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Very virulent infectious bursal disease virus: reduced pathogenicity in a rare natural segment-B-reassorted isolate.

The purpose of this study was to compare the molecular epidemiology of infectious bursal disease virus (IBDV) segments A and B of 50 natural or vaccine IBDV strains that were isolated or produced between 1972 and 2002 in 17 countries from four continents, with phenotypes ranging from attenuated to very virulent (vv). These strains were subjected to sequence and phylogenetic analysis based on partial sequences of genome segments A and B. Although there is co-evolution of the two genome segments (70 % of strains kept the same genetic relatives in the segment A- and B-defined consensus trees), several strains (26 %) were identified with the incongruence length difference test as exhibiting a significantly different phylogenetic relationship depending on which segment was analysed. This suggested that natural reassortment could have occurred. One of the possible naturally occurring reassortant strains, which exhibited a segment A related to the vvIBDV cluster whereas its segment B was not, was thoroughly sequenced (coding sequence of both segments) and submitted to a standardized experimental characterization of its acute pathogenicity. This strain induced significantly less mortality than typical vvIBDVs; however, the mechanisms for this reduced pathogenicity remain unknown, as no significant difference in the bursal lesions, post-infectious antibody response or virus production in the bursa was observed in challenged chickens.

Animals↗

Mutations in the Drosophila gene extradenticle affect the way specific homeo domain proteins regulate segmental identity.

We characterized a gene, extradenticle, which seems to interact with a specific subset of Drosophila homeo domain proteins, possibly affecting their target specificity. This interpretation is based on an examination of the zygotic and maternal effect phenotypes of extradenticle mutations. In embryos with reduced levels of extradenticle gene product, anterior and posterior segmental transformations occur. Segmental identity in Drosophila is mediated by the products of the Antennapedia and bithorax complexes. These homeo domain proteins are thought to regulate different target genes specifically in each segment, resulting in different morphologies. extradenticle alters segmental identity without affecting the pattern of expression of homeotic genes. Genetic tests demonstrate that in extradenticle mutants, the homeotic proteins are functional and act in their normal segmental domains, yet segmental identities are altered. Even when homeotic proteins are ectopically expressed under the control of a heterologous promoter, extradenticle mutations affect their consequences. Thus, in the absence of sufficient extradenticle product, altered segmental morphology results from alteration of the functional consequences of specific homeo domain proteins, possibly through alterations in their target gene specificity. extradenticle is also expressed maternally. Complete removal of extradenticle, maternally and zygotically, leads to specific alterations in segmentation, many of which result from failure to maintain the expression of the homeo domain protein engrailed.

Animals↗

Defense Responses in Infected and Elicited Cucumber (Cucumis sativus L.) Hypocotyl Segments Exhibiting Acquired Resistance.

Segments from dark-grown cucumber (Cucumis sativus L.) hypocotyls were used to study defense reactions occurring upon fungal infection and induced by elicitors in the same tissue. The segments were rendered resistant to infection by Colletotrichum lagenarium either by growing the seedlings in the presence of dichloroisonicotinic acid (DCIA) or by preincubation of the cut segments with DCIA, salicylic acid (SA), or 5-chlorosalicylic acid (5CSA). This resistance appears to be due mainly to inhibition of fungal penetration into epidermal cells. In the resistant hypocotyl segments, the fungus induced, at the time of attempted penetration, an increased deposition of phenolics, which were visualized by autofluorescence. These phenolics were located mainly in the epidermal cell wall around and in the emerging papillae below appressoria and were quantified either as lignin-like polymers by the thioglycolic acid method or as 4-OH-benzaldehyde, 4-OH-benzoic, or 4-coumaric acid liberated upon treatment with alkali at room temperature. Pretreatment with DCIA, SA, and 5CSA induced little chitinase activity, but this activity greatly increased in resistant tissues upon subsequent infection. These observations indicate that resistance is associated with an improved perception of the pathogen stimulus resulting in the enhanced induction of diverse defense reactions. When the cut segments were pretreated with DCIA, SA, or 5CSA and then split and incubated with chitosan fragments, the deposition of cell wall phenolics was also enhanced. These pretreated and split segments also exhibited an increase in the rapid production of activated oxygen species induced by an elicitor preparation from Phytophthora megasperma f. sp. Glya. Pretreatment of the segments with methyl jasmonate neither induced resistance nor enhanced induction of cell wall phenolics upon fungal infection, although we observed in the corresponding split segments some increase in chitosan-induced cell wall phenolics and in elicitor-induced rapid production of activated oxygen species.

