Decision-making in imminent extreme premature births: perceived shared decision-making, parental decisional conflict and decision regret.

OBJECTIVE: To describe levels of perceived shared decision making (SDM), decisional conflict (DC), and decision regret (DR) in prenatal counseling by pregnant women, partners, neonatologists, and obstetricians regarding decision-making around imminent extreme premature birth in which a decision about palliative comfort care versus early intensive care had to be made. STUDY DESIGN: Multicenter, cross-sectional study using surveys to determine perceived SDM at imminent extreme premature birth in parents and physicians, and to determine DC and DR in parents. RESULTS: In total, 73 participants from 22 prenatal counseling sessions were included (21 pregnant women, 20 partners, 14 obstetricians, 18 neonatologists). High perceived levels of SDM were found (median 82,2), and low levels of DC (median 23,4) and DR at one month (median 12, 5). CONCLUSIONS: Reported levels of self-perceived SDM in the setting of prenatal counseling in extreme prematurity were high, by both the parents and the physicians. Levels of DC and DR were low.

Home-based telemonitoring versus hospital admission in high risk pregnancies: a qualitative study on women's experiences.

BACKGROUND: Hospital admission during pregnancy complications is considered to be an event of significant impact. Besides conventional in-clinic maternal and fetal monitoring, recent technologies enable home-based telemonitoring with self-measurements in high risk pregnancy. This study is part of a feasibility pilot to explore the usability and acceptability of telemonitoring and aims to gain insight in the experiences and preferences of high risk pregnant women concerning the novel strategy of telemonitoring, opposed to women who were hospitalized in pregnancy. METHODS: Using secured Facebook Groups, we conducted four online focus groups: two focus groups with women who were admitted during pregnancy (n = 11) and two with women who received home telemonitoring in the pilot phase (n = 11). The qualitative data were analyzed thematically. RESULTS: Four major themes emerged from both participant groups: [1] care experience, [2] emotions regarding pregnancy, [3] privacy and [4] impact on daily life. Different views were reported on all four themes, resulting in a direct comparison of experiences during hospitalization and telemonitoring. Most admitted patients reported a growing sense of boredom and anxiety during their clinical admission. Lack of privacy on ward was a great concern, as it affected their contact with hospital staff and family. This issue was not reported amongst telemonitored women. These participants still felt like a patient at times but responded that the comfort of their own home and bed was pleasant. Only a minority of telemonitored participants reported being anxious at times at home, while not having a physician or nurse nearby. Being at home resulted in less travel time for partners or family for hospital visits, which had its positive effects on family life. CONCLUSIONS: Telemonitoring of a high-risk pregnancy provides an innovative manner to monitor fetal and maternal condition from home. Compared to the experiences of hospital admission in high risk pregnancy, it allows women to be in a comforting and private environment during an anxious time in their lives. As future studies should further investigate the safety and cost effectiveness of this novel strategy, women's views on the preference of telemonitoring need to be taken into consideration.

Tomato marchitez virus, a new plant picorna-like virus from tomato related to tomato torrado virus.

A new virus was isolated from a tomato plant from the state of Sinaloa in Mexico. This plant showed symptoms locally known as 'marchitez disease': severe leaf necrosis, beginning at the base of the leaflets, and necrotic rings on the fruits. A virus was isolated from the infected plant consisting of isometric particles with a diameter of approximately 28 nm. The viral genome consists of two (+)ssRNA molecules of 7221 (RNA1) and 4898 nts (RNA2). The viral capsid contains three coat proteins of 35, 26 and 24 kDa, respectively. The abovementioned characteristics: symptoms, morphology, number and size of coat proteins, and number of RNAs are similar to those of the previously described tomato torrado virus (ToTV). Sequence analysis of the entire viral genome shows that this new virus is related to, but distinct from, ToTV and that these members of two obviously new virus species belong to the recently proposed plant virus genus Torradovirus. For this new virus, the name tomato marchitez virus (ToMarV) is proposed.

