bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA barcoding Australia's fish species
Authors: Ward Robert D , Zemlak Tyler S , Innes Bronwyn H , Last Peter R , Hebert Paul D.N
Published: September 2005

Two hundred and seven species of fish, mostly Australian marine fish, were sequenced (barcoded) for a 655 bp region of the mitochondrial cytochrome oxidase subunit I gene (cox1). Most species were represented by multiple specimens, and 754 sequences were generated. The GC content of the 143 species of teleosts was higher than the 61 species of sharks and rays (47.1% versus 42.2%), largely due to a higher GC content of codon position 3 in the former (41.1% versus 29.9%). Rays had higher GC than sharks (44.7% versus 41.0%), again largely due to higher GC in the 3rd codon position in the former (36.3% versus 26.8%). Average within-species, genus, family, order and class Kimura two parameter (K2P) distances were 0.39%, 9.93%, 15.46%, 22.18% and 23.27%, respectively. All species could be differentiated by theircox1sequence, although single individuals of each of two species had haplotypes characteristic of a congener. Although DNA barcoding aims to develop species identification systems, some phylogenetic signal was apparent in the data. In the neighbour-joining tree for all 754 sequences, four major clusters were apparent: chimaerids, rays, sharks and teleosts. Species within genera invariably clustered, and generally so did genera within families. Three taxonomic groups—dogfishes of the genusSqualus, flatheads of the family Platycephalidae, and tunas of the genusThunnus—were examined more closely. The clades revealed after bootstrapping generally corresponded well with expectations. Individuals from operational taxonomic units designated asSqualusspecies B through F formed individual clades, supporting morphological evidence for each of these being separate species. We conclude thatcox1sequencing, or ‘barcoding’, can be used to identify fish species.
Deciphering amphibian diversity through DNA barcoding: chances and challenges
Authors: Vences Miguel , Thomas Meike , Bonett Ronald M , Vieites David R
Published: September 2005

Amphibians globally are in decline, yet there is still a tremendous amount of unrecognized diversity, calling for an acceleration of taxonomic exploration. This process will be greatly facilitated by a DNA barcoding system; however, the mitochondrial population structure of many amphibian species presents numerous challenges to such a standardized, single locus, approach. Here we analyse intra- and interspecific patterns of mitochondrial variation in two distantly related groups of amphibians, mantellid frogs and salamanders, to determine the promise of DNA barcoding with cytochrome oxidase subunit I (cox1) sequences in this taxon. High intraspecificcox1divergences of 7–14% were observed (18% in one case) within the whole set of amphibian sequences analysed. These high values are not caused by particularly high substitution rates of this gene but by generally deep mitochondrial divergences within and among amphibian species. Despite these high divergences,cox1sequences were able to correctly identify species including disparate geographic variants. The main problems withcox1barcoding of amphibians are (i) the high variability of priming sites that hinder the application of universal primers to all species and (ii) the observed distinct overlap of intraspecific and interspecific divergence values, which implies difficulties in the definition of threshold values to identify candidate species. Common discordances between geographical signatures of mitochondrial and nuclear markers in amphibians indicate that a single-locus approach can be problematic when high accuracy of DNA barcoding is required. We suggest that a number of mitochondrial and nuclear genes may be used as DNA barcoding markers to complementcox1.
Land plants and DNA barcodes: short-term and long-term goals
Authors: Chase Mark W , Salamin Nicolas , Wilkinson Mike , Dunwell James M , Kesanakurthi Rao Prasad , Haidar Nadia , Savolainen Vincent
Published: September 2005

Land plants have had the reputation of being problematic for DNA barcoding for two general reasons: (i) the standard DNA regions used in algae, animals and fungi have exceedingly low levels of variability and (ii) the typically used land plant plastid phylogenetic markers (e.g.
rbcL
,
trnL-F
, etc.) appear to have too little variation. However, no one has assessed how well current phylogenetic resources might work in the context of identification (versus phylogeny reconstruction). In this paper, we make such an assessment, particularly with two of the markers commonly sequenced in land plant phylogenetic studies, plastid
rbcL
and internal transcribed spacers of the large subunits of nuclear ribosomal DNA (ITS), and find that both of these DNA regions perform well even though the data currently available in GenBank/EBI were not produced to be used as barcodes and BLAST searches are not an ideal tool for this purpose. These results bode well for the use of even more variable regions of plastid DNA (such as, for example,
psbA-trnH
) as barcodes, once they have been widely sequenced. In the short term, efforts to bring land plant barcoding up to the standards being used now in other organisms should make swift progress. There are two categories of DNA barcode users, scientists in fields other than taxonomy and taxonomists. For the former, the use of mitochondrial and plastid DNA, the two most easily assessed genomes, is at least in the short term a useful tool that permits them to get on with their studies, which depend on knowing roughly which species or species groups they are dealing with, but these same DNA regions have important drawbacks for use in taxonomic studies (i.e. studies designed to elucidate species limits). For these purposes, DNA markers from uniparentally (usually maternally) inherited genomes can only provide half of the story required to improve taxonomic standards being used in DNA barcoding. In the long term, we will need to develop more sophisticated barcoding tools, which would be multiple, low-copy nuclear markers with sufficient genetic variability and PCR-reliability; these would permit the detection of hybrids and permit researchers to identify the ‘genetic gaps’ that are useful in assessing species limits.
Microcoding: the second step in DNA barcoding
Authors: Summerbell R.C , Lévesque C.A , Seifert K.A , Bovers M , Fell J.W , Diaz M.R , Boekhout T , de Hoog G.S , Stalpers J , Crous P.W
Published: September 2005

