Herpesviridae) have large genomes that encode hundreds of proteins. Apart from amino acid mutations, protein domain acquisitions, duplications and losses are also common modes of evolution. HV domain repertoires differ across species, and only a core set is shared among all species, aspect that raises a question: How have HV domain repertoires diverged while keeping some similarities? To answer such question, we used profile Hidden Markov Models (HMMs) to search for domains in all possible translated open reading frames (ORFs) of fully sequenced HV genomes. With at least 274 domains being identified, we built a matrix of domain counts per species, and applied a parsimony method to reconstruct the ancestral states of these domains along the HV phylogeny. It revealed events of domain gain, duplication, and loss over more than 400 millions of years, where Alpha-, Beta-, and GammaHVs expanded and condensed their domain repertoires at distinct rates. Most of the acquired domains perform ‘Modulation and Control’, ‘Envelope’, or ‘Auxiliary’ functions, categories that showed high flexibility (number of domains) and redundancy (number of copies). Conversely, few gains and duplications were observed for domains involved in ‘Capsid assembly and structure’, and ‘DNA Replication, recombination and metabolism’. Among the forty-one primordial domains encoded by Herpesviridae ancestors, twenty-eight are still found in all present-day HVs. Because of their distinct evolutionary strategies, HV domain repertoires are very specific at the subfamily, genus and species levels. Differences in domain composition may not only explain HV host range and tissue tropism, but also provide hints to the origins of HVs.Herpesviruses (HVs, Family:  Herpesviridae (hereinafter referred simply as ‘herpesviruses’ or ‘HVs’), answering this question becomes even harder, specially taking into consideration their genomic complexity.Viruses are known by their unorthodox modes of evolution and outstanding strategies of immune evasion and cell infection. Their biological origins have long been debated, with many theories being proposed, without a consensus , probablHerpesviridae is the best-characterized family of large double-stranded DNA viruses  of domains at all internal nodes of the tree, including its root. This analysis allowed us to reconstruct parsimonious scenarios explaining the evolution of HV domain families by events of domain gain, loss, and duplication. It revealed that current herpesviral species encode a core set of twenty-eight domains inherited from their ancestors since ∼400 million years ago. By classifying the HV domains in functional groups, it was possible to determine that domains implied in virus–host interactions were the main elements acquired by these HVs, especially in early periods of their evolution. Our results were interpreted in light of the current hypothesis explaining genomic and viral evolution, and shed light on the possible origins of HVs.In this study, we extracted all possible ORFs from 75 fully sequenced genomes of members of the E-values were defined using a conservative cutoff of 0.001, as used in previous studies , and the total number of taxa (spp) per subfamily  and the end of the Cretaceous (K), similar numbers of losses and duplications of domains of distinct functions were observed. As the number of sampled taxa differs in Alpha-, Beta-, and GammaHVs, to compare the impact of domain gains, losses, and duplications in the evolution of their repertoires, we established event rates by normalizing the total number of events (e) by the tMRCA of −3 gains per Myr per species), nearly twice as high as AlphaHVs (8.15 × 10−3). The levels of domain losses in all subfamilies were very similar (around 5 × 10−3), but lower than gains and duplications. Finally, rates of domain duplications varied among the three HV groups, with BetaHVs showing the highest rate (11.19 × 10−3 duplications per Myr per species). Overall, rates of events expanding the repertoire of Herpesviridae (gains + duplications) were nearly four times higher (19.22 × 10−3) than those causing repertoire contraction .For domain gains, these results revealed that Beta- and GammaHVs evolved under higher rates of acquisitions  encoded a minimal set of forty-one domains  and the Quaternary (Q) periods . In thesDuplications of such domains were not as common as in other functional categories, being kept at very low levels until the Cretaceous . Only frSimplexvirus lost zf-RING_2 (a Ring finger domain) at least three times. Finally, at the same period, GammaHVs from different genera lost one of their multiple copies of Herpes_ORF11, a domain found in dUTPase proteins.Losses were uncommon among ‘Auxiliary’ domains; however, a few of them were lost in more than one occasion, in distinct time intervals. The domain dNK (Deoxynucleoside kinase), for example, was lost in most Alpha- and BetaHVs along the Triassic–Jurassic interval. Later, between the Paleogene and Quaternary periods, AlphaHVs from the genus Herpesviridae  Parvo_NS1, a domain encoded by Parvovirus non-structural protein NS1 homologs, involved in viral genome replication (GO:0019079), found specifically in HHV6 and MuHV2; (2) YqaJ, a viral recombinase domain independently acquired by MyHV8 and MuHV8; and (3) Rep_N, found in HHV6, but originally encoded by Adeno-associated virus, found in proteins involved in viral replication and integration.As observed for ‘Capsid’ domains, the functional category ‘DNA Replication, recombination and metabolism’ represents the second most conserved and less flexible set of domains of HVs. Apart from thirteen domains already present in the primordial repertoire of ncestors , few DNADuplications in this functional category were mostly restricted to three domain families: Herpes_UL42, encoded by DNA polymerase processivity factors of AlphaHVs; Herpes_PAP, found in polymerase accessory proteins of BetaHVs; and more importantly, dUTPase, observed as two copies in several GammaHVs.Herpesviridae, such as Ribonuc_red_lgN and Ribonuc_red_sm, which are ribonucleotide reductase domains; and Herpes_TK, a herpesviral thymidine kinase. These domains are mostly absent in all BetaHVs since the Permian and Triassic periods.Losses of DNA-related domains, albeit uncommon, were recurrent mainly in BetaHVs , which lCytomegalovirus  and Herpes_IE1 . In GammaHVs, the main domains with duplicates were: Sushi domain, mostly found in multiple copies in Rhadinovirus, and; the death effector domain, mainly encoded by members of Rhadinovirus and Percavirus.Duplications of modulation domains were frequent all over the evolution of HVs, and most of them involved domains specific to a single HV subfamily. AlphaHVs, for example, obtained duplicates that are now ubiquitous among them, as the Zinc finger domains zf-C3HC4, zf-C3HC4_2, and zf-C3HC4_3; and the viral regulatory domains Herpes_IE68, and Herpes_ICP4_C. BetaHVs, on the other hand, obtained extra copies of domains found only in a few viral lineages, as the immediate early protein domains HHV6-IE , US2, and Gene66. Around the same periods, BetaHVs acquired a set of domains to date found only among them: Herpes_U5; Herpes_U55; DUF587; and DUF570. GammaHVs also gained their subfamily-specific domains, in particular Herpes_BBRF1 and DUF717, now present in most members of that group. Finally, most probably in the Devonian, ancestors of Beta- and GammaHVs acquired three domains still conserved in all their species: Herpes_UL87; Herpes_UL92; and Herpes_UL95.Although domains classified with other/unknown functions do not make a functional category Roseolovirus, multiple duplications of DUF865 took place, and now such viruses encode from eight to twenty-two copies of this uncharacterized domain. Finally, losses involved mostly domains unique to AlphaHVs , tracking gains, losses, and duplications of domains from distinct functions along the viral phylogeny. Since all possible ORFs were scanned for domains disregarding their sizes, initial codons, or whether or not they were annotated in the original genome annotations, our approach detected a large set of at least 274 domains encoded by members of the Herpesviridae family. It was only possible due to the sensitivity of HMM profile screenings, which allows the detection of remote homology between sequences, a task that conventional methods of sequence comparison are unable to achieve  in the repertoire of The rates of domain losses for each HV subfamily were very similar, although the functional categories of lost domains differed across these viral groups . AlthougApart from dosage sensitivity, another aspect favoring the loss of duplicates is the ‘dominant negative effect’, a phenomenon that emerges when a second copy of a protein interferes with the functioning of its original version . Such meIn the evolution of ‘Tegument’ domains, losses were very common, while their gains exceptionally decreased over time. Tegument proteins are known to establish a myriad of interactions among each other in the space between the capsid and the viral envelope, strategic position that allow them to perform structural and/or modulatory roles upon cell entry . Inside Finally, considering the hypothesis of regressive evolution in a context of neutrality, genetic drift can also operate removing domains according to their levels of conditional dispensability. A genetic element can be considered dispensable when the environmental pressures justifying their maintenance in the genome cease to exist, generating a condition where keeping the gene/domain is not strictly advantageous . This phThe evolutionary origins of viruses remain unclear despite decades of debates, raising distinct (and to some extent opposing) hypothesis to explain their origins, such as: (1) the ‘Escape’ hypothesis, which considers viruses as genetic elements that escaped from cells and expanded themselves by gene acquisitions; (2) the ‘Regressive’ hypothesis, which claims that viruses derive from free-living unicellular organisms that lost several genes, thus becoming obligate intracellular parasites; and (3) the ‘virus-first’ hypothesis, which asserts that viruses originated before cellular organisms, and co-evolved with them .Herpesviridae family, at least thirteen domains were lost along their evolution, proving not to be strictly essential. Despite that, events of domain accretion outnumbered losses, and HV genomes expanded progressively by capturing or generating domains de novo to perform specific functions . Using the ‘trace ancestral character’ function implemented in Mesquite , this maing iTOL , highlighttps://github.com/andersonbrito/openData/tree/master/brito_2019_HVsDomainsAll accession numbers of genomes, domains and GO terms mentioned in this study are listed in veaa001_Supplementary_DataClick here for additional data file.