Identification and characterisation of tomato torrado virus, a new plant picorna-like virus from tomato.

A new virus was isolated from tomato plants from the Murcia region in Spain which showed symptoms of 'torrado disease'; very distinct necrotic, almost burn-like symptoms on leaves of infected plants. The virus particles are isometric with a diameter of approximately 28 nm. The viral genome consists of two (+)ssRNA molecules of 7793 (RNA1) and 5389 nts (RNA2). RNA1 contains one open reading frame (ORF) encoding a predicted polyprotein of 241 kDa that shows conserved regions with motifs typical for a protease-cofactor, a helicase, a protease and an RNA-dependent RNA polymerase. RNA2 contains two, partially overlapping ORFs potentially encoding proteins of 20 and 134 kDa. These viral RNAs are encapsidated by three proteins with estimated sizes of 35, 26 and 23 kDa. Direct protein sequencing mapped these coat proteins to ORF2 on RNA2. Phylogenetic analyses of nucleotide and derived amino acid sequences showed that the virus is related to but distinct from viruses belonging to the genera Sequivirus, Sadwavirus and Cheravirus. This new virus, for which the name tomato torrado virus is proposed, most likely represents a member of a new plant virus genus.

Characterization of a new densovirus infecting the green peach aphid Myzus persicae.

A new icosahedral DNA virus was isolated from aphids (Myzus persicae) that showed abnormal growth and development. The purified virus particles have a diameter of 20 nm and contain a single-stranded DNA molecule of approximately 5.7 kb. The viral particles are composed of five structural proteins (92, 85, 68, 64, and 57 kDa). As the main biophysical properties of this virus are similar to those of the members of the genus Densovirus it was tentatively named Myzus persicae densovirus (MpDNV). A PCR-based detection method and a polyclonal antiserum raised against MpDNV allowed the detection of the virus in a single-infected aphid. MpDNV is immunologically related to Junonia coenia densovirus, but not to other members of the subfamily Densovirinae. Biological assays showed that MpDNV could be both transmitted transovarially and horizontally via honeydew and saliva. MpDNV was able to infect whiteflies but not other aphid species tested.

A new virus infecting Myzus persicae has a genome organization similar to the species of the genus Densovirus.

The genomic sequence of a new icosahedral DNA virus infecting Myzus persicae has been determined. Analysis of 5499 nt of the viral genome revealed five open reading frames (ORFs) evenly distributed in the 5' half of both DNA strands. Three ORFs (ORF1-3) share the same strand, while two other ORFs (ORF4 and ORF5) are detected in the complementary sequence. The overall genomic organization is similar to that of species from the genus DENSOVIRUS: ORFs 1-3 most likely encode the non-structural proteins, since their putative products contain conserved replication motifs, NTP-binding domains and helicase domains similar to those found in the NS-1 protein of parvoviruses. The deduced amino acid sequences from ORFs 4 and 5 show sequence similarities with the structural proteins of the members of the genus DENSOVIRUS: These data indicate that this virus is a new species of the genus Densovirus in the family PARVOVIRIDAE: The virus was tentatively named Myzus persicae densovirus.

Sequence analysis and genomic organization of Aphid lethal paralysis virus: a new member of the family Dicistroviridae.

The complete nucleotide sequence of the genomic RNA of an aphid-infecting virus, Aphid lethal paralysis virus (ALPV), has been determined. The genome is 9812 nt in length and contains two long open reading frames (ORFs), which are separated by an intergenic region of 163 nt. The first ORF (5' ORF) is preceded by an untranslated leader sequence of 506 nt, while an untranslated region of 571 nt follows the second ORF (3' ORF). The deduced amino acid sequences of the 5' ORF and 3' ORF products respectively showed similarity to the non-structural and structural proteins of members of the newly recognized genus Cripavirus (family Dicistroviridae). On the basis of the observed sequence similarities and identical genome organization, it is proposed that ALPV belongs to this genus. Phylogenetic analysis showed that ALPV is most closely related to Rhopalosiphum padi virus, and groups in a cluster with Drosophila C virus and Cricket paralysis virus, while the other members of this genus are more distantly related. Infectivity experiments showed that ALPV can not only infect aphid species but is also able to infect the whitefly Trialeurodes vaporariorum, extending its host range to another family of the order Hemiptera.