After the process of DNA barcoding has become well advanced in a group of organisms, as it has in the economically important fungi, the question then arises as to whether shorter and literally more barcode-like DNA segments should be utilized to facilitate rapid identification and, where applicable, detection. Through appropriate software analysis of typical full-length barcodes (generally over 500 base pairs long), uniquely distinctive oligonucleotide ‘microcodes’ of less than 25 bp can be found that allow rapid identification of circa 100–200 species on various array-like platforms. Microarrays can in principle fulfill the function of microcode-based species identification but, because of their high cost and low level of reusability, they tend to be less cost-effective. Two alternative platforms in current use in fungal identification are reusable nylon-based macroarrays and the Luminex system of specific, colour-coded DNA detection beads analysed by means of a flow cytometer. When the most efficient means of rapid barcode-based species identification is sought, a choice can be made either for one of these methodologies or for basic high-throughput sequencing, depending on the strategic outlook of the investigator and on current costs. Arrays and functionally similar platforms may have a particular advantage when a biologically complex material such as soil or a human respiratory secretion sample is analysed to give a census of relevant species present.
The unholy trinity: taxonomy, species delimitation and DNA barcoding
Authors: DeSalle Rob , Egan Mary G , Siddall Mark
Published: September 2005

Recent excitement over the development of an initiative to generate DNA sequences for all named species on the planet has in our opinion generated two major areas of contention as to how this ‘DNA barcoding’ initiative should proceed. It is critical that these two issues are clarified and resolved, before the use of DNA as a tool for taxonomy and species delimitation can be universalized. The first issue concerns how DNA data are to be used in the context of this initiative; this is the DNA barcode reader problem (or barcoder problem). Currently, many of the published studies under this initiative have used tree building methods and more precisely distance approaches to the construction of the trees that are used to place certain DNA sequences into a taxonomic context. The second problem involves the reaction of the taxonomic community to the directives of the ‘DNA barcoding’ initiative. This issue is extremely important in that the classical taxonomic approach and the DNA approach will need to be reconciled in order for the ‘DNA barcoding’ initiative to proceed with any kind of community acceptance. In fact, we feel that DNA barcoding is a misnomer. Our preference is for the title of the London meetings—Barcoding Life. In this paper we discuss these two concerns generated around the DNA barcoding initiative and attempt to present a phylogenetic systematic framework for an improved barcoder as well as a taxonomic framework for interweaving classical taxonomy with the goals of ‘DNA barcoding’.
DNA-based species delineation in tropical beetles using mitochondrial and nuclear markers
Authors: Monaghan Michael T , Balke Michael , Gregory T. Ryan , Vogler Alfried P
Published: September 2005

DNA barcoding has been successfully implemented in the identification of previously described species, and in the process has revealed several cryptic species. It has been noted that such methods could also greatly assist in the discovery and delineation of undescribed species in poorly studied groups, although to date the feasibility of such an approach has not been examined explicitly. Here, we investigate the possibility of using short mitochondrial and nuclear DNA sequences to delimit putative species in groups lacking an existing taxonomic framework. We focussed on poorly known tropical water beetles (Coleoptera: Dytiscidae, Hydrophilidae) from Madagascar and dung beetles (Scarabaeidae) in the genus
Canthon
from the Neotropics. Mitochondrial DNA sequence variation proved to be highly structured, with >95% of the observed variation existing between discrete sets of very closely related genotypes. Sequence variation in nuclear 28S rRNA among the same individuals was lower by at least an order of magnitude, but 16 different genotypes were found in water beetles and 12 genotypes in
Canthon
, differing from each other by a minimum of two base pairs. The distribution of these 28S rRNA genotypes in individuals exactly matched the distribution of mtDNA clusters, suggesting that mtDNA patterns were not misleading because of introgression. Moreover, in a few cases where sequence information was available in GenBank for morphologically defined species of
Canthon
, these matched some of the DNA-based clusters. These findings demonstrate that clusters of close relatives can be identified readily in the sequence variation obtained in field collected samples, and that these clusters are likely to correspond to either previously described or unknown species. The results suggest that DNA-assisted taxonomy will not require more than a short fragment of mtDNA to provide a largely accurate picture of species boundaries in these groups. Applied on a large scale, this DNA-based approach could greatly improve the rate of species discovery in the large assemblages of insects that remain undescribed.
Defining operational taxonomic units using DNA barcode data
Authors: Blaxter Mark , Mann Jenna , Chapman Tom , Thomas Fran , Whitton Claire , Floyd Robin , Abebe Eyualem
Published: September 2005