Journal Article↗

Effects of growth regulators on ribonucleic acid metabolism of barley leaf segments.

Illumination or gibberellic acid treatment of etiolated barley leaf segments stimulates unrolling and results in an increased level of RNA. In contrast, segments treated with abscisic acid do not unroll and have a lower content of RNA. Gibberellic acid treatment enhanced the capacity of segments to incorporate radioactivity from (32)P-orthophosphate into all the RNA components detected by gel electrophoresis; abscisic acid greatly restricted the incorporation of precursors into all the RNA fractions. In conjunction with a changed capacity for RNA synthesis it was observed that abscisic acid-treated segments had a lowered soluble DNA-dependent RNA polymerase level in comparison to gibberellic acid-treated or illuminated segments. However, the influence of growth regulators on RNA polymerase content of the segments was associated with general effects on protein level rather than a specific effect on the synthesis of polymerase enzyme. Ribosomal preparations from gibberellic acid-treated segments had a greater percentage of polysomes and a greater capacity for amino acid incorporation in vitro into proteins than similar preparations from abscisic acid-treated segments.

Journal Article↗

Bayesian image segmentation using local iso-intensity structural orientation.

Image segmentation is a fundamental problem in early computer vision. In segmentation of flat shaded, nontextured objects in real-world images, objects are usually assumed to be piecewise homogeneous. This assumption, however, is not always valid with images such as medical images. As a result, any techniques based on this assumption may produce less-than-satisfactory image segmentation. In this work, we relax the piecewise homogeneous assumption. By assuming that the intensity nonuniformity is smooth in the imaged objects, a novel algorithm that exploits the coherence in the intensity profile to segment objects is proposed. The algorithm uses a novel smoothness prior to improve the quality of image segmentation. The formulation of the prior is based on the coherence of the local structural orientation in the image. The segmentation process is performed in a Bayesian framework. Local structural orientation estimation is obtained with an orientation tensor. Comparisons between the conventional Hessian matrix and the orientation tensor have been conducted. The experimental results on the synthetic images and the real-world images have indicated that our novel segmentation algorithm produces better segmentations than both the global thresholding with the maximum likelihood estimation and the algorithm with the multilevel logistic MRF model.

Algorithms↗

Shape statistics variational approach for the outer contour segmentation of left ventricle MR images.

Segmentation of left ventricles is one of the important research topics in cardiac magnetic resonance (MR) imaging. The segmentation precision influences the authenticity of ventricular motion reconstruction. In left ventricle MR images, the weak and broken boundary increases the difficulty of segmenting the outer contour precisely. In this paper, we present an improved shape statistics variational approach for the outer contour segmentation of left ventricle MR images. We use the Mumford-Shah model in an object feature space and incorporate the shape statistics and an edge image to the variational framework. The introduction of shape statistics can improve the segmentation with broken boundaries. The edge image can enhance the weak boundary and thus improve the segmentation precision. The generation of the object feature image, which has homogenous "intensities" in the left ventricle, facilitates the application of the Mumford-Shah model. A comparison of mean absolute distance analysis between different contours generated with our algorithm and that generated by hand demonstrated that our method can achieve a higher segmentation precision and a better stability than various approaches. It is a semiautomatic way for the segmentation of the outer contour of the left ventricle in clinical applications.

Algorithms↗

A versatile segmentation procedure.

In this paper, a new method for the segmentation of natural images is proposed. Original images g(x, y) are first regularized by using a self-adaptive implementation of the Mumford-Shah functional so that the two parameters alpha and gamma controlling the smoothness and fidelity, automatically adapt to the local scale and contrast of g(x, y). From the regularized image u(x, y) which is piecewise smooth, it is possible to obtain a piecewise constant image sN(x, y) representing a segmentation of the original image g(x, y). Indeed, sN(X, y) is the union of N closed regions, having a constant grey level, preserving thin bars and trihedral junctions present in the original image g(x, y). If the number N of closed regions is too high, closed regions can be merged by minimizing a functional which depends on a parameter n. When n is set equal to 1, a coarse segmentation is obtained with a few tens of distinct regions. With larger values of n, finer segmentations are obtained with about a hundred distinct regions. Therefore, by selecting the value of n it is possible to obtain segmentations at different resolutions. The proposed method for image segmentation was evaluated in two cases where a ground truth segmentation is available. The proposed procedure for image segmentation is rather versatile and depends on only one parameter n and seems suitable for higher level processing, such as categorization, recognition, and scene understanding.