Nucleotide sequence and genomic organization of an ophiovirus associated with lettuce big-vein disease.

The complete nucleotide sequence of an ophiovirus associated with lettuce big-vein disease has been elucidated. The genome consisted of four RNA molecules of approximately 7.8, 1.7, 1.5 and 1.4 kb. Virus particles were shown to contain nearly equimolar amounts of RNA molecules of both polarities. The 5'- and 3'-terminal ends of the RNA molecules are largely, but not perfectly, complementary to each other. The virus genome contains seven open reading frames. Database searches with the putative viral products revealed homologies with the RNA-dependent RNA polymerases of rhabdoviruses and Ranunculus white mottle virus, and the capsid protein of Citrus psorosis virus. The gene encoding the viral polymerase appears to be located on the RNA segment 1, while the nucleocapsid protein is encoded by the RNA3. No significant sequence similarities were observed with other viral proteins. In spite of the morphological resemblance with species in the genus Tenuivirus, the ophioviruses appear not to be evolutionary closely related to this genus nor any other viral genus.

Development of a multiplex AmpliDet RNA assay for simultaneous detection and typing of potato virus Y isolates.

A multiplex AmpliDet RNA assay was developed for the specific detection of potato virus Y (PVY), and for the differentiation of the PVY(N), PVY(O/C) strains and the tuber necrotic isolates (PVY(NTN)). The assay is based on the generic amplification of a region within the coat protein coding region of all known PVY isolates by nucleic acid sequence-based amplification (NASBA) and strain-specific detection by molecular beacons. PVY(NTN)-specific diagnosis is achieved by detecting PVY(N) and PVY(O)-specific sequences flanking a recombination site that is associated with the tuber necrotic pathotype. The assay exhibited good specificity toward the various PVY strains in both single and mixed infections. The technique was validated by the use of 47 PVY isolates originating from six countries. The results of the AmpliDet RNA assay were identical or consistent with those of biological characterisation in the decisive majority of cases.

Studies on the role of the minor capsid protein in transport of Beet western yellows virus through Myzus persicae.

Beet western yellows virus (BWYV), family Luteoviridae, is an icosahedral plant virus which is strictly transmitted by aphids in a persistent and circulative manner. Virions cross two cellular barriers in the aphid by receptor-based mechanisms involving endocytosis and exocytosis. Particles are first transported across intestinal cells into the haemolymph and then across accessory salivary gland cells for delivery to the plant via saliva. We identified the midgut part of the digestive tract as the site of intestinal passage by BWYV virions. To analyse the role in transmission of the minor capsid component, the readthrough (RT) protein, the fate of a BWYV RT-deficient non-transmissible mutant was followed by transmission electron microscopy in the vector Myzus persicae. This mutant was observed in the gut lumen but was never found inside midgut cells. However, virion aggregates were detected in the basal lamina of midgut cells when BWYV antiserum was microinjected into the haemolymph. The presence of virions in the haemolymph was confirmed by a sensitive molecular technique for detecting viral RNA. Thus, transport of the mutant virions through intestinal cells occurred but at a low frequency. Even when microinjected into the haemolymph, the RT protein mutant was never detected near or in the accessory salivary gland cells. We conclude that the RT protein is not strictly required for the transport of virus particles through midgut cells, but is necessary for the maintenance of virions in the haemolymph and their passage through accessory salivary gland cells.

Development of a multiplex AmpliDet RNA for the simultaneous detection of Potato leafroll virus and Potato virus Y in potato tubers.