The scale of diversity of life on this planet is a significant challenge for any scientific programme hoping to produce a complete catalogue, whatever means is used. For DNA barcoding studies, this difficulty is compounded by the realization that any chosen barcode sequence is not the gene ‘for’ speciation and that taxa have evolutionary histories. How are we to disentangle the confounding effects of reticulate population genetic processes? Using the DNA barcode data from meiofaunal surveys, here we discuss the benefits of treating the taxa defined by barcodes without reference to their correspondence to ‘species’, and suggest that using this non-idealist approach facilitates access to taxon groups that are not accessible to other methods of enumeration and classification. Major issues remain, in particular the methodologies for taxon discrimination in DNA barcode data.
An integrated approach to fast and informative morphological vouchering of nematodes for applications in molecular barcoding
Authors: De Ley Paul , De Ley Irma Tandingan , Morris Krystalynne , Abebe Eyualem , Mundo-Ocampo Manuel , Yoder Melissa , Heras Joseph , Waumann Dora , Rocha-Olivares Axayácatl , Jay Burr A.H , Baldwin James G , Thomas W. Kelley
Published: September 2005

Molecular surveys of meiofaunal diversity face some interesting methodological challenges when it comes to interstitial nematodes from soils and sediments. Morphology-based surveys are greatly limited in processing speed, while barcoding approaches for nematodes are hampered by difficulties of matching sequence data with traditional taxonomy. Intermediate technology is needed to bridge the gap between both approaches. An example of such technology is video capture and editing microscopy, which consists of the recording of taxonomically informative multifocal series of microscopy images as digital video clips. The integration of multifocal imaging with sequence analysis of the D2D3 region of large subunit (LSU) rDNA is illustrated here in the context of a combined morphological and barcode sequencing survey of marine nematodes from Baja California and California. The resulting video clips and sequence data are made available online in the database NemATOL (http://nematol.unh.edu/). Analyses of 37 barcoded nematodes suggest that these represent at least 32 species, none of which matches available D2D3 sequences in public databases. The recorded multifocal vouchers allowed us to identify most specimens to genus, and will be used to match specimens with subsequent species identifications and descriptions of preserved specimens. Like molecular barcodes, multifocal voucher archives are part of a wider effort at structuring and changing the process of biodiversity discovery. We argue that data-rich surveys and phylogenetic tools for analysis of barcode sequences are an essential component of the exploration of phyla with a high fraction of undiscovered species. Our methods are also directly applicable to other meiofauna such as for example gastrotrichs and tardigrades.
Critical factors for assembling a high volume of DNA barcodes
Authors: Hajibabaei Mehrdad , deWaard Jeremy R , Ivanova Natalia V , Ratnasingham Sujeevan , Dooh Robert T , Kirk Stephanie L , Mackie Paula M , Hebert Paul D.N
Published: September 2005

Large-scale DNA barcoding projects are now moving toward activation while the creation of a comprehensive barcode library for eukaryotes will ultimately require the acquisition of some 100 million barcodes. To satisfy this need, analytical facilities must adopt protocols that can support the rapid, cost-effective assembly of barcodes. In this paper we discuss the prospects for establishing high volume DNA barcoding facilities by evaluating key steps in the analytical chain from specimens to barcodes. Alliances with members of the taxonomic community represent the most effective strategy for provisioning the analytical chain with specimens. The optimal protocols for DNA extraction and subsequent PCR amplification of the barcode region depend strongly on their condition, but production targets of 100K barcode records per year are now feasible for facilities working with compliant specimens. The analysis of museum collections is currently challenging, but PCR cocktails that combine polymerases with repair enzyme(s) promise future success. Barcode analysis is already a cost-effective option for species identification in some situations and this will increasingly be the case as reference libraries are assembled and analytical protocols are simplified.
A likelihood ratio test for species membership based on DNA sequence data
Authors: Matz Mikhail V , Nielsen Rasmus
Published: September 2005

DNA barcoding as an approach for species identification is rapidly increasing in popularity. However, it remains unclear which statistical procedures should accompany the technique to provide a measure of uncertainty. Here we describe a likelihood ratio test which can be used to test if a sampled sequence is a member of ana priorispecified species. We investigate the performance of the test using coalescence simulations, as well as using the real data from butterflies and frogs representing two kinds of challenge for DNA barcoding: extremely low and extremely high levels of sequence variability.
Characterization of Striped jack nervous necrosis virus subgenomic RNA3 and biological activities of its encoded protein B2
Authors: Iwamoto Tokinori , Mise Kazuyuki , Takeda Atsushi , Okinaka Yasushi , Mori Koh-Ichiro , Arimoto Misao , Okuno Tetsuro , Nakai Toshihiro
Published: September 2005

Striped jack nervous necrosis virus (SJNNV), which infects fish, is the type species of the genus Betanodavirus. This virus has a bipartite genome of positive-strand RNAs, designated RNAs 1 and 2. A small RNA (ca. 0·4 kb) has been detected from SJNNV-infected cells, which was newly synthesized and corresponded to the 3′-terminal region of RNA1. Rapid amplification of cDNA ends analysis showed that the 5′ end of this small RNA (designated RNA3) initiated at nt 2730 of the corresponding RNA1 sequence and contained a 5′ cap structure. Substitution of the first nucleotide of the subgenomic RNA sequence within RNA1 selectively inhibited production of the positive-strand RNA3 but not of the negative-strand RNA3, which suggests that RNA3 may be synthesized via a premature termination model. The single RNA3-encoded protein (designated protein B2) was expressed in Escherichia coli, purified and used to immunize a rabbit to obtain an anti-protein B2 polyclonal antibody. An immunological test showed that the antigen was specifically detected in the central nervous system and retina of infected striped jack larvae (Pseudocaranx dentex), and in the cytoplasm of infected cultured E-11 cells. These results indicate that SJNNV produces subgenomic RNA3 from RNA1 and synthesizes protein B2 during virus multiplication, as reported for alphanodaviruses. In addition, an Agrobacterium co-infiltration assay established in transgenic plants that express green fluorescent protein showed that SJNNV protein B2 has a potent RNA silencing-suppression activity, as discovered for the protein B2 of insect-infecting alphanodaviruses.
Prion clearance in bigenic mice
Authors: Safar Jiri G. , DeArmond Stephen J. , Kociuba Katarzyna , Deering Camille , Didorenko Svetlana , Bouzamondo-Bernstein Essia , Prusiner Stanley B. , Tremblay Patrick
Published: September 2005