Hepatitis B virus (HBV) is an important but difficult to study human pathogen. Most basics of the hepadnaviral life-cycle were unraveled using duck HBV (DHBV) as a model although DHBV has a capsid protein (CP) comprising ~260 rather than ~180 amino acids. Here we present high-resolution structures of several DHBV capsid-like particles (CLPs) determined by electron cryo-microscopy. As for HBV, DHBV CLPs consist of a dimeric α-helical frame-work with protruding spikes at the dimer interface. A fundamental new feature is a ~ 45 amino acid proline-rich extension in each monomer replacing the tip of the spikes in HBV CP. In vitro, folding of the extension takes months, implying a catalyzed process in vivo. DHBc variants lacking a folding-proficient extension produced regular CLPs in bacteria but failed to form stable nucleocapsids in hepatoma cells. We propose that the extension domain acts as a conformational switch with differential response options during viral infection. In humans, chronic infections with HBV cause ~800,000 deaths per year . They lack an X protein, a regulator of cccDNA transcription  structure. Typical small type CPs comprise an N-terminal ~140 aa assembly domain  and an aThe assembly domains feature five α-helices . α3 and Early cryo-EM reconstructions of DHBc suggested a T = 4 arrangement similar to HBc CLPs but with laterally widened spikes . ExtensiHere we discovered that over several months of storage this loop, now termed extension domain, adopted a defined fold. This enabled us to determine the structure of whole DHBc CLPs at a resolution of 3.7 Å enabling de novo model building. Functional studies revealed that the extension domain´s ability to fold is dispensable for recombinant CLP assembly but correlates with capsid stability in hepatoma cells. We propose that plasticity of the extension domain represents a large-type CP-specific evolutionary adaptation.E. coli self-assembles into CLPs which package bacterial RNA (E. coli optimized DHBc gene encoding the DHBV16 CP sequence (Uniprot accession: P0C6J7.1). CLPs for cryo-EM analysis were enriched by two sequential ammonium sulfphate precipitations (DHBV CP (DHBc) expressed in rial RNA . Here, irial RNA  with an itations  followeditations .After two weeks of storage at 4°C we exchanged the sucrose-containing buffer against a cryo-EM compatible buffer with low solute concentration and vitrified the samples. Micrographs showed distinct particles of spherical shape, aggregates of such particles and some proteo-liposomes . 2D-clasTo test this hypothesis, we analysed the individual asymmetric units by 3D-classification. The class averages  showed 4The analysis of the individual asymmetric units required accurate determination of the orientations of an ordered asymmetric unit of only some 80 kDa, which is at the lower size limit that can be confidently analysed. We speculated that our analysis might have been limited by the imaging conditions , which were chosen for entire capsids  rather tMuch to our surprise 2D-class averages showed crisper projection averages  and the We were able to build a de novo model of DHBc’s assembly domain into the 3D-map of the asymmetric unit of DHBc , Table 2Analysis with Intersurf  showed tIn HBc which lacks an extension domain a disulphide-bridge between Cys61 on opposite monomers  can add The pockets at the base of the spikes close to the N-termini of chains A and D contained density that was not accounted for by the so far described model  magenta.While the intra-dimer contacts differed significantly between HBc and DHBc, the inter-dimer packing in the C-terminal hand-region was largely conserved. The last resolved residues in our model agreed well with the biochemically derived C-terminal border of the DHBc assembly domain around position 187 . At the E. coli. After 2 weeks of storage in sucrose at 4°C the respective CLPs were prepared for cryo-EM. These CLPs displayed similar overall heterogeneity as CLPs without co-expressed FkpA . Sedimentation, NAGE and negative staining EM data all indicated the formation of orderly CLPs, prompting a closer look by cryo-EM image  with the respective DHBc protein expressed from a separate plasmid vector. This excludes inadvertent effects on the virus genome. Mutant E109R_R124E was also tested in the cis-context. Controls included the non-complemented core- genome and a variant with a mutated YMDD motif in the polymerase active site (pCD16_YMhD), allowing pgRNA encapsidation but not reverse transcription  versus wt-DHBc, which reproduced the previously seen NAGE blot data for capsids and capsid-borne DNA . Eukaryotic DHBc expression vectors were based on the cytomegalovirus immediate early (CMV-IE) enhancer/promoter regulated plasmid pAAV-MCS (Stratagene). The parental vector pCD16 contains a 1.1x DHBV16 genome, also under CMV-IE control; in pCD16-core- the initiator ATG codon of the DHBc ORF was changed to ACG. Mutations were introduced via PCR using appropriate primers and the Q5 Site-directed Mutagenesis Kit or the NEBuilder HiFi DNA Assembly Master Mix (both NEB).Bacterial DHBc vectors were based on either pET28a2-DHBc  carryingE. coli Top10 cells, using primers FkpA_Nco+ 5´-CACCATCACGCCATGGCTGAAGCTGCAAAACCTG and FkpA_AvrNot- 5´- GACTCGAGTGCGGCCGCCTAGGGTTTTTTAGCAGAGTCTGCGGCTTTCG (ATG start codon and complement to TAG stop codon underlined) providing terminal restriction sites to replace the Tet-promoter controlled serine-arginine-rich protein kinase (SRPK1) ORF in vector pRSF_Tet-SRPK1_T7-HBc183opt . Using a Microfluidizer, the cells were disrupted by 2 passages at 1,500 bar. Insoluble material was removed by centrifugation at 2,000 g and 4°C for 90 min. Then, ammonium sulfphate was added to the supernatant to 40% saturation at pH 7.5 at 0°C. The resulting precipitate was isolated by centrifugation, re-solubilized, and re-precipitated by adding ammonium sulfphate to 30% final saturation. The new precipitate was dissolved and subjected to density-gradient centrifugation  in an SW 32 Ti rotor (Beckman Coulter) at 4°C and 125755 g for 4 hr. After centrifugation the topmost 5 ml of the gradient were discarded and the remaining volume was manually fractionated into thirty-three 1 ml fractions. Fractions were analyzed by SDS-PAGE and NAGE as described, except that nucleic acids were stained using SYBR Safe dye .For cryo-EM analysis of DHBc particles a modified procedure was used, intended to improve homogeneity of the samples. To this end, BL21*(DE3) cells transformed with plasmid pRSF_T7-DHBcOpt were grown in terrific broth and induced for 16 hr at 18°C. Cells were harvested and the cell pellet was re-suspended (20% w/v) in TN300+ buffer  and concentrated to 100 µl at 4°C and 2,500 g in a concentrator with a molecular weight cut-off of 100 kDa. The concentrate was five-fold diluted and re-centrifuged. This procedure was repeated until the sucrose concentration dropped below 0.03%. The final protein concentrations as determined by the Bradford assay were 2.6 mg/ml for the 2 weeks stored sample and 3.2 mg/ml for the 10 months stored sample. For the vitrification, grids ) were rendered hydrophilic by glow discharging in air at a pressure of 29 Pa for 2 min at medium power with a Plasma Cleaner . Then, 3.5 µl of DHBc solution was pipetted onto the grids and they were plunge frozen in liquid ethane with a Vitrobot mark IV (FEI-Thermo Fisher Scientific). The settings for the Vitrobot were 3 s blot time, 45 s wait time, blot force 0 at a temperature of 4°C and 100% humidity. The vitrified samples were stored in liquid nitrogen until use.200 µl of the peak fractions containing about 30% sucrose were mixed with 300 µl TN50 (50 mM TrisHCl at pH 7.5 for the 2 weeks old sample) or TN20 buffer  and movies were acquired with EPU in integrating mode for DHBc and DHBc+FkpA prepared after 2 weeks and in counting mode for DHBc prepared after 10 months, DHBc+FkpA prepared after 2 weeks and DHBc R124EΔ .−1) for auto-refinement of the 1st data set (2 weeks). For the other data set (10 months) the current best map of DHBc was used as starting reference. During iterative auto-refinement icosahedral-symmetry was applied. Afterwards, particles were classified into 3 classes by 3D-classification without further refinement of orientations. The most populated 3D-class was further refined with auto-refine using only local orientation searches. This was followed by refinement of the contrast transfer function (ctf). For this, particles were re-extracted with a larger box of 800 × 800 Px² without phase-flipping followed by ctf-refinement per particle and beam-tilt refinement for the whole data set. For beam-tilt refinement, we defined beam-tilt classes for each day of acquisition to compensate for a possible drift of beam-tilt over time. For DHBcR124EΔ and DHBc+FkpA we also defined separate beam-tilt classes for each acquisition position in a hole. Ctf-refinement was followed by another round of local auto-refinement. Ctf-refinement only improved resolution of the maps of DHBc after 10 months and DHBcR124EΔ for the other maps no improvement was observed. In these cases we continued with the data without ctf-refinement.Data of the different samples were processed separately, following similar protocols. In brief: Movies were dose-weighted and motion corrected with MotionCor2 . Dose weThe resolution was determined during the post-processing