesized from virion DNA suggested that SCSV has a segmented genome composed of 3 or 4 different species of circular ssDNA each of about 850-880 nucleotides. To further investigate the complexity of the SCSV genome, we have isolated the replicative form DNA from infected pea and from it prepared putative full-length clones representing the SCSV genome segments. Analysis of these clones by restriction mapping indicated that clones representing at least 4 distinct genomic segments were obtained. This method is thus suitable for generating an extensive genomic library of novel ssDNA viruses containing multiple genome segments such as SCSV and banana bunchy top virus. The N-terminal amino acid sequence and amino acid composition of the coat protein of SCSV were determined. Comparison of the amino acid sequence with partial DNA sequence data, and the distinctly different restriction maps obtained for the full-length clones suggested that only one of these clones contained the coat protein gene. The results confirmed that SCSV has a functionally divided genome composed of several distinct ssDNA circles each of about 1 kb.

Vaccinia virus DNA replication: two hundred base pairs of telomeric sequence confer optimal replication efficiency on minichromosome templates.

Vaccinia virus is a complex DNA virus that exhibits significant genetic and physical autonomy from the host cell. Most if not all of the functions involved in replication and transcription of the 192-kb genome are virally encoded. Although significant progress has been made in identifying trans-acting factors involved in DNA synthesis, the mechanism of genome replication has remained poorly understood. The genome is a linear duplex with covalently closed hairpin termini, and it has been presumed that sequences and/or structures within these termini are important for the initiation of genome replication. In this report we describe the construction of minichromosomes containing a central plasmid insert flanked by hairpin termini derived from the viral genome and their use as replication templates. When replication of these minichromosomes was compared with a control substrate containing synthetic hairpin termini, specificity for viral telomeres was apparent. Inclusion of > or = 200 bp from the viral telomere was sufficient to confer optimal replication efficiency, whereas 65-bp telomeres were not effective. Chimeric 200-bp telomeres containing the 65-bp terminal element and 135 bp of ectopic sequence also failed to confer efficient replication, providing additional evidence that telomere function is sequence-specific. Replication of these exogenous templates was dependent upon the viral replication machinery, was temporally coincident with viral replication, and generated covalently closed minichromosome products. These data provide compelling evidence for specificity in template recognition and utilization in vaccinia virus-infected cells.

Sequence and organization of barley yellow dwarf virus genomic RNA

The nucleotide sequence of the genomic RNA of barley yellow dwarf virus, PAV serotype was determined except for the 5′-terminal base, and its genome organization deduced. The 5,677 nucleotide genome contains five large open reading frames (ORFs). The genes for the coat protein (1) and the putative viral RNA-dependent RNA polymerase were identified. The latter shows a striking degree of similarity to that of carnation mottle virus (CarMV). By comparison with corona- and retrovirus RNAs, it is proposed that a translational frameshift is involved in expression of the polymerase. An ORF encoding an Mr 49,797 protein (50K ORF) may be translated by in-frame readthrough of the coat protein stop codon. The coat protein, an overlapping 17K ORF, and a 3′ 6.7K ORF are likely to be expressed via subgenomic mRNAs. © 1988 IRL Press Limited.

The global distribution of Banana bunchy top virus reveals little evidence for frequent recent, human-mediated long distance dispersal events

Banana bunchy top virus (BBTV; family Nanoviridae, genus Babuvirus) is a multi-component single-stranded DNA virus, which infects banana plants in many regions of the world, often resulting in large-scale crop losses. Weanalyzed 171 banana leaf samples from fourteen countries and recovered, cloned, and sequenced 855 complete BBTV components including ninety-four full genomes. Importantly, full genomes were determined from eight countries, where previously no full genomes were available (Samoa, Burundi, Republic of Congo, Democratic Republic of Congo, Egypt, Indonesia, the Philippines, and the USA [HI]). Accounting for recombination and genome component reassortment, we examined the geographic structuring of global BBTV populations to reveal that BBTV likely originated in Southeast Asia, that the current global hotspots of BBTV diversity are Southeast Asia/Far East and India, and that BBTV populations circulating elsewhere in the world have all potentially originated from infrequent introductions. Most importantly, we find that rather than the current global BBTV distribution being due to increases in human-mediated movements of bananas over the past few decades, it is more consistent with a pattern of infrequent introductions of the virus to different parts of the world over the past 1,000 years.