The clearance of prions from the brain was investigated in bigenic mice designated Tg(tTA : PrP+/0)3, in which expression of the cellular prion protein (PrPC) was regulated by oral doxycycline administration. With suppression of PrPCexpression, the incubation time for RML prions was prolonged almost threefold from ∼150 to ∼430 days. To determine the clearance rate of disease-causing PrPSc, bigenic mice were given oral doxycycline beginning 98 days after inoculation with RML prions and sacrificed at various time points over the subsequent 56 days. The half-life (t1/2) for PrPScwas ∼1·5 days in mouse brain, in reasonable agreement with the apparentt1/2of 30 h that was determined in a separate study for scrapie-infected mouse neuroblastoma (ScN2a) cells in culture. Both protease-sensitive and -resistant conformers of PrPScwere cleared at the same rate. Thet1/2value for PrPCclearance from brain was ∼18 h, which was considerably longer than thet1/2of 5 h found in ScN2a cells. The capability of the brain to clear prions raises the possibility that PrPScis normally made at low levels and continually cleared, and that PrPScmay have a function in cellular metabolism. Moreover, these bigenic mice make it possible to determine both components of PrPScaccumulation, i.e. the rates of formation and clearance, for various strains of prions exhibiting different incubation times.
Attenuation and immunogenicity in mice of temperature-sensitive influenza viruses expressing truncated NS1 proteins
Authors: Falcón Ana M. , Fernandez-Sesma Ana , Nakaya Yurie , Moran Thomas M. , Ortín Juan , García-Sastre Adolfo
Published: September 2005

It was previously shown that two mutant influenza A viruses expressing C-terminally truncated forms of the NS1 protein (NS1-81 and NS1-110) were temperature sensitive in vitro. These viruses contain HA, NA and M genes derived from influenza A/WSN/33 H1N1 virus (mouse-adapted), and the remaining five genes from human influenza A/Victoria/3/75 virus. Mice intranasally infected with the NS1 mutant viruses showed undetectable levels of virus in lungs at day 3, whereas those infected with the NS1 wild-type control virus still had detectable levels of virus at this time. Nevertheless, the temperature-sensitive mutant viruses induced specific cellular and humoral immune responses similar to those induced by the wild-type virus. Mice immunized with the NS1 mutant viruses were protected against a lethal challenge with influenza A/WSN/33 virus. These results indicate that truncations in the NS1 protein resulting in temperature-sensitive phenotypes in vitro correlate with attenuation in vivo without compromising viral immunogenicity, an ideal characteristic for live attenuated viral vaccines.
Isolation and characterization of a new type of chlorovirus that infects an endosymbiotic Chlorella strain of the heliozoon Acanthocystis turfacea
Authors: Bubeck Julia A. , Pfitzner Artur J. P.
Published: September 2005

A novel virus, named Acanthocystis turfacea Chlorella virus (ATCV), that infects endosymbiotic Chlorella algae of the heliozoon Acanthocystis turfacea was isolated from freshwater samples. Electron microscopic analysis of ATCV revealed that the viral capsid has a distinct icosahedral shape with a diameter of 140–190 nm. Filamentous structures extending from some of the virus vertices, which may aid attachment of the virus to host cells, were also observed. The capsid is made up of one major coat protein of about 50 kDa and contains a large dsDNA genome. ATCV is a member of the genus Chlorovirus, which belongs to the family Phycodnaviridae, a group of large, icosahedral, dsDNA-containing viruses that infect algae and are ubiquitous in natural environments. However, ATCV is clearly distinct from the prototype Chlorovirus, Paramecium bursaria Chlorella virus (PBCV-1), in some aspects of its genome structure and gene content and therefore must be regarded as a member of a new group of Chlorella viruses.
Expression, localization and effects on virulence of the cysteine-rich 8 kDa protein of Potato mop-top virus
Authors: Lukhovitskaya N. I. , Yelina N. E. , Zamyatnin A. A. , Schepetilnikov M. V. , Solovyev A. G. , Sandgren M. , Morozov S. Yu. , Valkonen J. P. T. , Savenkov E. I.
Published: September 2005