step for masked maps after gold-standard refinement.Starting with the results of the final refinement of the capsids, particles were symmetry expanded (‘relion_particle_symmetry_expand’) followed by re-extraction (128 × 128 Px²), phase-flipping and re-centering to the center of one asymmetric unit. A 3D-map of the re-extracted particle fragments was calculated with ‘relion_reconstruct’ and a mask was generated from the model of the asymmetric unit with Chimera vop zone  followedTCCAACATCGAGGTCGTAAAC and 5'-GTACCGCAAGGGAAAGATGAA', also as described  and maintained as described . Speciesescribed . Transfeescribed . For trah format  DNA amouh format .CGCCTGAGTAGTATGCTCGC and MycoHom(-) 5´-GCGGTGTGTACAAAACCCGA amplify an about 520 bp segment from genomic DNA coding for the 16S rRNA of various mycoplasma species. As internal control, a pUC plasmid was constructed in which a 188 bp heterologous sequence replaces a 67 bp HpaI - BstBI fragment within a cloned 700 bp 16S rRNA gene fragment from M. yeatzii that contains the primer binding sites; on this template the primers amplify a 638 bp fragment. Standard 50 µl PCR reactions using KAPA HiFi polymerase (Kapa Biosystems) were set up as follows: 33 µl H2O; 10 µl 5x KAPA HiFi buffer with Mg2+; 1.5 µl 10 mM dNTP; 1.5 µl of each 10 µM primer; 0.5 µl (1 U/µl) KAPA HiFi polymerase; plus 3 × 10E3 or 10E4 copies of the internal control template. Cycling conditions: (1) Initial denaturation, 98°C 2 min; (2) cycling denaturation, 98°C, 10 s; (3) primer annealing, 58°C, 15 s; (4) extension, 72°C, 30 s; total 34 cycles from step (4) to  final extension, 72°C, 5 min. For analysis, 5 µl of the reactions were separated by electrophoresis in 1.3% agarose gels alongside a 50 bp or 100 bp DNA ladder marker. DNAs were visualized by ethidium bromide staining. Samples generating an about 500 bp amplicon in addition to the 638 bp control amplicon were considered positive; however, all cells used in this study scored negative in this assay.Potential mycoplasma contamination was monitored every fifth passage using a home-made PCR test, similar to a published procedure . Primers32P-labeled DHBV DNA probe  has been used widely as a model to study the hepatitis B virus (HBV) – a human virus infects over 250 million people worldwide. This work revealed the structure of DHBV core protein, which is substantially larger than that of HBV. The study generates important new information regarding the assembly and replication of HBV-like viruses (hepadnaviruses).Decision letter after peer review:eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Cynthia Wolberger as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Slowly folding surface extension in the prototypic avian hepatitis B virus capsid governs stability" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.eLife papers without additional data, even if they feel that they would make the manuscript stronger. Thus the revisions requested below mainly address clarity and presentation.We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we are asking editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:The duck HBV (DHBV) has been used widely as a model to study the human pathogen, the hepatitis B virus (HBV). A striking difference between HBV and DHBV is that the DHBV capsid protein is substantially larger than that of HBV (ca. 260 vs. ca. 180 amino acid residues). The three significant accomplishments of this paper are (i) the observation that DHBV capsid protein is partially unfolded and yet still assembles, (ii) the protein will slowly refold and that can be catalyzed by the presence of proline isomerase, and (iii) the actual structure of the protruding domain.Essential revisions:There are some points to be addressed or discussed to further strengthen the manuscript, for example:It is well known that HBc lacks the extension domain and yet is fully functional in human and avian cells. Conversely, DHBc with the extra domain is also functional in both human and avian cells. If there were indeed a host-cell specific determinant for the folding/functions of the extension domain in DHBV, as implied by the authors, it would be interesting to test the various extension domain deletion/substitution mutants in human cells, in addition to avian cells. As it stands now, little could be learnt regarding the exact roles of this extension in viral replication from the phenotype analysis of these mutants in avian cells, due to the apparently severe deleterious effect on capsid assembly.Figure 5. The authors should check levels of the mutant DHBV capsid protein subunits using SDS-PAGE and western blotting. Did the subunits still accumulate? Was the defect in subunit stability, assembly, or the stability of the assembled capsids?The question of which chaperone is responsible for capsid protein refolding in vivo is not answered in this paper. The effect of proline isomerase indicates that refolding can be catalyzed. However the limited effect of proline isomerase suggests that some other chaperone(s) may be involved. Also, the interaction of the capsid protein with the surface protein may also contribute to folding. The possibilities should be discussed with an open mind.The evolutionary connections from avian to other HBVs is important to understanding virus evolution. The authors should also relate the DHBV structure to other insert containing sequences instead of just the limited sequence alignment of Figure 1—figure supplement 1D.Following please find more comments from the two reviewers.Reviewer #1:The duck HBV (DHBV) has been used widely as a model to study the human pathogen, the hepatitis B virus (HBV). A striking difference between HBV and DHBV is that the DHBV capsid protein is substantially larger than that of HBV (ca. 260 vs. ca. 180 amino acid residues). The authors report high-resolution structures of several DHBV capsid-like particles (CLPs) determined by electron cryo-microscopy. These structures show that DHBV CLPs are generally similar to those of HBV, consisting of a dimeric α-helical backbone with protruding spikes at the dimer interface. The ca. 45 amino acid proline-rich extension unique to the DHBV capsid forms an extra domain near the spike as compared to HBV. The authors noticed that folding of the extension domain seemed to take months in vitro, which could accelerated by prolyl peptidyl isomerase, consistent with a catalyzed process in vivo. DHBc variants lacking a folding-proficient extension domain were competent to assemble capsids in bacteria but failed to form stable nucleocapsids in avian cells. The authors propose that the extension domain acts as a conformational switch with differential response options during viral infection.This is an interesting study that has a strong rationale and generates important new information regarding the assembly and replication of HBV-like viruses (hepadnaviruses). The authors should consider the following points in revising the manuscript.Reviewer #2:In this paper the authors show the structure of Duck Hepatitic B Virus Core Protein and variants. DHBc is a dimeric protein that assembles into T=4 capsids. While closely related to the human homolog, DHBc has a bulky insert at the dimer interface. The authors show:1) The insert is disordered in freshly purified protein2) Can slowly fold, over several months, to an ordered formE. coli with a prolyl isomerase yields capsids that are somewhat better folded.3) Co-expression in 4) Compensating charged residue mutations support a stable insert. Non-compensated mutations do not. The resulting proteins with a misfolded insert do not support assembly of functional capsid.5) Deletion of the disordered residues results DHBcR124E∆ which has a well-ordered assembly domain and shows how the helical dimer interface is preservedThe most striking feature of the results is the folded-unfolded transition of the insert as discussed by the authors. This insert is present in lizard and fish variants of HBV but not in mammalian variants. There is an evolutionary argument. The authors do not discuss whether the insert may also interact with receptors or other host-protein.The authors include a peculiar discussion of a gap at the dimer interface which may be "pocket factor" binding site. Has a pocket factor ever been observed or is this a speculation? The authors should clarify if this discussion is entirely speculative.The quality of the reconstructions is high. However many of the figures are too small and busy. The text is clear with a few exceptions. The structure of the insert will facilitate future experimentation and will raise interest in non-mammalian HBV viruses. The puzzle of the folded to unfolded transition is striking. Co-expression of DHBc with proline isomerase yields a marginally better reconstruction. In the model structures, are prolines in the cis conformation? Would any chaperone do? The conclusion that the delay in folding is due to proline isomerization is ambiguous. Essential revisions:There are some points to be addressed or discussed to further strengthen the manuscript, for example:It is well known that HBc lacks the extension domain and yet is fully functional in human and avian cells. Conversely, DHBc with the extra domain is also functional in both human and avian cells. If there were indeed a host-cell specific determinant for the folding/functions of the extension