A novel isothermal multiplex AmpliDet RNA system is described for the simultaneous amplification and detection of Potato leafroll virus (PLRV) and Potato virus Y (PVY) in seed potatoes. The risk of contamination by carry-over during diagnostic screening is eliminated by performing the reaction in a single closed tube. The viruses present in a sample are identified using differently coloured molecular beacons directed to a selected virus-specific sequence within the amplicon formed during amplification. With this system, as little as 10 fg of purified PLRV or PVY can be detected. The presence of both viruses in a sample is detected by the multiplex assay within a high range of virus concentrations. The reliability of the multiplex assay was compared with the enzyme-linked immunosorbent assay for detection of PLRV- or PVY-antigens in potato tubers. The multiplex assay detected clearly the viruses present originally in the potato tubers in all samples, demonstrating its potential for routine diagnostic work and high-throughput screening.

Mechanical transmission of poleroviruses.

Previously, transmission of poleroviruses has relied solely on the use of their aphid vectors. Biolistic inoculation allowed for the first time the mechanical transmission of Beet western yellows virus (BWYV) and Potato leafroll virus (PLRV) to several host plants. Inoculation with purified preparations and viral RNA extracts of PLRV resulted in 30-50% systemically infected Nicotiana occidentalis P1 plants and 15-30% infected Nicotiana clevelandii plants. Particle bombardment was also used successfully to infect N. clevelandii plants with in vitro RNA transcripts of full-length cDNA of BWYV.

Rapid and Sensitive Detection of Apple stem pitting virus in Apple Trees Through RNA Amplification and Probing with Fluorescent Molecular Beacons.

ABSTRACT Currently, detection of Apple stem pitting virus (ASPV; genus Foveavirus) in apple trees for certification purposes occurs by woody indexing. This method requires a minimum of 12 to 24 weeks in greenhouse testing to up to 2 years in field testing. In this paper, the development of a single tube AmpliDet RNA system for the rapid gel-free detection of ASPV in apple tree tissues is described. The system relies on the specific amplification of the viral RNA by nucleic acid sequence-based amplification and the simultaneous fluorescent detection of the amplification product through molecular beacons. A sensitivity of a minimum of 100 molecules of transcript RNA was obtained by the ASPV-specific AmpliDet RNA. All biologically characterized ASPV isolates from a field trial and 12 of 14 isolates from a plant virus collection were readily detected with this AmpliDet RNA system. In addition, the efficiency of this method for detecting ASPV in 'Golden Delicious' and 'Gravenstein' apple trees was compared throughout the year with mechanical inoculation onto Nicotiana occidentalis 37B, a candidate indicator for ASPV. This revealed that only AmpliDet RNA consistently detected the virus in bark tissue, irrespective of the season. Season-specific tissues such as buds, petals, and fruits, but not leaves, also were reliable sources for detection of ASPV by the AmpliDet RNA system.

Identifying the determinants in the equatorial domain of Buchnera GroEL implicated in binding Potato leafroll virus.

Luteoviruses avoid degradation in the hemolymph of their aphid vector by interacting with a GroEL homolog from the aphid's primary endosymbiotic bacterium (Buchnera sp.). Mutational analysis of GroEL from the primary endosymbiont of Myzus persicae (MpB GroEL) revealed that the amino acids mediating binding of Potato leafroll virus (PLRV; Luteoviridae) are located within residues 9 to 19 and 427 to 457 of the N-terminal and C-terminal regions, respectively, of the discontinuous equatorial domain. Virus overlay assays with a series of overlapping synthetic decameric peptides and their derivatives demonstrated that R13, K15, L17, and R18 of the N-terminal region and R441 and R445 of the C-terminal region of the equatorial domain of GroEL are critical for PLRV binding. Replacement of R441 and R445 by alanine in full-length MpB GroEL and in MpB GroEL deletion mutants reduced but did not abolish PLRV binding. Alanine substitution of either R13 or K15 eliminated the PLRV-binding capacity of the other and those of L17 and R18. In the predicted tertiary structure of GroEL, the determinants mediating virus binding are juxtaposed in the equatorial plain.