Algorithms↗

Exercise-provoked ST-segment depression and prognosis in patients recovering from acute myocardial infarction. Significance and pitfalls.

The importance of maximal versus submaximal exercise testing and the significance of heart failure on the prognostic value of exercise-provoked ST-segment depression > or = 0.1 mV was studied in 143 patients recovering from acute myocardial infarction. Patients were exercise tested prior to discharge and follow up lasted for up to 18 months (mean 17 months). End-point was first major event (i.e. first non-fatal reinfarction or death). A symptom-limited exercise test was superior to a heart-rate-limited test in detecting ST-segment depressions (27% vs. 20%: P < 0.5), and patients with ST-segment depression at lower heart rates did not have an increased risk of subsequent events compared with patients with ST-segment depression at higher heart rates (14% vs. 27%; NS). Heart failure surpassed ST-segment depression as a risk predictor (34% vs. 18%). Based on a meta-analysis including 13 studies (1987 patients) exercise-provoked ST-segment depression possessed an increased risk of subsequent major events (P < 0.0001; risk ratio = 1.90; 95% confidence limits 1.43,2.51). Thus, ST-segment depression provoked by a symptom-limited test selects patients with an increased risk of subsequent major events. In patients with a history of heart failure exercise-provoked ST-segment depression is of limited value.

Aged↗

Chemical synthesis of phosphorylated peptides of the carboxy-terminal domain of human p53 by a segment condensation method.

A segment condensation method was developed for the chemical synthesis of large (> 90 amino acid) phosphopeptides and was used to produce phosphorylated and non-phosphorylated derivatives of the C-terminal tetramerization and regulatory domains of human p53 (residues 303-393). Efficient condensation synthesis of the 91 residue p53 domain was achieved in two steps. The non-phosphorylated N-terminal segment p53(303-334) (1) and its derivative phosphorylated at serine 315 (1P315), and the non-phosphorylated middle segment p53(335-360) (2), were synthesized as partially protected peptide thioesters in the solid phase using Boc chemistry. The C-terminal segment p53(361-393) (3) and its derivative phosphorylated at serine 392 (3P392) were synthesized as partially protected peptides in the solid phase using Fmoc chemistry. Phosphoamino acid was incorporated into the N-terminal segment (1P315) at the residue corresponding to p53 serine 315 as Boc-Ser(PO3(Bzl)2)-OH during synthesis. Serine 392 in the C-terminal segment was selectively phosphorylated after synthesis by phosphitylation followed by oxidation. A derivative phosphorylated at serine 378 was synthesized in a one-step condensation of the unphosphorylated N-terminal segment (1) and the phosphorylated long C-terminal segment p53(335-393) (2-3P378). Yields of the ligated peptides after removal of the protecting groups and HPLC purification averaged 60% for the first condensation and 35% for the second condensation. All five p53 peptides exhibited monomer-tetramer association as determined by analytical ultracentrifugation. Circular dichroism spectroscopy revealed that phosphorylation at Ser315 increased the alpha-helical content, which was abolished when Ser392 also was phosphorylated, suggesting an interaction between N-terminal and C-terminal residues of the C-terminal domain of p53.

Amino Acid Sequence↗

Calcium ion dependency of ethylene production in segments of primary roots of Zea mays.