domain in DHBV, as implied by the authors, it would be interesting to test the various extension domain deletion/substitution mutants in human cells, in addition to avian cells. As it stands now, little could be learnt regarding the exact roles of this extension in viral replication from the phenotype analysis of these mutants in avian cells, due to the apparently severe deleterious effect on capsid assembly.Figure 5. The authors should check levels of the mutant DHBV capsid protein subunits using SDS-PAGE and western blotting. Did the subunits still accumulate? Was the defect in subunit stability, assembly, or the stability of the assembled capsids?We agree that the biological function of the extension domain in the large-type Cp remains an intriguing open question, and that investigating the impact of a different, e.g. human, cell environment is a potentially promising approach for new insight. An example is our demonstration, some time ago that the avian DHBV produces much more cccDNA in human hepatoma cells than human HBV. These phenotypic differences might indeed relate to the presence vs. absence of the extension domain in the respective Cps. Hence these and further host-factor focussed investigations are certainly worthwhile but in our view are beyond the scope of the current structure-focussed study.However, we experimentally followed up the reviewer suggestion to address the fate of the unassembled subunits of the key mutants DHBcR124E and DHBcR124E∆ which failed to generate stable, replication-competent nucleo-capsids in chicken LMH cells – whereas DHBc WT as well as the charge-reversal mutant DHBc_E109R_R124E did. This was initially shown by DHBc capsid and DHBV DNA detection after NAGE . As direct SDS-PAGE immunoblotting of cell lysates did not yield conclusive data, we adopted an immunoprecipitation (IP) protocol to enrich potentially low amounts and/or partially degraded Cp subunits . To cover as many DHBc epitopes as possible we used a polyclonal rabbit antiserum  both for the IP and the subsequent immunoblot (IB) detection step. To minimize signals from the IP antibody chains on the blot we then used an Fc-part specific anti-rabbit secondary antibody-peroxidase conjugate in the final visualization step (which does not react strongly with the antibody light chains in the relevant 25 kDa range of the gel), producing the data now shown in Figure 5—figure supplement 2C. To corroborate the previously seen nucleocapsid phenotypes in the very same source samples as used for the IP/IB analysis we also subjected about 5% of each lysate to the NAGE-based DHBV capsid and capsid-associated DNA assays .As before the extension-less variant DHBcR124EΔ gave a very weak NAGE capsid signal  and this correlated with a weak signal in the SDS-PAGE IB, comparable to the E109R_R124E lo sample that had received one fifth the amount of DHBc plasmid DNA of the others. Hence the lower steady-state level of DHBcR124EΔ subunits (be it from less efficient production or more efficient degradation) might explain the lower levels of capsids detected in the NAGE IB; however, as previously surmised, the complete lack of detectable DHBV DNA supports a replicative defect of the mutant . This assumption is now further strengthened by the detectability of viral DNA in the DHBcR124EΔ lo sample, which produced a similarly low capsid signal.E. coli derived faster-than-wt migrating R124E capsids were clearly detectable on the same blot). The IP/SDS-PAGE/IB protocol revealed the presence of substantial amounts of DHBc-related signals, however, besides little full-length protein the faster mobility of the other bands indicates heavy, variant-specific proteolytic processing, as similar products are completely absent from the DHBc WT and E109R_R124E samples.The most striking new data concern variant DHBc_R124E which, as before, gave no capsid and no capsid-DNA signal in the NAGE blots  causes higher sensitivity to proteolysis, or vice versa. However, although these new data do not answer all of the reviewer´s respective questions they directly support the previously only assumed defect in subunit stability when no salt bridge can form between positions 109 and 124 and when the extension domain does not fold.We have added these new data as Figure 5—figure supplement 2, and added statements on the increased proteolysis susceptibility of DHBc_R124E in eukaryotic cells in the Results  and the Discussion section (subsection “A temporarily mobile spike extension – defining feature of hepadnaviruses employing large type CPs?”) of the main text. We also added additional information to the Materials and methods .The question of which chaperone is responsible for capsid protein refolding in vivo is not answered in this paper. The effect of proline isomerase indicates that refolding can be catalyzed. However the limited effect of proline isomerase suggests that some other chaperone(s) may be involved. Also, the interaction of the capsid protein with the surface protein may also contribute to folding. The possibilities should be discussed with an open mind.E. coli FkpA PPiase. Typically, PPiases are among the most abundant proteins. Therefore, we would expect that the extension domain folds catalysed by a general PPiase of the host cells.Yes, it is true that we cannot say which chaperone is (or which chaperones are) acting in vivo. However, we respectfully disagree with the reviewer about the "limited effect" of the PPiase. While after two weeks and 2 months (not shown) without co-expression of a PPiase, most DHBc subunits in capsids have still unfolded extension domains this has changed to the majority of extension domains being folded within 2 weeks when co-expressed with a PPiases. A general PPiase is sufficient for the catalysis as it works with the both the mutant capsids are less stable. In line with a reduced stability of capsids harbouring CPs with disordered extension domains, we observed that DHBcR124E∆ and DHBcR124E CLPs burst in thin, vitrified buffer whereas DHBc CLPs remains stable. Moreover, the new cell culture data (see above) illustrate a clearly enhanced proteolysis susceptibility of the R124E variant in cell culture.Another effect on the folding propensity of the extension domain is the formation of salt bridges (R124-E109) and hydrophobic interactions (H109-I99). These interactions are unlikely to require additional chaperones for formation and might happen instantaneously after the prolines are in the correct isomerization state. More importantly the significance of these stabilizing interactions can be interrogated by mutations: Mutants with a destabilized extension domain (DHBc R124E) behave very similar in cell culture to mutants with an entirely missing extension domain (DHBc R124E∆) as they do not form stable capsids that support replication. Considering that the folded extension domain almost doubles the intra-dimer interface it is conceivable that Thus proline isomerization probably acts by delaying the folding of the extension domain, whereas interactions within the extension domain are likely to fine-tune capsid stability during the viral life cycle. We have broadened the Discussion along these lines (subsection “A temporarily mobile spike extension – defining feature of hepadnaviruses employing large type CPs?”)The evolutionary connections from avian to other HBVs is important to understanding virus evolution. The authors should also relate the DHBV structure to other insert containing sequences instead of just the limited sequence alignment of Figure 1—figure supplement 1D.While avian large-type CPs have a well preserved extension domain, the sequence conservation of the extension domain to large-type CPs in other species is less well preserved. However, a large number of prolines in the extension domains of other host species is maintained. This is what we wanted to show with the alignment. Detailed, sequence based discussions of evolutionary aspects have been made by Lauber et al., 2017, and Dill et al., 2016, which is given in the Introduction.Based on the weak sequence conservation we would not necessarily expect that amphibian or lizard extension domains have a very similar structure – although structural data in Lauber et al., 2017, suggest that small-type CPs of nackedna viruses adopt an HBV CP-like fold despite highly divergent primary sequence. We have modelled the structure of Tibetan Frog CPs based on our DHBc structure. This suggests indeed a similar fold of the extension domain with maintenance of αe2 but not of αe1 and the positioning of the prolines at similar strategic positions as in avian viruses . HoweverHowever, large-type CPs go together with small-type surface proteins. We think that the large type CPs have evolved two potential surface protein binding sites per spike compared to only one in small type CPs. Support for this idea comes from the observation that the tip of the small-type HBc CPs matches in its overall fold and arrangement of loops the upper part of one extension domain. We have added the information on the size of the surface proteins in the Introduction (second paragraph) and have discussed the aspect of two potential binding sites per spike in the Discussion (subsection “A temporarily mobile spike extension – defining feature of hepadnaviruses employing large type CPs?”). We also added a figure to illustrate the point .