Potato mop-top virus (PMTV) RNA3 contains a triple gene block (TGB) encoding viral movement proteins and an open reading frame for a putative 8 kDa cysteine-rich protein (CRP). In this study, PMTV CRP was shown to be expressed in the course of virus infection, and a PMTV CRP-specific subgenomic RNA was mapped. CRP has previously been shown to be dispensable for infection of PMTV in Nicotiana benthamiana. In this study, PMTV CRP was found to increase the severity of disease symptoms when expressed from Potato virus X or Tobacco mosaic virus in N. benthamiana and Nicotiana tabacum, suggesting that the protein affects virulence of the virus or might suppress a host defence mechanism. However, PMTV CRP did not show RNA silencing suppression activity in three assays. Host responses to the PMTV CRP expression from different viral genomes ranged from an absence of response to extreme resistance at a single cell level and were dependent on the viral genome. These findings emphasized involvement of viral proteins and/or virus-induced cell components in the plant reaction to CRP. PMTV CRP was predicted to possess a transmembrane segment. CRP fused to the green fluorescent protein was associated with endoplasmic reticulum-derived membranes and induced dramatic rearrangements of the endoplasmic reticulum structure, which might account for protein functions as a virulence factor of the virus.
Nucleolar localization of potato leafroll virus capsid proteins
Authors: Haupt Sophie , Stroganova Tanya , Ryabov Eugene , Kim Sang Hyon , Fraser Gill , Duncan George , Mayo Mike A. , Barker Hugh , Taliansky Michael
Published: September 2005

Potato leafroll virus (PLRV) encodes two capsid proteins, major protein (CP) and minor protein (P5), an extended version of the CP produced by occasional translational ‘readthrough’ of the CP gene. Immunogold electron microscopy showed that PLRV CP is located in the cytoplasm and also localized in the nucleus, preferentially targeting the nucleolus. The nucleolar localization of PLRV CP was also confirmed when it was expressed as a fusion with green fluorescent protein (GFP) via an Agrobacterium vector. Mutational analysis identified a particular sequence within PLRV CP involved in nucleolar targeting [the nucleolar localization signal (NoLS)]. Minor protein P5 also contains the same NoLS, and was targeted to the nucleolus when it was expressed as a fusion with GFP from Agrobacterium. However, P5–GFP lost its nucleolar localization in the presence of replicating PLRV.
Selection of human immunodeficiency virus type 1 R5 variants with augmented replicative capacity and reduced sensitivity to entry inhibitors during severe immunodeficiency
Authors: Repits Johanna , Öberg Monica , Esbjörnsson Joakim , Medstrand Patrik , Karlsson Anders , Albert Jan , Fenyö Eva Maria , Jansson Marianne
Published: September 2005

Early in human immunodeficiency virus 1 (HIV-1) infection CCR5-using (R5) viruses predominate. With disease progression, approximately 50 % of infected individuals develop viruses able to use CXCR4. In the present work, the evolution of the biological properties of HIV-1 was studied in patients who retain viruses with an R5 phenotype despite AIDS onset. A panel of primary R5 HIV-1 isolates sequentially obtained at an asymptomatic stage and after AIDS diagnosis was examined. The viruses were selected based on our previous observation that R5 variants with reduced sensitivity to RANTES inhibition may appear during disease progression. Biological properties of the early and late R5 viruses, including infectivity, replicative capacity, impact of cationic polymer and sensitivity to inhibition by the entry inhibitors T-20 and TAK-779, were evaluated. R5 viruses isolated after AIDS onset displayed elevated replicative capacity and infectivity, and did not benefit from cationic polymer assistance during infection. Late R5 isolates also exhibited reduced sensitivity to inhibition by T-20 and TAK-779, even though the included patients were naïve to treatment with entry inhibitors and the isolates had not acquired mutations within the gp41 HR1 region. In addition, CD4+ T-cell counts at the time of R5 virus isolation correlated with infectivity, replicative capacity and sensitivity to inhibition by entry inhibitors. The results indicate that R5 HIV-1 variants with augmented replicative capacity and reduced sensitivity to entry inhibitors may be selected for during severe immunodeficiency. At a time when the clinical use of entry inhibitors is increasing, this observation could be of importance in the optimal design of such treatments.
Epitopes in the P2 domain of norovirus VP1 recognized by monoclonal antibodies that block cell interactions
Authors: Lochridge Vance P. , Jutila Kathryn L. , Graff Joel W. , Hardy Michele E.
Published: September 2005

Noroviruses cause the majority of epidemic outbreaks of acute viral gastroenteritis worldwide. Human norovirus strains do not grow in cell culture, but recent carbohydrate binding, sequence and structural analyses have begun to define functional domains in the norovirus capsid that may be conserved among multiple antigenic types. The purpose of this study was to localize domains and define sequences in the major capsid protein VP1 that are important for cell interactions. Monoclonal antibodies to genogroups GI.1 and GII.2 reference strains Norwalk virus and Snow Mountain virus, respectively, were generated that blocked binding of recombinant virus-like particles to Caco-2 intestinal cells and inhibited haemagglutination. Peptides that mimicked the mAb binding epitopes were selected from a phage-displayed random nonapeptide library. Anti-recombinant Norwalk virus mAb 54.6 and anti-recombinant Snow Mountain virus mAb 61.21 recognized epitopes located in the protruding P2 domain of VP1. The epitope recognized by mAb 61.21 contained amino acids that are completely conserved among norovirus strains across genogroups, including strains isolated from swine, bovine and murine species. This study identifies the first epitope involved in inhibition of norovirus–cell interactions and supports increasing evidence that interactions between noroviruses and host cells rely on structures in the P2 domain of VP1.
Contribution of the charged residues of hepatitis C virus glycoprotein E2 transmembrane domain to the functions of the E1E2 heterodimer
Authors: Ciczora Yann , Callens Nathalie , Montpellier Claire , Bartosch Birke , Cosset François-Loïc , De Beeck Anne Op , Dubuisson Jean
Published: September 2005