We investigated the effect of Ca2+ on ethylene production in 2-cm long apical segments from primary roots of corn (Zea mays L., B73 x Missouri 17) seedlings. The seedlings were raised under different conditions of Ca2+ availability. Low-Ca and high-Ca seedlings were raised by soaking the grains and watering the seedlings with distilled water or 10 mM CaCl2, respectively. Segments from high-Ca roots produced more than twice as much ethylene as segments from low-Ca roots. Indoleacetic acid (IAA; 1 micromole) enhanced ethylene production in segments from both low-Ca and high-Ca roots but auxin-induced promotion of ethylene production was consistently higher in segments from high-Ca roots. Addition of 1-aminocyclopropane-1-carboxylic acid (ACC) to root segments from low-Ca seedlings doubled total ethylene production and the rate of production remained fairly constant during a 24 h period of monitoring. In segments from high-Ca seedlings ACC also increased total ethylene production but most of the ethylene was produced within the first 6 h. The data suggest that Ca2+ enhances the conversion of ACC to ethylene. The terminal 2 mm of the root tip were found to be especially important to ethylene biosynthesis by apical segments and, experiments using 45Ca2+ as tracer indicated that the apical 2 mm of the root is the region of strongest Ca2+ accumulation. Other cations such as Mn2+, Mg2+, and K+ could largely substitute for Ca2+. The significance of these findings is discussed with respect to recent evidence for gravity-induced Ca2+ redistribution and its relationship to the establishment of asymmetric growth during gravitropic curvature.

Amino Acids↗

Accurate synapse regeneration despite ablation of the distal axon segment.

In each body ganglion of the leech Hirudo medicinalis there is a single S-cell. After an S-cell axon is severed, it regenerates along its surviving distal segment and reconnects with its synaptic target, the axon of the neighbouring S-cell. In approximately half the cases the regenerating axon forms a temporary electrical synapse specifically with the distal segment, which remains active and connected to the target, thereby functioning as a splice until regeneration is complete. To determine whether the distal axon segment is required for successful regeneration, distal segments of severed S-cell axons were ablated by intracellular injection of bacterial protease. Fifty-seven preparations were examined from 2 to 212 days after injection of the axon segment. The extent of S-cell axon regeneration was assessed electrophysiologically by intracellular and extracellular recording, and anatomically by intracellular injection of markers followed by light microscopy and electron microscopy. The S-cell axons regenerated successfully in almost 90% of animals examined after 2 weeks or more. In a further four animals the target S-cell was ablated in addition to the distal axon segment, permanently disrupting conduction along the S-cell pathway. Nevertheless, the regenerating axon grew along its usual pathway and there was no evidence that alternative connections were formed. It is concluded that, although the distal axon segment can provide a means for rapid functional repair, the segment is not required for reliable regeneration of the axon along its usual pathway and accurate formation of an electrical synapse.

Animals↗

Early development and segment formation in the centipede, Strigamia maritima (Geophilomorpha).

Geophilomorph centipedes exhibit a number of unique characteristics that make them of particular developmental and evolutionary interest. Segment numbers in geophilomorphs are higher than in any other centipedes, ranging from 27 to 191. They may be constant within a species, presenting in extreme form the "counting" problem in development, or they may vary--a situation that provides us with the opportunity to study naturally occurring variation in segment numbers. All their segments are generated during embryogenesis, a situation unlike that in the more basal centipede orders, which generate only a fraction of their 15 trunk segments in the embryo and develop the rest postembryonically. Here we provide a foundation for further developmental studies of the Geophilomorpha, building on the one study that has been conducted to date, on the coastal species Strigamia maritima. Development begins with the migration of nuclei to the surface of the egg, which then condense to form an embryonic rudiment of more than 20,000 cells, covering an entire hemisphere. During early development, the embryo can be divided into two distinct areas: a large terminal disc of apparently undifferentiated tissue and the germ-band, which has a clear anteroposterior axis and differentiated segments. The germ-band forms from the anterior of the terminal disc and extends anteriorly as the disc contracts. New segments are formed at the posterior margin of the germ-band. Once the process of segmentation ends, the germ-band folds and sinks into the yolk. We note that the classic description of centipede development, by Heymons more than a century ago, contains a fundamental error in the identification of the axes and hence in the interpretation of early segmentation.

Animals↗

Short and long germ segmentation: unanswered questions in the evolution of a developmental mode.

The insect body plan is very well conserved, yet the developmental mechanisms of segmentation are surprisingly varied. Less evolutionarily derived insects undergo short germ segmentation where only the anterior segments are specified before gastrulation whereas the remaining posterior segments are formed during a later secondary growth phase. In contrast, derived long germ insects such as Drosophila specify their entire bodies essentially simultaneously. These fundamental embryological differences imply potentially divergent molecular patterning events. Numerous studies have focused on comparing the expression and function of the homologs of Drosophila segmentation genes between Drosophila and different short and long germ insects. Here we review these comparative data with special emphasis on understanding how short germ insects generate segments and how this ancestral mechanism may have been modified in derived long germ insects such as Drosophila. We break down the larger issue of short versus long germ segmentation into its component developmental problems and structure our discussion in order to highlight the unanswered questions in the evolution of insect segmentation.