Thermal, antioxidant, physicochemical properties, degree of puffing and rollability of flour and tortillas were determined. Tortillas were produced from maize and Bambara Groundnut (BGN) flours at the ratio of 100:0, 95:5, 90:10, 85:15 and 80:20, respectively. Compositing maize with BGN flour showed an improvement on the proximate composition of maize flour and tortillas; however, carbohydrate content of tortillas significantly decreased with the addition of BGN in blends from 77.07 to 55.22. The temperatures of gelatinisation such as onset temperature (To) of flour blends increased from 57.50 to 71.95 °C, peak temperature (Tp) from 74.94 to 76.74 °C and the end temperature (Te) from 81.72 to 91.58 °C. Composite flours and tortillas had higher values of polyphenolic compounds and antioxidant activities than the control sample. Textural properties of control tortillas were higher than that of composite tortillas. Increase in the levels of BGN flour improved the weight and thickness of tortillas. However, diameter and spread ratio decreased. Degree of puffing and rollability of tortillas increased with the incorporation levels of BGN flour.The effect of substituting maize ( Moringa oleífera flour, amaranth flour, unripe banana and cassava flours, flaxseed flour and iron and other micronutrients [Consumers always demand food products that contain high nutritional value as well as furnishing extra health benefits. Therefore, there is a need for the food industry to develop new products with added potential health benefits. Tortilla is a flat unfermented bread prepared either from maize or wheat and has been widely consumed as the staple bakery food in Mexico and some Central America countries for centuries . Howeverutrients ,6,7.Vigna subterranae (L). Verdc) is a legume crop originally from Africa. Bambara groundnut (BGN) is among the most adaptable plants and it withstands harsh conditions better than many crops [Bambara groundnut  among the onset temperature of all flour blends and they showed an increasing trend with an increase of BGN flour. The inclusion of BGN flour at all levels resulted in an improved onset temperature of maize-BGN flour blends. The presence of mucilage in the BGN flour might have contributed to the increase of onset temperature of maize-BGN flour blends. Mucilage contains protein and complex polysaccharides that could compete with starch for moisture leading to higher onset starch gelatinisation temperatures in the flour blends [r blends . Other cr blends .p < 0.05. The peak temperature obtained follows the same trend reported for other flour such as cowpea (67–78 °C) [Peak temperature ranged from 74.94 to 78.45 °C, control and BGN-maize flour blends showed significant difference at 7–78 °C) . However7–78 °C)  and this7–78 °C) . Moreove4 had the highest value of gelatinisation enthalpy and this shows that BGN starch is more stable during baking and it increased the thermal stability of flour blends [The end temperature ranged from 81.72 to 91.58 °C and there was variation in the values. Higher gelatinisation end temperature in control sample is attributed to its larger granule size. Larger granules tend to have poorer hydration and swelling capacity than smaller granules. On the other hand, the low end temperature in BGN-maize flour blends could be due to large amounts of short amylopectin chains present in the starch of the flour blends . The entr blends .Gelatinisation enthalpy indicates the thermal stability of starch. Values of ΔH at different levels of composite flour blends might be attributed to the size of starch granules and amylopectin ratio. BGN flour interacted with the matrix of maize flour which caused a release of water and matric hardening resulting in a higher gelatinisation temperature. Higher values of gelatinisation enthalpy of composite flours suggest mild processing conditions because thermal treatment produces starch gelatinisation with a higher degree of disorder. In addition, higher values of gelatinisation enthalpy will result in tortillas with denser cell walls that will require higher force to rupture . Texture4). The low moisture content of control and composite flour blends is a good indication of microbiological stability of the flour during storage and can also lead to a reduction in the stalking tendency in baked goods [4) and there was a significant difference among all tortillas samples (p < 0.05). The control tortillas had the lowest moisture content than the composite tortillas. The significant increase in the moisture content of tortillas with the increase of BGN flour could be attributed to the nature of BGN which increased the overall water holding capacity of the tortillas. The moisture values are within the range (35–50%) reported for tortillas, depending on the conditions of nixtamalisation and variety of maize. However, higher moisture content of between 45%-50% makes the tortillas very susceptible to microbial spoilage. Moulds and yeasts become visible on tortillas stored at room temperature after 48 h but this can be prevented by keeping tortillas in an alkaline pH (>9) [The proximate composition of maize flour and tortillas added with BGN flour is shown in ed goods . The moied goods . The moi pH (>9) . These rs p < 0.0. The con4). The ash content was related to the amount of BGN flour added and this shows that BGN is rich in minerals than maize. The ash content of tortillas varied from 1.04 to 1.63%. The control sample was significantly different (p < 0.05) from all the samples and it had the smallest quantity of ash (1.04%) compared with composite tortillas. However, the ash content of tortillas was lower than that of flour blends and this could have been caused by heat during baking of tortillas since some minerals such as potassium, copper and zinc are decreased during cooking/high temperatures [The ash content of flour ranged from 1.23 (control) to 3.08% (BGNeratures .4). The increase in the protein content of composite flours with the quantity of BGN flour added is due to that BGN is a complete balanced food high in protein content rich with lysine and methionine while maize lacks lysine [4) and there was significant difference (p < 0.05) among all the tortillas samples except BGN2 and BGN3. The heat during the baking process induces oxidation through the generation of radicals [2 and BGN3. There was an increasing trend on the protein content of tortillas with the increase in the inclusion levels of BGN flour. There was a slight decrease on the protein content of the composite tortillas when compared with the flour blends. This might be due to the heat during the baking of tortillas because amino acid chains are broken down and protein denatures at high temperatures. Similar results were reported by Vazquez-Rodrigues et al. [There was a significant increase in the protein content of composite flour samples from 3.32 (control) to 7.24% . The high fat content in composite tortillas could be attributed to that BGN comparatively has more fat than maize [Moringa oleifera flour.The fat content of composite flour blends ranged from 1.74 to 2.95% with control having the lowest value. The fat content of the composite flour blends increased with the incorporation of BGN flour in the blends. The fat content of tortillas ranged from 1.73% (control) to 2.93% (BGNan maize . Diets wan maize . There wp < 0.05) among all the samples except between control and BGN1. The fibre content of tortillas ranged from 2.09 to 4.03% with the control sample having the lowest value. The fibre content of the flour blends was slightly higher than that of tortillas and this could be due to the heat during processing of tortillas as it has been reported that cooking/heating reduces the fibre content of food [The fibre content of the flour blends varied from 2.03 to 3.53% and there was a significant difference  of flour blends and tortillas is presented in z et al.  on tortip < 0.05) except BGN1 and BGN2 of tortillas which were not significantly different. TFC values ranged from 0.14 to 0.588 mg CE/g and 0.03 to 0.47 mg CE/g for both maize-BGN flour blends and tortillas, respectively. Both composite flour blends and tortillas showed an increasing trend with the increase in BGN flour inclusion. However, there was a decrease in the flavonoids content of composite tortillas compared to flour blends and this could be due to the breaking down of flavonoids and glycosides extraction by heat and steam during baking [1 and BGN2 tortillas was also similar. Moreover, the decrease in TFC of composite tortillas might be due epolymerisation of polyphenols and decarboxylation of phenolic acids that occur during thermal treatment [The total flavonoids content (TFC) of both composite flour blends and tortillas varied significantly (g baking . Such prg baking . This mareatment . Howeverreatment . The resreatment  that thep < 0.05) in control and composite flour blends and tortillas. Control flour and tortillas had lower radical scavenging ability than that added with BGN flour. The increase in DPPH activity of composite blend flours and tortillas is due to the higher radical scavenging activity of BGN flour. The development of melanoidins during baking further increased the radical scavenging ability of composite tortillas. In addition, the increase in DPPH activity of composite tortillas could be attributed to the heat during baking which contributed to bioactive compounds such as ferulic acid from matric interacting with some antioxidant substances that were dissolved in water during dough formation [The antioxidant activity of maize flour and tortillas incorporated with BGN flour at various levels was evaluated by DPPH and FRAP methods . The incormation . A higheThe Ferric Reducing Ability of Plasma (FRAP) activity of the composite flour blends varied from 0.071 to 0.156 mg GAE/g and for tortillas ranged from 0.106 to 0.183 mg GAE/g, respectively. The control flour and tortillas had lower FRAP activity than that added with BGN flour. The inclusion of BGN flour increased the FRAP activity of maize-BGN flour blends and tortillas.The increment of antioxidant activity of flours and tortillas is associated with the increase of TPC when incorporating BGN flour. Therefore, the BGN flour could be used as an alternative source of antioxidants that would enhance the nutritional characteristics of the tortillas. These findings suggest that consumption of BGN complemented product could offer some health benefits especially thermal processed products because they contain a lot of antioxidants reducing power. Sharma and Gujral  reportedp ≤ 0.05) was observed for control and composite tortillas. The results show that the control sample had a