Isolation and characterization of APSE-1, a bacteriophage infecting the secondary endosymbiont of Acyrthosiphon pisum.

A bacteriophage infecting the secondary endosymbiont of the pea aphid Acyrthosiphon pisum was isolated and characterized. The phage was tentatively named bacteriophage APSE-1, for bacteriophage 1 of the A. pisum secondary endosymbiont. The APSE-1 phage particles morphologically resembled those of species of the Podoviridae. The complete nucleotide sequence of the bacteriophage APSE-1 genome was elucidated, and its genomic organization was deduced. The genome consists of a circularly permuted and terminally redundant double-stranded DNA molecule of 36524 bp. Fifty-four open reading frames, putatively encoding proteins with molecular masses of more than 8 kDa, were distinguished. ORF24 was identified as the gene coding for the major head protein by N-terminal amino acid sequencing of the protein. Comparison of APSE-1 sequences with bacteriophage-derived sequences present in databases revealed the putative function of 24 products, including the lysis proteins, scaffolding protein, transfer proteins, and DNA polymerase. This is the first report of a phage infecting an endosymbiont of an arthropod.

Faba bean necrotic yellows virus (genus Nanovirus) requires a helper factor for its aphid transmission.

Purified faba bean necrotic yellows virus (FBNYV; genus Nanovirus) alone is not transmissible by its aphid vector, Acyrthosiphon pisum, regardless of whether it is acquired from artificial diets or directly microinjected into the aphid's hemocoel. The purified virus contains all of the genetic information required for its infection cycle as it readily replicated in cowpea protoplasts and systemically infected Vicia faba seedlings that were biolistically inoculated using gold particles coated with intact virions or viral DNA. The bombarded plants not only developed the typical disease syndrome, thus indicating that FBNYV is the sole causal agent of the disease, but also served as a source from which the virus was readily acquired and transmitted by A. pisum. The defect of the purified virus in aphid transmissibility suggests that FBNYV requires a helper factor (HF) for its vector transmission that is either nonfunctional or absent in purified virus suspensions. The requirement for an HF was confirmed in complementation experiments using two distinct isolates of the virus. These experiments revealed that aphids transmitted the purified virus isolate from artificial diets only when they had fed previously on plants infected with the other FBNYV isolate. Also, microinjected FBNYV, which persisted to the same extent in A. pisum as naturally acquired virus, was transmissible when aphids had acquired the HF from infected plants. This suggests that one of the functions of the HF in the transmission process is to facilitate virus transport across the hemocoel-salivary gland interface.

A GroEL homologue from endosymbiotic bacteria of the whitefly Bemisia tabaci is implicated in the circulative transmission of tomato yellow leaf curl virus.

Evidence for the involvement of a Bemisia tabaci GroEL homologue in the transmission of tomato yellow leaf curl geminivirus (TYLCV) is presented. A approximately 63-kDa protein was identified in B. tabaci whole-body extracts using an antiserum raised against aphid Buchnera GroEL. The GroEL homologue was immunolocalized to a coccoid-shaped whitefly endosymbiont. The 30 N-terminal amino acids of the whitefly GroEL homologue showed 80% homology with that from different aphid species and GroEL from Escherichia coli. Purified GroEL from B. tabaci exhibited ultrastructural similarities to that of the endosymbiont from aphids and E. coli. In vitro ligand assays showed that tomato yellow leaf curl virus (TYLCV) particles displayed a specific affinity for the B. tabaci 63-kDa GroEL homologue. Feeding whiteflies anti-Buchnera GroEL antiserum before the acquisition of virions reduced TYLCV transmission to tomato test plants by >80%. In the haemolymph of these whiteflies, TYLCV DNA was reduced to amounts below the threshold of detection by Southern blot hybridization. Active antibodies were recovered from the insect haemolymph suggesting that by complexing the GoEL homologue, the antibody disturbed interaction with TYLCV, leading to degradation of the virus. We propose that GroEL of B. tabaci protects the virus from destruction during its passage through the haemolymph.