Animals↗

Statistical cerebrovascular segmentation in three-dimensional rotational angiography based on maximum intensity projections.

Segmentation of three-dimensional rotational angiography (3D-RA) can provide quantitative 3D morphological information of vasculature. The expectation maximization-(EM-) based segmentation techniques have been widely used in the medical image processing community, because of the implementation simplicity, and computational efficiency of the approach. In a brain 3D-RA, vascular regions usually occupy a very small proportion (around 1%) inside an entire image volume. This severe imbalance between the intensity distributions of vessels and background can lead to inaccurate statistical modeling in the EM-based segmentation methods, and thus adversely affect the segmentation quality for 3D-RA. In this paper we present a new method for the extraction of vasculature in 3D-RA images. The new method is fully automatic and computationally efficient. As compared with the original 3D-RA volume, there is a larger proportion (around 20%) of vessels in its corresponding maximum intensity projection (MIP) image. The proposed method exploits this property to increase the accuracy of statistical modeling with the EM algorithm. The algorithm takes an iterative approach to compiling the 3D vascular segmentation progressively with the segmentation of MIP images along the three principal axes, and use a winner-takes-all strategy to combine the results obtained along individual axes. Experimental results on 12 3D-RA clinical datasets indicate that the segmentations obtained by the new method exhibit a high degree of agreement to the ground truth segmentations and are comparable to those produced by the manual optimal global thresholding method.

Algorithms↗

An improved MLC segmentation algorithm and software for step-and-shoot IMRT delivery without tongue-and-groove error.

We present an improved multileaf collimator (MLC) segmentation algorithm, denoted by SLS(NOTG) (static leaf sequencing with no tongue-and-groove error), for step-and-shoot intensity-modulated radiation therapy (IMRT) delivery. SLS(NOTG) is an improvement over the MLC segmentation algorithm called SLS that was developed by Luan et al. [Med. Phys. 31(4), 695-707 (2004)], which did not consider tongue-and-groove error corrections. The aims of SLS(NOTG) are (1) shortening the treatment times of IMRT plans by minimizing their numbers of segments and (2) minimizing the tongue-and-groove errors of the computed IMRT plans. The input to SLS(NOTG) is intensity maps (IMs) produced by current planning systems, and its output is (modified) optimized leaf sequences without tongue-and-groove error. Like the previous SLS algorithm [Luan et al., Med. Phys. 31(4), 695-707 (2004)], SLS(NOTG) is also based on graph algorithmic techniques in computer science. It models the MLC segmentation problem as a weighted minimum-cost path problem, where the weight of the path is the number of segments and the cost of the path is the amount of tongue-and-groove error. Our comparisons of SLS(NOTG) with CORVUS indicated that for the same intensity maps, the numbers of segments computed by SLS(NOTG) are up to 50% less than those by CORVUS 5.0 on the Elekta LINAC system. Our clinical verifications have shown that the dose distributions of the SLS(NOTG) plans do not have tongue-and-groove error and match those of the corresponding CORVUS plans, thus confirming the correctness of SLS(NOTG). Comparing with existing segmentation methods, SLS(NOTG) also has two additional advantages: (1) SLS(NOTG) can compute leaf sequences whose tongue-and-groove error is minimized subject to a constraint on the maximum allowed number of segments, which may be desirable in clinical situations where a treatment with the complete correction of tongue-and-groove error takes too much time, and (2) SLS(NOTG) can be used to minimize a more general type of error called the tongue-or-groove error.

Algorithms↗

Accuracy and variability assessment of a semiautomatic technique for segmentation of the carotid arteries from three-dimensional ultrasound images.