higher hardness, springiness, gumminess, cohesiveness and chewiness values than the tortillas containing BGN flour. The observed difference could be attributed to the fact that the control tortillas contain high starch (amylose and amylopectin) content. A three dimensional network is created by gelation of amylose right after baking. Amylose associates to form an insoluble network that causes a flexible gel structure in maize tortillas [1. These values are higher compared to that reported by Chel-Guerrero et al. [Moringa oleifera flour.ortillas . In addiortillas . The haro et al.   to 14.60 cm (control).The physical properties of tortillas prepared from maize-BGN flour blends are presented in 4). The decrease in diameter might have influenced the increase in thickness of the tortillas. Baljeet et al. [The thickness of the tortillas ranged from 2.17 (control) to 3.07 mm . The puffing degree and rollability increased as more BGN flour was added to the formulation and full puffing of the tortillas was observed in BGN4 sample. The observed variations in puffing degree might be attributed to the fact that puffing takes place during the baking of tortillas owing to the creation and aggregation of steam between the two layers of the tortillas [The results of puffing degree and rollability of tortillas are presented in ortillas . Initialortillas .Rollability of the composite tortillas increased as more BGN flour was added to the formulation although the cohesiveness of the tortillas added with BGN was low when compared with the control tortillas. Basic ingredients such as water and fat are considered to be dough conditioners because they directly impact the way the dough is formed and its final structure; this improves the softness and rollability of tortillas . This mimasa” (corn dough) was obtained from Azteca Mexican Products CC, Midrand, Gauteng Province, South Africa. All chemicals were purchased from Merck, Centurion, South Africa, and were of analytical grade.Creamy white BGN, shortening and salt were obtained from a local market. Commercial “w/v) for 12 h at room temperature. BGN were manually de-hulled and oven dried at 60 °C for 12 h. A disc attrition mill  was used to mill BGN into flour and sifted using a 355 μm sieve.Approximately 2 kg of Bambara groundnut (BGN) was sorted to remove foreign materials, damaged and spoiled groundnuts. They were soaked in 5 L of tap water . Composite flours were prepared to make dough and were thoroughly mixed to obtain uniformity. The composite flour samples were sealed in polyethylene bags and stored at 4 °C for further use.Composite flour was prepared by mixing BGN with maize flours in various ratios . This was followed by adding water for “masa” formation to make tortillas. The dough was moulded into 50 g round pieces. The pieces were pressure pressed into 4 mm thick tortillas. A hot plate with shortenings was preheated to 250 °C and the tortillas were baked for 30 s on the first side, overturned and then baked for another 40 s on the second side, flipped and baked for another 10 s on the first side. A rack was used to cool the baked tortillas for 1 min and they were packaged in polyethylene bags and stored in a desiccator for subsequent analysis. The tortillas made from 100% maize dough were used as control.The composite dough prepared from maize and BGN flours was used to produce ratios of 5, 10, 15 and 20% (o (onset temperature), Tp (peak temperature), Te (end temperature) and ΔH . Samples were analysed in triplicates and the obtained data was calculated using Pyris software .The gelatinisation behaviour of composite flour blends from maize and BGN were measured as described by Sun et al.  using diComposite flour samples were analysed for proximate composition: moisture, ash, protein, fat and crude fibre were determined using hot air oven, Muffle furnace, micro-Kjedahl Nx6.25, soxhlet extraction and Fibre Tech following the method of Association of Official Analytical Chemists . CarbohySamples were extracted with acidified methanol . Approximately one gram of sample was added to 5 mL of acidified methanol at room temperature for 2 h in a water bath (Model no: WBH 601 Labcon) with constant shaking at every 10 min. The extract was separated in a centrifuge for 15 min at 4500 rpm. Supernatant was then collected.2CO (7.5% concentration) was added to the mixture. Aluminium foil was used to cover the mixture before incubation at 50 °C for a period of 5 min. With the use of UV spectrophotometer microplate reader , the absorbance of the samples was read at 760 nm. The solvent used for extraction was acetone and the standard phenol compound used was Gallic acid. The results obtained were recorded as gallic acid equivalents from calibration curve [Folin–Ciocalteu colorimetric method was used to determine the total polyphenols of flour blends and tortillas. Briefly, 0.2 g of flour blends or tortillas was weighed and mixed with 2 mL of acetone. The mixture was incubated for a period of 1 h . The mixture was centrifuged at a speed of 6000 rpm for 5 min at a temperature of 4 °C. About 109 µL of Folin Ciocalteu solution was added to 9 µL of a centrifuged sample placed in a microplate. Approximately 180 µL of Naon curve .The total flavonoid content of samples was determined using a colorimetric method with few modifications. Approximately 0.5 mL aliquots extracts were deposited into 15 mL conical tube which comprised 4 mL distilled water with 0.15 mL of  mg CE/g .The antioxidant activity (DPPH Assay) of flour blends and tortillas was assessed using the method of Anyasi et al. . BrieflyThe method of Oyaizu  was follThe textural profile of the tortillas was analysed using texture analyser TAXTplus  attached to a 50 kg load cell with different probes, following the method described by Bejosano et al. . The texTortillas’ diameter was determined by averaging two perpendicular measurements on each of the three tortillas for a total of six observations per treatment. A venier calliper was used to measure the average thickness (mm) and this was done by stacking twelve (12) tortillas on top of each other. The digital weight balance was used to measure the weight of the tortillas. The method of Winstone  was usedThe method described by Cuevas-Martínez et al.  was adopThe method of Cepeda et al.  was usedp < 0.05 which was considered statistically significant.All measurements were performed in triplicate and results obtained were expressed as mean standard error deviation. The analysis of variance (ANOVA) was carried out using SPSS version 24 . The mean was separated using the Duncan multiple range tests, 4 sample. The study concluded that the substitution of maize with BGN flour produced tortillas with improved nutritional characteristics and better degree of puffing and rollability.The results showed that thermal properties  of composite flour samples were higher than those of the control flour. However, the end temperature values decreased. Proximate compositions of both maize flour and tortillas were improved by the addition of BGN flour and there was a significant increase on the protein content of both flour samples and tortillas. Polyphenolic compounds and antioxidant activity of flour samples and tortillas incorporated with BGN flour showed an increase. In terms of the texture, the control tortillas had higher hardness, springiness, gumminess, cohesiveness and chewiness values than the tortillas containing BGN flour. Weight and thickness of tortillas added with BGN flour were higher than control tortillas. However, diameter and spread ratio decreased. The puffing degree and rollability increased as more BGN flour was added to the formulation and full puffing of the tortillas was observed in the BGN


The paper presents an experimental study on microstructure changes in duplex steel after hot deformation. Duplex steels and cast steels are characterized by a multiphase microstructure. They are relatively new materials with great contributions to the many fields of industries. Due to the fact of deforming two different phase austenite and ferrite those materials have a complex plasticity. This work is a continuation and complementation of previous works and is a significant supplement to information presented in them. The article concerns precipitation phenomena and changes in the microstructure of two grades of ferritic-austenitic steels: X2CrNiMoN25-7-4 and X2CrNiMoCuN25-6-3. Those steels have a very similar chemical composition, differing by only 2.5% copper content. An important aspect presented in the work is we observed that adding 2.5% copper prevented precipitation of the destructive sigma phase during the hot deformation. Steels and other iron alloys are very well known as construction materials, are widely-used and in the near future this will not change. Among the resistant-to-corrosion steels  and castTaking under consideration such conditions, duplex steels and cast steels with a comparable proportion of the basic phases—ferrite and austenite—present a better set of mechanical properties compared to traditionally used ferritic or austenitic steels. The main area of application are the chemical industry, construction of warehouses, tanks of vessels transporting products with high chemical activity  or equipment used: in the energy production, pulp, paper, food and petrochemical industries ,15,16,17The subjects of the study were two grades of duplex steels X2CrNiMoN25-7-4 and X2CrNiMoCuN25-6-3, which chemical compositions are presented in Making of Y-shaped castings with a wall thickness of 25.0 mm ;Analysis of the microstructure of the materials in the raw state;Solution-annealing to remove the primary precipitated sigma phase;Hot deformation using the Gleeble 3800 Physical Simulation System with the Hydrawedge module;Analysis of materials microstructure after the plastic deformation process.The conducted experimental researches were divided into the following stages:γ’ known also as γ2.The microstructure of analyzed materials was revealed with the metallographic reagent Mi21Fe . The main advantage of this reagent was marking on different colors phases, what allows and helps easily analysis of microstructure. The structure of materials were analyzed with use of the light microscope and the phase content was determined using the Nis-Elements D application. The evaluation of the raw state microstructure showed that the material was characterized by presence of ferritic–austenitic grains with a small presence of 2%–2.5% of the σ phase. According to literature study ,28,29,30In the first stage of studies were made deformation of the raw state. In some cases of hot deformed samples, the cracks were observed. This was a basis for