Recognition and receptors in virus transmission by arthropods.

Fundamental knowledge of the molecular mechanisms underlying virus transmission by arthropods is a prerequisite for the creation of new strategies to modulate vector competence. There have been several recent advances in identifying the viral and vector determinants involved in virus recognition, attachment and retention.

Potato leafroll virus binds to the equatorial domain of the aphid endosymbiotic GroEL homolog.

A GroEL homolog with a molecular mass of 60 kDa, produced by the primary endosymbiotic bacterium (a Buchnera sp.) of Myzus persicae and released into the hemolymph, has previously been shown to be a key protein in the transmission of potato leafroll virus (PLRV). Like other luteoviruses and pea enation mosaic virus, PLRV readily binds to extracellular Buchnera GroEL, and in vivo interference in this interaction coincides with reduced capsid integrity and loss of infectivity. To gain more knowledge of the nature of the association between PLRV and Buchnera GroEL, the groE operon of the primary endosymbiont of M. persicae (MpB groE) and its flanking sequences were characterized and the PLRV-binding domain of Buchnera GroEL was identified by deletion mutant analysis. MpB GroEL has extensive sequence similarity (92%) with Escherichia coli GroEL and other members of the chaperonin-60 family. The genomic organization of the Buchnera groE operon is similar to that of the groE operon of E. coli except that a constitutive promoter sequence could not be identified; only the heat shock promoter was present. By a virus overlay assay of protein blots, it was shown that purified PLRV bound as efficiently to recombinant MpB GroEL (expressed in E. coli) as it did to wild-type MpB GroEL. Mutational analysis of the gene encoding MpB GroEL revealed that the PLRV-binding site was located in the so-called equatorial domain and not in the apical domain which is generally involved in polypeptide binding and folding. Buchnera GroEL mutants lacking the entire equatorial domain or parts of it lost the ability to bind PLRV. The equatorial domain is made up of two regions at the N and C termini that are not contiguous in the amino acid sequence but are in spatial proximity after folding of the GroEL polypeptide. Both the N- and C-terminal regions of the equatorial domain were implicated in virus binding.

Nucleotide sequence and genomic organization of Acyrthosiphon pisum virus.

The nucleotide sequence of the genomic RNA of Acyrthosiphon pisum virus was determined. The APV genome is 10,016 nucleotides in length, excluding the 3'-end poly(A) track, and contains two large open reading frames (ORFs), encoding proteins of 296,340 and 63,279 Da. The ORF1 is preceded by an untranslated leader sequence of 267 nucleotides. The ORF1 product contains sequence motifs characteristic of RNA-dependent RNA polymerases, chymotrypsin-like proteases, and helicases. Interviral sequence comparison revealed significant similarities with viruses belonging to the so-called picornavirus superfamily. The ORF2 is most likely expressed by a -1 translational frameshift and is followed by an untranslated sequence of 222 nucleotides. Internal amino acid sequences of three capsid proteins (66K, 34K, 23/24K) were determined. Comparison of the obtained amino acid sequences with the APV sequence disclosed that the structural proteins are located in the 3'-terminal half of the genome. The 34K protein is encoded by the ORF1, while the 66K protein contains both ORF1-(34K) and ORF2-derived sequences and is probably expressed by a translational frameshift. The 23/24K proteins most likely arise by proteolytic breakdown of the 34K protein. Although the deduced APV genomic organization in some aspects resembles that of the picornaviruses, its overall genomic organization indicates that APV is a distinct species only distantly related to the Picornaviridae.