Epstein-Barr Virus Large Tegument Protein BPLF1 Contributes to Innate Immune Evasion through Interference with Toll-Like Receptor Signaling

Viral infection triggers an early host response through activation of pattern recognition receptors, including Toll-like receptors (TLR). TLR signaling cascades induce production of type I interferons and proinflammatory cytokines involved in establishing an anti-viral state as well as in orchestrating ensuing adaptive immunity. To allow infection, replication, and persistence, (herpes)viruses employ ingenious strategies to evade host immunity. The human gamma-herpesvirus Epstein-Barr virus (EBV) is a large, enveloped DNA virus persistently carried by more than 90% of adults worldwide. It is the causative agent of infectious mononucleosis and is associated with several malignant tumors. EBV activates TLRs, including TLR2, TLR3, and TLR9. Interestingly, both the expression of and signaling by TLRs is attenuated during productive EBV infection. Ubiquitination plays an important role in regulating TLR signaling and is controlled by ubiquitin ligases and deubiquitinases (DUBs). The EBV genome encodes three proteins reported to exert in vitro deubiquitinase activity. Using active site-directed probes, we show that one of these putative DUBs, the conserved herpesvirus large tegument protein BPLF1, acts as a functional DUB in EBV-producing B cells. The BPLF1 enzyme is expressed during the late phase of lytic EBV infection and is incorporated into viral particles. The N-terminal part of the large BPLF1 protein contains the catalytic site for DUB activity and suppresses TLR-mediated activation of NF-κB at, or downstream of, the TRAF6 signaling intermediate. A catalytically inactive mutant of this EBV protein did not reduce NF-κB activation, indicating that DUB activity is essential for attenuating TLR signal transduction. Our combined results show that EBV employs deubiquitination of signaling intermediates in the TLR cascade as a mechanism to counteract innate anti-viral immunity of infected hosts.

Biosafety of Recombinant Adeno-associated Virus Vectors.

It is hoped that the use of gene transfer technology to treat both monogenetic and acquired diseases may soon become a common therapy option in medicine. For gene therapy to achieve this objective, any gene delivery method will have to meet several criteria, including ease of manufacturing, efficient gene transfer to target tissue, long-term gene expression to alleviate the disease, and most importantly safety in patients. Viral vectors are an attractive choice for use in gene therapy protocols due to their relative efficiency in gene delivery. Since there is inherent risk in using viruses, investigators in the gene therapy community have devoted extensive efforts toward reengineering viral vectors for enhance safety. Here we review the approaches and technologies that are being evaluated for the use of recombinant vectors based upon adeno-associated virus (AAV) in the treatment of a variety of human diseases. AAV is currently the only known human DNA virus that is non-pathogenic and AAV-based vectors are classified as Risk Group 1 agents for all laboratory and animal studies carried out in the US. Although its apparent safety in natural infection and animals appears well documented, we examine the accumulated knowledge on the biology and vectorology of AAV, lessons learned from gene therapy clinical trials, and how this information is impacting current vector design and manufacturing with an overall emphasis on biosafety.

Phylogenetic analysis of beak and feather disease virus across a host ring-species complex

Pathogens have been hypothesized to play a major role in host diversity and speciation. Susceptibility of hybrid hosts to pathogens is thought to be a common phenomenon that could promote host population divergence and subsequently speciation. However, few studies have tested for pathogen infection across animal hybrid zones while testing for codivergence of the pathogens in the hybridizing host complex. Over 8 y, we studied natural infection by a rapidly evolving single-strand DNA virus, beak and feather diseases virus (BFDV), which infects parrots, exploiting a host-ring species complex (Platycercus elegans) in Australia. We found that host subspecies and their hybrids varied strikingly in both BFDV prevalence and load: both hybrid and phenotypically intermediate subspecies had lower prevalence and load compared with parental subspecies, while controlling for host age, sex, longitude and latitude, as well as temporal effects. We sequenced viral isolates throughout the range, which revealed patterns of genomic variation analogous to Mayr's ring-species hypothesis, to our knowledge for the first time in any host-pathogen system. Viral phylogeny, geographic location, intraspecific host density, and parrot community diversity and composition did not explain the differences in BFDV prevalence or load between subpopulations. Overall, our analyses suggest that functional host responses to infection, or force of infection, differ between subspecies and hybrids. Our findings highlight the role of host hybridization and clines in altering host-pathogen interactions, dynamics that can have important implications for models of speciation with gene flow, and offer insights into how pathogens may adapt to diverging host populations.

Araçatuba virus: A vaccinialike virus associated with infection in humans and cattle

We describe a vaccinialike virus, Araçatuba virus, associated with a cowpoxlike outbreak in a dairy herd and a related case of human infection. Diagnosis was based on virus growth characteristics, electron microscopy, and molecular biology techniques. Molecular characterization of the virus was done by using polymerase chain reaction amplification, cloning, and DNA sequencing of conserved orthopoxvirus genes such as the vaccinia growth factor (VGF), thymidine kinase (TK), and hemagglutinin. We used VGF-homologous and TK gene nucleotide sequences to construct a phylogenetic tree for comparison with other poxviruses. Gene sequences showed 99% homology with vaccinia virus genes and were clustered together with the isolated virus in the phylogenetic tree. Araçatuba virus is very similar to Cantagalo virus, showing the same signature deletion in the gene. Araçatuba virus could be a novel vaccinialike virus or could represent the spread of Cantagalo virus.