the microstructure analysis and some selected results are shown in −1. Quantitative analysis allowed to determine its share at the level of 10%–15% depending on the deformation temperature. The volume fraction of the phase increases with temperature decrease. Based on the obtained data, it was found that, the limit temperature below which the alloy should not be deformed was 1100 °C and it was definitely safer to apply lower deformation rates.Evaluation of the microstructure showed that below 1100 °C, can appear the initiation of phase σ precipitation, what was observed for a deformation rate of 10.0 sHot deformation tests were also carried out for the X2CrNiMoCuN25-6-3 alloy, which was presented earlier works ,3,14. Crγ’ also named γ2 phase [As it can be seen the material without cooper was characterized by presence of the austenite and ferrite. In the duplex steel with addition of cooper was also present the γ2 phase ,29,30,32In the further part of the work, deformation tests were carried out for both tested alloys according to the parameters presented in −1. The obtained results (no crack observed) showed that the X2CrNiMoN25-7-4 steel was characterized by good plasticity in the tested temperature range for true deformation 0.2 and 0.3 for both deformation rates 1.0 and 10 s−1.The deformation according to variant 16 led to the crack of the sample. It should be emphasized, that however in this case a total relative deformation of 1.2 was achieved in a relatively short time and with a strain rate of 10 s−1 at 850 °C . The only observed precipitations of sigma phase were observed during initial tests which concerned no solution-annealed material. In those tests the amount of precipitated σ phase during crystallization was preserved and after deformation its amount was similar. After solution-annealing process in the microstructure of the steel with 2.5% of copper, deformed in the 850 °C, none σ phase precipitation were observed. It should be mentioned that deformation in this range of temperature can provide the grain refining and changes in texture as it is presented in [The plasticity of the X2CrNiMoCuN25-6-3 steel was similar to the X2CrNiMoN25-7-4 steel. Although both materials cracks during performed tests. In the microstructure of the grade with copper was not observed σ phase. This steel was characterized by the presence of not only main phases like austenite and ferrite but also the ented in . Lack of


The experimental images obtained by the camera were post-processed using an in-house image processing code developed on the plat-form of MATLAB software. The results show that particle-free droplets can undergo prolate (deformation in the applied electric field direction) or oblate deformation (deformation that is perpendicular to the direction of the applied electric field) of the droplet interface, whereas the low-conductivity particles can be manipulated at the emulsion interface to form a ‘belt’, ‘helmet’ or ‘cup’ morphologies. A densely particle-covered droplet may not restore to its initial spherical shape due to ‘particle jamming’ at the interface, resulting in the formation of unique droplet shapes. Densely particle-covered droplets behave like droplets covered with a thin particle sheet, a capsule. The deformation of such droplets is explored using a simulation model under a range of electric capillary numbers . The results obtained are then compared with the theory and experimental findings. It was shown that the proposed simulation model can serve as a tool to predict the deformation/distortion of both the particle-free and the densely particle-covered droplets within the small deformation limit. We believe that this study could provide new findings for the fabrication of complex-shaped species and colloidosomes.The electrohydrodynamic deformation of an emulsion droplet with a clean and particle-covered interface was explored. Here, the electrohydrodynamic deformation was numerically and experimentally demonstrated under the stimuli of moderate and strong electric fields. The numerical method involves the coupling of the Navier–Stokes equation with the level set equation of interface tracking and the governing equations of so-called leaky dielectric theory. The simulation model developed for a clean interface droplet was then extended to a capsule model for densely particle-covered droplets. The experiments were conducted using various combinations of immiscible oils and particle suspensions while the electric field strength ~10 S) and the electrical conductivity ratio (R) amongst the droplet and the outside liquids.A mixture of two or more immiscible liquids results in the formation of emulsions ,2. The eR > S, if the degree of deformation  exceeds a value of 1.5, the droplet becomes unstable and breaks at the ends [The earliest experiments on a neutrally buoyant emulsion droplet under an electric field were conducted in 1953 . Later, Colloidal particles do have a robust affinity to get adsorbed at the liquid–liquid interface and this phenomenon can stabilize the emulsions ,29. The Ha and Yang  developeIn this article, a numerical simulation model is established as a consolidated model that can predict the low-limit deformation of both the particle-free and the densely particle-covered droplets. Here, we used COMSOL Multiphysics for the implementation of various equations governing the problem. We numerically described a particle monolayer surrounding the emulsion interface. We also performed experiments using the basic experimental facility and compared the experimental results with the theory and the simulation results. D) of the droplet. The physical properties of the dielectric oils used in this research are given in The experimental scheme for observing the droplet deformation is as shown in 3 of the particle suspension in the form of a droplet was dispensed into the castor oil to investigate the dynamics of asphaltene particles under an electric field. The suspensions of kaolinite clay were also formed using the same procedure.The suspension of asphaltene particles  was prepared by dissolving asphaltenes in silicone oil with a concentration of 1.0 wt%. The process of particle synthesis is given in the V) was applied to the right side of the domain while the left side was grounded. All the other walls were electrically insulated. No slip condition was implemented on all the walls of the domain. The initial fluid interface for fluid flow, and the continuity of electric potential and the continuity of electric current for electrostatics were imposed at the interface. By using the finite element method, the electrohydrodynamic problem under consideration was solved. Here, the Navier–Stokes equation was coupled with the equations of the electrostatics’ theory. The flow was considered to be laminar, two phase and incompressible. The numerical domain with complete boundary conditions is as shown in The velocity of the flow equation ,36,37. Iequation ,39 givenThe hydrodynamic properties such as density The governing equation of the electric field for leaky dielectric fluid is given asThe electrical properties such as conductivity G) for the particle monolayer.The total electric force is given asWe first performed the domain and grid independency tests to ensure the reliability of the simulation results based on the convergence criterion. We increased the grid resolution near the interface introducing a rectangular virtual domain using virtual operations in COMSOL Multiphysics . This method increased the accuracy of interface tracking without much increase in the computational loads. The final grid element size in the virtual domain and the remaining domain was set as 0.044 mm and 0.150 mm, respectively. Furthermore, the domain size of length and height equal to 3.35 L and 3 L, respectively, (L being the droplet diameter) were chosen as it avoided the effects of any domain truncation. The details about the grid independency and the domain independency tests are given in D) is calculated asP and W are the dimensions of the distorted droplet in the parallel and perpendicular directions with respect to the applied electric field. Leaky dielectric fluids behave differently as compared to dielectric fluids under an electric field. A small amount of charge is accumulated at the interface which is not present in perfect dielectrics. The deformation (eCa) is defined as the fraction of electric stress to capillary stress, acting at the liquid–liquid interface of the droplet of radius r: ε2 is the outside liquid electrical permittivity and oE is the external unperturbed electric field. γ is surface tension of the liquid–liquid interface. Taylor’s theory [Here, the electric capillary number (s theory  for predeqD) is given as [sCa is the modified electric capillary number due to the existence of particles at the interface.The particle monolayer developed at the droplet interface can be modeled as an incompressible material, called the capsule model and the deformation (given as (14)Deq=oErε02/γ), which is the ratio of the electrical stresses (oEε02) countered by the capillary stresses (γ/r). The simulation results were then validated with the experimental findings. Owing to the slight density difference between the liquids, the resulting droplet gravitational velocity was much slower than the electrohydrodynamic flow speed. For example; for the case of the silicone droplet in castor oil, the gravitational velocity −5 m/s and the electrohydrodynamic flow velocity was  was less than the conductivity ratio (R), the upper and the lower half of the droplet developed the opposite charge of that of the input (+) and the ground electrode (−). As the opposite charge attracted, the droplet was prolate deformed and the flow was directed from the equator towards the poles. Conversely, when the S > R, the upper and the lower half of the droplet developed the same charge as that of the input (+) and the ground electrode (−), and the droplet was oblate deformed and the flow direction was now reversed i.e., from the poles towards the equator of the droplet. It should be noted that it is also possible to get prolate deformation with a flow direction from the poles towards the equator under a certain set of parametric conditions [A particle-free droplet was observed to show prolate  or oblate deformation (deformation that is perpendicular to the electric field direction) under the electric field. This is as shown in nditions .The experiments were performed with different immiscible liquids refer to  as shownS > R holds, the particles were driven to the interface by applying weak electric field pulses (oE ~ 0.1 kV mm−1) for about 2 min. By doing so, the electrohydrodynamic