The envelope glycoproteins of Hepatitis C virus (HCV), E1 and E2, form a heterodimer that is retained in the endoplasmic reticulum (ER). The transmembrane (TM) domains play a major role in E1E2 heterodimerization and in ER retention. Two fully conserved charged residues in the middle of the TM domain of E2 (Asp and Arg) are crucial for these functions. Replacement of the Asp residue by a Leu impaired E1E2 heterodimerization, whereas the Arg-to-Leu mutation had a milder effect. Both Asp and Arg residues were shown to contribute to the ER retention function of E2. In addition, the entry function of HCV envelope glycoproteins was affected by these mutations. Together, these data indicate that the charged residues present in the TM domain of E2 play a major role in the biogenesis and the entry function of the E1E2 heterodimer. However, the Asp and Arg residues do not contribute equally to these functions.
The cotton rat provides a useful small-animal model for the study of influenza virus pathogenesis
Authors: Ottolini Martin G. , Blanco Jorge C. G. , Eichelberger Maryna C. , Porter David D. , Pletneva Lioubov , Richardson Joann Y. , Prince Gregory A.
Published: September 2005

Influenza A virus continues to cause annual epidemics. The emergence of avian viruses in the human population poses a pandemic threat, and has highlighted the need for more effective influenza vaccines and antivirals. Development of such therapeutics would be enhanced by the use of a small-animal model that is permissive for replication of human influenza virus, and for which reagents are available to dissect the host response. A model is presented of nasal and pulmonary infection in adult inbred cotton rats (Sigmodon hispidus) that does not require viral ‘adaptation’. It was previously demonstrated that animals infected intranasally with 107TCID50of a recent H3N2 influenza, A/Wuhan/359/95, have increased breathing rates. In this report it is shown that this is accompanied by weight loss and decreased temperature. Virus replication peaked within 24 h in the lung, with peak titres proportional to the infecting dose, clearing by day 3. Replication was more permissive in nasal tissues, and persisted for 6 days. Pulmonary pathology included early bronchiolar epithelial cell damage, followed by extensive alveolar and interstitial pneumonia, which persisted for nearly 3 weeks. Interleukin 1 alpha (IL1α), alpha interferon (IFN-α), IL6, tumour necrosis factor alpha (TNF-α), GROαand MIP-1βmRNA were elevated soon after infection, and expression coincided with virus replication. A biphasic response was observed for RANTES, IFN-γ, IL4, IL10 and IL12-p40, with increased mRNA levels early during virus replication followed by a later increase that coincided with pulmonary inflammation. These results indicate that cotton rats will be useful for further studies of influenza pathogenesis and immunity.
Virulence and competitiveness of Cydia pomonella granulovirus mutants: parameters that do not match
Authors: Arends Hugo M. , Winstanley Doreen , Jehle Johannes A.
Published: September 2005

The LD50, median survival time (ST50) and virus production are virulence parameters that are commonly used to describe the biological characteristics of viruses. In this study, these parameters were determined for Cydia pomonella granulovirus (CpGV-M) and two naturally occurring mutants (CpGV-MCp4 and -MCp5) that carry Tc1-like insect transposable elements. The three virus genotypes were similar in their LD50, ST50 and virus production. However, the mutant genotypes MCp4 and MCp5 were very effectively out-competed by CpGV-M in direct competition experiments, where Cydia pomonella larvae were co-infected with known ratios of occlusion bodies or budded virus of CpGV-M and one of the two mutants. It was demonstrated that MCp5 and MCp4 could not be sustained in the virus population when the progeny viruses of different co-infections were used as inocula to infect next passage larvae. These results show that the virulence parameters LD50, ST50 and virus production alone do not adequately reflect the competitiveness of the virus and are thus not suitable to describe virus population dynamics.
Characterization and genetic variability of Hepatitis A virus genotype IIIA
Authors: Stene-Johansen Kathrine , Jonassen Tom Øystein , Skaug Kjell
Published: September 2005

Molecular epidemiological studies of hepatitis A outbreaks in Norway showed the emergence of Hepatitis A virus (HAV) genotype IIIA in association with parenteral transmission among haemophiliacs and intravenous drug users. The complete genomic sequence of one of these outbreak isolates, NOR-21, was determined. This is the first complete genomic sequence of HAV genotype IIIA. Phylogenetic analysis showed that genotype IIIA/NOR-21 was genetically distinct from the other human and simian genotypes. Phylogenetic analysis of the nucleotide sequences clearly distinguished the different HAV genotypes, regardless of the genomic region used for analysis, whereas the amino acid sequences showed a more vague distinction between human HAV genotypes I and II. In particular, the inferred phylogeny based on the capsid proteins showed that the human HAV strains were related more closely to each other than to the simian strains. The greatest variability and clearest distinction between genotypes were observed for the polymerase gene. The outbreak isolates of HAV genotype IIIA in this study showed greater nucleotide variability than is generally seen in outbreaks of genotype I. This high nucleotide variability, which may be characteristic of this HAV genotype, the mode of transmission in this outbreak or parallel introductions, is discussed.
IgA-coated particles of Hepatitis A virus are translocalized antivectorially from the apical to the basolateral site of polarized epithelial cells via the polymeric immunoglobulin receptor
Authors: Dotzauer Andreas , Brenner Meike , Gebhardt Ulrike , Vallbracht Angelika
Published: September 2005