Porcine circovirus type 2 DNA influences cytoskeleton rearrangements in plasmacytoid and monocyte-derived dendritic cells

Functional disruption of dendritic cells (DC) is an important strategy for viral pathogens to evade host defences. In this context, porcine circovirus type 2 (PCV2), a single-stranded DNA virus, impairs plasmacytoid DC (pDC) and conventional DC activation by certain viruses or Toll-like receptor (TLR) ligands. This inhibitory capacity is associated with the viral DNA, but the impairment does not affect all signalling cascades; TLR7 ligation by small chemical molecules will still induce interleukin-6 (IL-6) and tumour necrosis factor-α secretion, but not interferon-α or IL-12. In this study, the molecular mechanisms by which silencing occurs were investigated. PP2, a potent inhibitor of the Lyn and Hck kinases, produced a similar profile to the PCV2 DNA interference with cytokine secretion by pDC, efficiently inhibiting cell activation induced through TLR9, but not TLR7, ligation. Confocal microscopy and cytometry analysis strongly suggested that PCV2 DNA impairs actin polymerization and endocytosis in pDC and monocyte-derived DC, respectively. Altogether, this study delineates for the first time particular molecular mechanisms involved in PCV2 interference with DC danger recognition, which may be responsible for the virus-induced immunosuppression observed in infected pigs.

The Apis mellifera filamentous virus genome

A complete reference genome of the Apis mellifera Filamentous virus (AmFV) was determined using Illumina Hiseq sequencing. The AmFV genome is a double stranded DNA molecule of approximately 498,500 nucleotides with a GC content of 50.8%. It encompasses 247 non-overlapping open reading frames (ORFs), equally distributed on both strands, which cover 65% of the genome. While most of the ORFs lacked threshold sequence alignments to reference protein databases, twenty-eight were found to display significant homologies with proteins present in other large double stranded DNA viruses. Remarkably, 13 ORFs had strong similarity with typical baculovirus domains such as PIFs (per os infectivity factor genes: pif-1, pif-2, pif-3 and p74) and BRO (Baculovirus Repeated Open Reading Frame). The putative AmFV DNA polymerase is of type B, but is only distantly related to those of the baculoviruses. The ORFs encoding proteins involved in nucleotide metabolism had the highest percent identity to viral proteins in GenBank. Other notable features include the presence of several collagen-like, chitin-binding, kinesin and pacifastin domains. Due to the large size of the AmFV genome and the inconsistent affiliation with other large double stranded DNA virus families infecting invertebrates, AmFV may belong to a new virus family.

Dynamics of Apis mellifera filamentous virus (AmFV) infections in honey bees and relationships with other parasites

Apis mellifera filamentous virus (AmFV) is a large double stranded DNA virus of honey bees, but its relationship with other parasites and prevalence are poorly known. We analyzed individual honey bees from three colonies at different times post emergence in order to monitor the dynamics of the AmFV gut colonization under natural conditions. Prevalence and loads of microsporidia and trypanosomes were also recorded, as well as five common honey bee RNA viruses. The results show that a high proportion of bees get infected with AmFV during the first week post-emergence (75%) and that AmFV DNA levels remained constant. A similar pattern was observed for microsporidia while trypanosomes seem to require more time to colonize the gut. No significant associations between these three infections were found, but significant positive correlations were observed between AmFV and RNA viruses. In parallel, the prevalence of AmFV in France and Sweden was assessed from pooled honey bee workers. The data indicate that AmFV is almost ubiquitous, and does not seem to follow seasonal patterns, although higher viral loads were significantly detected in spring. A high prevalence of AmFV was also found in winter bees, without obvious impact on overwintering of the colonies.

Virus del papiloma humano : presentación oral y su relación con el carcinoma espinocelular

El virus del papiloma humano (VPH) (HPV en su sigla en inglés) es un virus ADN con especial afinidad por células epiteliales, tanto cutáneas como mucosas, que incluyen el epitelio del cérvix, región anogenital y orofaríngea. Existen más de 120 subtipos de VPH subdivididos en dos grupos según su bajo o alto riesgo de inducir actividad oncogénica. En cavidad oral la manifestación clínica benigna de la infección es el papiloma plano.Sin embargo, un 12% de los carcinoma~ o rofaríngeos son causados por este virus. Se ha demostrado que la infección por VPH tiene un papel independiente como factor de riesgo para el desarrollo de carcinoma espinocelular en cavidad oral.

Abundance of bacteria and virus during the METEOR cruise M87/1

In temperate, subpolar and polar marine systems, the classical perception that bacteria are carbon limited by end of winter and respond in activity and abundance to the production of new carbon during the diatom spring bloom and post bloom. Contrary to this view, we here document an strong increase in bacterial abundance and activity (latter measured by increasing high nuclei acid (HNA) to low nuclei acid (LNA) bacteria ratio) during the winter-spring transition, where phytoplankton smaller than 10 µm dominate. Further DNA-virus were enumerated and revealed the virus to bacteria ratio (VBR) to be decreasing during winter-spring transition, indicating that the virus did not increase in number accordingly to bacteria. During repeated visits to stations in the deep Icelandic and the Norwegian Basins and the shallow Shetland Shelf (26 March to 29 April 2012), we investigated the abundance of bacteria and the succession of HNA:LNA bacteria and VBR. Water samples were collected from CTD rosette .10 L Niskin bottles and fixed in glutaraldehyde (final conc. 5%), flash frozen in liquid Nitrogen and stored at -80°C until analysis.