flow inside the droplet pushed the particles towards the interface. These particles were then dragged by the flow at the interface towards the equator. Once entrapped at the interface, the electric field was removed. The particle distribution was then randomized at the interface and particle-covered droplets were produced. The schematic of the whole process is as shown in S < R, the particles seemed unaffected by the electric field and did not move towards the emulsion interface. Particle interfacial dynamics under weak electric fields are as shown in In the case of suspensions for which It is pertinent to mention here that though the particle-free droplets regain their spherical shape (low surface energy configuration) after the removal of the electric field, the particle-covered droplets may or may not. Experiments were also performed with kaolinite clay suspensions as shown in S >R was subjected to an electric field; it behaves differently compared with a particle-free droplet. Here, only the droplets with high particle concentrations/coverage were considered. For such droplets, a mono layer of closely packed particles covered the entire interface. It behaved like an elastic sheet capable of supporting and withstanding the anisotropic stresses and strains [sCa) defined in terms of a solid property such assCa) is defined as the ratio of electric stresses to the shear elastic stresses, and G is the elastic shear modulus. The value of G for the densely packed particle at the emulsion interface can be taken as 1.43 times the interfacial tension  and conductivity ratio (R) between the droplet and the outside liquid emerged as two important parameters deciding the direction of the droplet distortions, the direction of the flow patterns and/or the possibility of the fabrication of particle-covered droplets. Weak electric field pulses were used to move the particles to the emulsion interface. Depending on the particle concentrations, stable non-spherical colloidosomes can be formed. For predicting the deformation of the particle-covered droplet, we adopted a simplified approach of re-defining the interfacial tension (γ) in the simulation model in terms of the elastic shear modulus (G) of the particle mono layer. The experimental results for the particle-covered droplets were then compared with the theory and our simulation results. Our simulation model emerged as a consolidated model and effectively predicted the deformation for both the cases. Thus, it can be used as a tool to predict the deformation for both cases within the small deformation limit i.e., when the droplet instabilities such as breakups or electrorotation (Quincke rotation) are not produced.In this article, the electrohydrodynamic deformation of a nearly neutrally buoyant droplet with a clean and particle-covered interface were studied. The main novelty of the current work lied in implementing the simulation model as a consolidated model to predict the deformation of both the particle-free and the densely particle-covered droplets. First of all, we built a simulation model by coupling Navier–Stokes equation of fluid flow with governing equations of electrostatics theory. The interface was tracked by a conservative level set function. We tested the model against the theory and our experiments for particle-free droplets under varying conditions. The experiments were performed using a cuvette cell and an electric field was generated. Deformation was measured by an in-house code developed using the MATLAB image-processing tool. We then extracted the low conductivity asphaltene particles from a raw feedstock and observed their behavior at the droplet interface. The permittivity ratio (We expect that the developed model and experimental findings contribute to provide a guideline to control the emulsion droplet shape and particle assembly. If the current oils are replaced with UV-cured polymer solutions, the complex shape of the species or colloidosomes can be fabricated.


This paper presents rich new families of relative orbits for spacecraft formation flight generated through the application of continuous thrust with only minimal intervention into the dynamics of the problem. Such simplicity facilitates implementation for small, low-cost spacecraft with only position state feedback, and yet permits interesting and novel relative orbits in both two- and three-body systems with potential future applications in space-based interferometry, hyperspectral sensing, and on-orbit inspection. Position feedback is used to modify the natural frequencies of the linearised relative dynamics through direct manipulation of the system eigenvalues, producing new families of stable relative orbits. Specifically, in the Hill–Clohessy–Wiltshire frame, simple adaptations of the linearised dynamics are used to produce a circular relative orbit, frequency-modulated out-of-plane motion, and a novel doubly periodic cylindrical relative trajectory for the purposes of on-orbit inspection. Within the circular restricted three-body problem, a similar minimal approach with position feedback is used to generate new families of stable, frequency-modulated relative orbits in the vicinity of a Lagrange point, culminating in the derivation of the gain requirements for synchronisation of the in-plane and out-of-plane frequencies to yield a singly periodic tilted elliptical relative orbit with potential use as a Lunar far-side communications relay. The  After the advent of spacecraft rendezvous and docking, perhaps one of the earliest recognitions of the potential utility of spacecraft formation flight was in the form of a space-based interferometer, proposed by Sholomitsky et al. , and theThe guidance and control of a wide variety of formation-flying concepts have been comprehensively surveyed in the previous decade, encompassing such applications as hyperspectral sensing and fractionated spacecraft  could be created in the vicinity of libration points using continuous thrust Dusek . Later, Displacing the plane of an orbit using out-of-plane thrust was explored most prominently by Nock , Yashko In this paper, rich new families of relative trajectories for spacecraft formation flight are generated through the application of continuous thrust. In both the two-body Hill–Clohessy–Wiltshire frame and the circular restricted three-body problem, it is shown that simple position feedback can be used to modify the natural frequencies of the linearised dynamics through direct manipulation of the system eigenvalues and to thereby produce interesting and novel stable relative orbits. Whereas past authors have generally used a top-down engineering approach, designing active controllers with which to force a spacecraft onto a predetermined reference trajectory  of the system to produce interesting and novel new relative orbits. The primary aim of this section is to generate relative orbits with potential future applications in on-orbit inspection—an area where the advantages of modifying the frequencies of periodic motion are readily apparent.x-axis as following the radius vector from the central mass through the target, the z-axis following the orbital angular momentum vector, and the y-axis points in the along-track direction of the target’s motion around the central body. This reference frame forms the environment in which new relative orbits are generated throughout this section of the paper.Figure n is the angular velocity of the rotating frame. This is in turn given byR is the inertial-frame orbit radius.The well-known HCW equations, augmented with continuous thrust terms, are , and the vector y-axis of the circle. Both x-axis as measured in the anticlockwise direction about the circle’s central axis. It is taken that x-y-plane is clockwise), where n and t have their usual meaning. The inclusion of x-y plane and centred at the origin, T. From Eq. , using linearised dynamics for spacecraft relative motion. Whereas the previous section considered the generation of orbits with potential applications for on-orbit inspection, this section aims to derive the feedback gains necessary to synchronise the in-plane and out-of-plane frequencies to produce a stable, three-dimensional, singly periodic orbit with potential utility as an Earth–Moon awaguchi  and can awaguchi .t. The usual origin of the system is at the barycentre of the two primary masses, the axis X is aligned with the vector connecting the two primary masses (Y is perpendicular to it such that the X-Y plane is the Earth–Moon plane. However, since the aim is to examine the motion of a low-thrust spacecraft relative to a Lagrange point, it is useful to place the origin of the system at the Lagrange point in question, x is aligned with the X-axis, y is aligned with the Y-axis, and z completes the right-hand coordinate system. The linearised equations of relative motion in the vicinity of the Lagrange point are given by Farquhar (In Fig. Farquhar  38a\docue as Eq. . The syse as Eq. . Using tWith reference to Fig. z-axis motion is decoupled, it is not shown here.For arbitrary initial conditions, with A, B, C, and D are thenx-axis motion, and y-axis amplitude of the elliptical relative orbit around the Lagrange point. The out-of-plane motion, which is always periodic, has the solutionz-axis amplitude, Let rom Baig 44\documeion Baig  and depeIt can then be shown that 3-D periodic orbits are achieved when z-axis is For Farquhar : insteadmentclasspt{minimaContinuous low-thrust propulsion can augment the capabilities of spacecraft formations by allowing relative flight on forced Keplerian and non-Keplerian trajectories. Simple control strategies using only position feedback can be applied to generate rich new families of stable relative orbits in both the two-body and restricted three-body problems, whilst requiring only minimal intervention into the dynamics of the problem, to permit implementation aboard small, low-cost spacecraft. One main contribution of this paper is the parametric derivation of a forced circular orbit of arbitrary dimension, orientation, and period relative to a target on a circular two-body orbit. A second contribution is the derivation of thrust commands for out-of-plane period modulation, leading to the analytical description of a cylindrical relative orbit in the two-body rotating frame, with potential future applications in on-orbit inspection. A third contribution is the derivation of gain requirements for the synchronisation of in-plane and out-of-plane periods of a relative orbit around a Lagrange point in the restricted three-body problem, such that a stable, singly periodic 3-D trajectory can be accessed for the purposes of providing a constantly visible Earth–Moon