AlthoughHepatitis A virus(HAV) is transmitted by the faecal–oral route, its target for replication is the liver. Little is known of its interactions with cells of the gastrointestinal tract, and it is not known by which mechanisms HAV crosses the intestinal epithelium. In this study, it is shown that HAV associated with IgA is translocated from the apical to the basolateral compartment of polarized epithelial cells via the polymeric immunoglobulin receptor by IgA-mediated reverse transcytosis. The relevance of this mechanism, by which HAV–IgA complexes may overcome the intestinal barrier and contribute to infections of the liver, results from the fact that HAV–IgA complexes are infectious for hepatocytes and that significant amounts of intestinal HAV–IgA are present during acute infections, which are also partly transmitted. Besides supporting the primary infection, this mechanism may play a role in relapsing infections by establishing an enterohepatic cycle for HAV.
Phylogenetic analysis of isolates of Beet necrotic yellow vein virus collected worldwide
Authors: Schirmer Audrey , Link Didier , Cognat Valérie , Moury Benoît , Beuve Monique , Meunier Alexandre , Bragard Claude , Gilmer David , Lemaire Olivier
Published: September 2005

A study of molecular diversity was carried out on 136 sugar beets infected withBeet necrotic yellow vein virus(BNYVV,Benyvirus) collected worldwide. The nucleotide sequences of the RNA-2-encoded CP, RNA-3-encoded p25 and RNA-5-encoded p26 proteins were analysed. The resulting phylogenetic trees allowed BNYVV to be classified into groups that show correlations between the virus clusters and geographic origins. The selective constraints on these three sequences were measured by estimating the ratio between synonymous and non-synonymous substitution rates (ω) with maximum-likelihood models. The results suggest that selective constraints are exerted differently on the proteins. CP was the most conserved, with meanωvalues ranging from 0·12 to 0·15, while p26 was less constrained, with meanωvalues ranging from 0·20 to 0·33. Selection was detected in three amino acid positions of p26, withωvalues of about 5·0. The p25 sequences presented the highest meanωvalues (0·36–1·10), with strong positive selection (ω=4·7–54·7) acting on 14 amino acids, and particularly on amino acid 68, where theωvalue was the highest so far encountered in plant viruses.
Restoration of virulence of escape mutants of H5 and H9 influenza viruses by their readaptation to mice
Authors: Rudneva Irina A. , Ilyushina Natalia A. , Timofeeva Tatiana A. , Webster Robert G. , Kaverin Nikolai V.
Published: September 2005

Antigenic mapping of the haemagglutinin (HA) molecule of H5 and H9 influenza viruses by selecting escape mutants with monoclonal anti-HA antibodies and subjecting the selected viruses to immunological analysis and sequencing has previously been performed. The viruses used as wild-type strains were mouse-adapted variants of the original H5 and H9 isolates. Phenotypic characterization of the escape mutants revealed that the amino acid change in HA that conferred resistance to a monoclonal antibody was sometimes associated with additional effects, including decreased virulence for mice. In the present study, the low-virulence H5 and H9 escape mutants were readapted to mice. Analysis of the readapted variants revealed that the reacquisition of virulence was not necessarily achieved by reacquisition of the wild-type HA gene sequence, but was also associated either with the removal of a glycosylation site (the one acquired previously by the escape mutant) without the exact restoration of the initial wild-type amino acid sequence, or, for an H5 escape mutant that had no newly acquired glycosylation sites, with an additional amino acid change in a remote part of the HA molecule. The data suggest that such ‘compensating’ mutations, removing the damaging effects of antibody-selected amino acid changes, may be important in the course of influenza virus evolution.
Location of, immunogenicity of and relationships between neutralization epitopes on the attachment protein (G) of Hendra virus
Authors: White John R. , Boyd Victoria , Crameri Gary S. , Duch Christine J. , van Laar Ryan K. , Wang Lin-Fa , Eaton Bryan T.
Published: September 2005

Epitopes involved in a protective immune response to Hendra virus (HeV) (Henipavirus, Paramxyoviridae) were investigated by generating five neutralizing monoclonal antibodies (mAbs) to the virus attachment protein (G) of HeV (HeV G) and sequencing of the G gene of groups of neutralization-escape variants selected with each mAb. Amino acid substitutions occurred at eight distinct sites on HeV G. Relationships between these sites were investigated in binding and neutralization assays using heterologous combinations of variants and mAbs. The sites were also mapped to a proposed structural model for the attachment proteins of Paramyxoviridae. Their specific locations and the nature of their interactions with the mAb panel provided the first functional evidence that HeV G in fact resembled the proposed structure. Four sites (aa 183–185, 417, 447 and 570) contributed to a major discontinuous epitope, on the base of the globular head, that was similar to immunodominant virus neutralization sites found in other paramyxoviruses. Amino acid similarity between HeV and Nipah virus was relatively highly conserved at these sites but decreased significantly at the other sites identified in this study. These included another discontinuous epitope on the base of the head region defined by sites aa 289 and 324 and well separated epitopes on the top of the head at sites aa 191–195 and 385–356. The latter epitope corresponded to immunodominant neutralization sites found in Rinderpest virus and Measles virus.
Quantitative Modeling of Arabidopsis Development
Authors: Mündermann Lars , Erasmus Yvette , Lane Brendan , Coen Enrico , Prusinkiewicz Przemyslaw
Published: September 2005