In this paper, we report on a semiautomatic method for segmentation of three-dimensional (3D) carotid vascular ultrasound (US) images. Our method is based on a dynamic balloon model represented by a triangulated mesh. The mesh is manually placed within the interior of the carotid vessels, then is driven outward until it reaches the vessel wall by applying an inflation force to the mesh. Once the mesh is in close proximity to the vessel wall, it is further deformed using an image-based force, in order to better localize the boundary. Since the method requires manual initialization, there is inherent variability in the position and shape of the final segmented boundary. Using a 3D US image of a patient's carotids, we have examined the local variability in boundary position as the initialization position is varied throughout the interior of the carotid vessels in the 3D image. We have compared the semiautomatic segmentation method to a fully manual segmentation method, and found that the semiautomatic approach is less variable than the intraobserver variability for manual segmentation. We have furthermore examined the accuracy of the semiautomatic method by comparing the average surface to an "ideal" surface, determined by the average manually segmented surface. We have found, in general, good agreement between the semiautomatic and manual segmentation methods. For the 3D US image in question, the mean separation between the average segmented surface and the gold standard was found to be 0.35 mm. The two surfaces were determined to agree with each other, within uncertainty, at 65% of the mesh points comprising the two surfaces.

Biophysical Phenomena↗

Alterations in the hydrophilic segment of the maltose-binding protein (MBP) signal peptide that affect either export or translation of MBP.

Mutations that reduce the net positive charge within the hydrophilic segments of the signal peptides of several prokaryotic exported proteins can result in a reduction in the rate of protein export, as well as a reduction in protein synthesis (M. N. Hall, J. Gabay, and M. Shwartz, EMBO J. 2:15-19, 1983; S. Inouye, X. Soberon, T. Franceschini, K. Nakamura, K. Itakura, and M. Inouye, Proc. Natl. Acad. Sci. USA 79:3438-3441, 1982; J. W. Puziss, J. D. Fikes, and P. J. Bassford, Jr., J. Bacteriol. 171:2302-2311, 1989). This result has been interpreted as evidence that the hydrophilic segment is part of a mechanism that obligatorily couples translation to protein export. We have investigated the role of the hydrophilic segment of the Escherichia coli maltose-binding protein (MBP) signal peptide in the export and synthesis of MBP. Deletion of the entire hydrophilic segment from the MBP signal peptide resulted in a defect in MBP export, as well as a dramatic reduction in total MBP synthesis. Suppressor mutations that lie upstream of the malE coding region were isolated. These mutations do not affect MBP export but instead were shown to partially restore MBP synthesis by increasing the efficiency of MBP translational initiation. In addition, analysis of a series of substitution mutations in the second codon of certain malE alleles demonstrated that MBP export and synthesis can be independently affected by mutations in the hydrophilic segment. Finally, analysis of alterations in the hydrophilic segment of the ribose-binding protein signal peptide fused to the mature moiety of the MBP has revealed that the role of the hydrophilic segment in the export process can be functionally separated from any role in translation. Taken together, these results strongly suggest that the hydrophilic segment of the MBP signal peptide is not involved in a mechanism that couples MBP translation to export and argue against the presence of a mechanism that obligatorily couples translation to protein export in Escherichia coli.

ATP-Binding Cassette Transporters↗

Mapping of genomic segments of influenza B virus strains by an oligonucleotide microarray method.

Similar to other segmented RNA viruses, influenza viruses can exchange genome segments and form a wide variety of reassortant strains upon coreplication within a host cell. Therefore, the mapping of genome segments of influenza viruses is essential for understanding their phenotypes. In this work, we have developed an oligonucleotide microarray hybridization method for simultaneous genotyping of all genomic segments of two highly homologous strains of influenza B virus. A few strain-specific oligonucleotide probes matching each of the eight segments of the viral genomes of the B/Beijing/184/93 and B/Shangdong/7/97 strains were hybridized with PCR-amplified fluorescently labeled single-stranded DNA. Even though there were a few mismatches among the genomes of the studied virus strains, microarray hybridization showed highly significant and reproducible discrimination ability and allowed us to determine the origins of individual genomic segments in a series of reassortant strains prepared as vaccine candidates. Additionally, we were able to detect the presence of at least 5% of mixed genotypes in virus stocks even when conventional sequencing methods failed, for example, for the NS segment. Thus, the proposed microarray method can be used for (i) rapid and reliable genome mapping of highly homologous influenza B viruses and (ii) extensive monitoring of influenza B virus reassortants and the mixed genotypes. The array can be expanded by adding new oligoprobes and using more quantitative assays to determine the origin of individual genomic segments in series of reassortant strains prepared as vaccine candidates or in mixed virus populations.

Animals↗