We present an empirical model of Arabidopsis (Arabidopsis thaliana), intended as a framework for quantitative understanding of plant development. The model simulates and realistically visualizes development of aerial parts of the plant from seedling to maturity. It integrates thousands of measurements, taken from several plants at frequent time intervals. These data are used to infer growth curves, allometric relations, and progression of shapes over time, which are incorporated into the final three-dimensional model. Through the process of model construction, we identify the key attributes required to characterize the development of Arabidopsis plant form over time. The model provides a basis for integrating experimental data and constructing mechanistic models.
The Sireviruses, a Plant-Specific Lineage of the Ty1/copia Retrotransposons, Interact with a Family of Proteins Related to Dynein Light Chain 8
Authors: Havecker Ericka R. , Gao Xiang , Voytas Daniel F.
Published: September 2005

Plant genomes are rich in long terminal repeat retrotransposons, and here we describe a plant-specific lineage of Ty1/copia elements called the Sireviruses. The Sireviruses vary greatly in their genomic organization, and many have acquired additional coding information in the form of an envelope-like open reading frame and an extended gag gene. Two-hybrid screens were conducted with the novel domain of Gag (the Gag extension) encoded by a representative Sirevirus from maize (Zea mays) called Hopie. The Hopie Gag extension interacts with a protein related to dynein light chain 8 (LC8). LC8 also interacts with the Gag extension from a Hopie homolog from rice (Oryza sativa). Amino acid motifs were identified in both Hopie Gag and LC8 that are responsible for the interaction. Two amino acids critical for Gag recognition map within the predicted LC8-binding cleft. Two-hybrid screens were also conducted with the Gag extension encoded by the soybean (Glycine max) SIRE1 element, and an interaction was found with light chain 6 (LC6), a member of the LC8 protein family. LC8 and LC6 proteins are components of the dynein microtubule motor, with LC8 being a versatile adapter that can bind many unrelated cellular proteins and viruses. Plant LC8 and LC6 genes are abundant and divergent, yet flowering plants do not encode other components of the dynein motor. Although, to our knowledge, no cellular roles for plant LC8 family members have been proposed, we hypothesize that binding of LC8 proteins to Gag aids in the movement of retrotransposon virus-like particles within the plant cell or possibly induces important conformational changes in the Gag protein.
Drinking pattern among adolescents with immigrant and Norwegian backgrounds: A two‐way influence?
Authors: Amundsen Ellen J. , Rossow Ingeborg , Skurtveit Svetlana
Published: September 2005

Aims  To assess the association between drinking behaviour among adolescents with both immigrant and native backgrounds with aspects of acculturation.

Design  Cross‐sectional school survey among 15–16 year olds.

Setting and participants  All students, a total of 8361, in 10th grade during spring 2000 and 2001 in Oslo, Norway with response rate 88.3%. The sample consisted of 1213 students with an immigrant background and 4627 students with a Norwegian background.

Measurements  Indicators of the length of the acculturation process were assessed as first versus second generation and in terms of length of stay in Norway. The proportion of Muslim students in school was an indicator of social environment ‘dryness’. Drinking behaviour was assessed as drinking alcohol ever, drinking frequency and intoxication frequency.

Findings  A smaller proportion of immigrant students were current drinkers, frequent drinkers and drank to intoxication compared with adolescents with a Norwegian background. Adjusted two‐level analyses showed that alcohol drinking was less common among immigrant students with a short stay in Norway and with a large proportion of Muslim students in school. In those with a Norwegian background there was a larger proportion of abstainers, and those who drank did so less frequently and were less frequently intoxicated the larger the proportion of Muslim students there was in their school.

Conclusion  Drinking behaviour among adolescents in a multicultural and heterogeneous society seems to reflect a bi‐directional acculturation process where the majority population tend to adapt to the behaviours of the immigrant population which in turn, to a varying degree, tends to adapt to the behaviour of the majority population.
Young children's understandings of cigarette smoking
Authors: Freeman Dan , Brucks Merrie , Wallendorf Melanie
Published: September 2005

Aims We explore young children's attitudes toward, beliefs about, and life‐style associations with cigarette smoking using direct and indirect measures.

Design, setting and participants Second (n = 100) and fifth grade (n = 141) elementary school students (i.e. 7–8 and 10–11‐year‐olds) were excused from class and individually interviewed.

Methods Participants selected pictures in response to the questions: who would like to smoke cigarettes the most and who would like to smoke cigarettes the least? Their picture choices were probed using open‐ended prompts designed to elicit the beliefs and life‐style associations underlying their choices. Survey‐based measures of attitudes and beliefs were also collected.

Findings Second graders reported life‐style associations with cigarette smoking that were consistent with those of fifth graders. While their associations with smoking are generally negative, children appear to perceive that others feel that smoking makes them look cool and feel cool and also helps them to fit in. By fifth grade, many children believe that smoking can help to reduce stress and alleviate negative mood states. The presence of a smoker in the household does not appear to affect these associations, suggesting that they may be being shaped by external socialization agents.

Conclusion Young children appear to be developing understandings of cigarette smoking that go beyond knowing that cigarettes are products that are smoked. As some of their perceptions appear likely to predispose them for future experimentation, young children need to be included in prevention research so that age‐appropriate interventions can be developed.