Predicted Amino Acid Sequence Research Articles

Structure–activity relationships of the intramolecular disulphide bonds in LEAP2, an antimicrobial peptide from Acrossocheilus fasciatus

BackgroundThe liver-expressed antimicrobial peptide 2 (LEAP2) plays a pivotal role in the host’s immune response against pathogenic microorganisms. Numerous such antimicrobial peptides have recently been shown to mitigate infection risk in fish, and studying those harboured by the economically important fish Acrossocheilus fasciatus is imperative for enhancing its immune responses against pathogenic microorganisms. In this study, we cloned and sequenced LEAP2 cDNA from A. fasciatus to examine its expression in immune tissues and investigate the structure–activity relationships of its intramolecular disulphide bonds.ResultsThe predicted amino acid sequence of A. fasciatus LEAP2 was found to include a signal peptide, pro-domain, and mature peptide. Sequence analysis indicated that A. fasciatus LEAP2 is a member of the fish LEAP2A cluster and is closely related to Cyprinus carpio LEAP2A. A. fasciatus LEAP2 transcripts were expressed in various tissues, with the head kidney exhibiting the highest mRNA levels. Upon exposure to Aeromonas hydrophila infection, LEAP2 expression was significantly upregulated in the liver, head kidney, and spleen. A mature peptide of A. fasciatus LEAP2, consisting of two disulphide bonds (Af-LEAP2-cys), and a linear form of the LEAP2 mature peptide (Af-LEAP2) were chemically synthesised. The circular dichroism spectroscopy result shows differences between the secondary structures of Af-LEAP2 and Af-LEAP2-cys, with a lower proportion of alpha helix and a higher proportion of random coil in Af-LEAP2. Af-LEAP2 exhibited potent antimicrobial activity against most tested bacteria, including Acinetobacter guillouiae, Pseudomonas aeruginosa, Staphylococcus saprophyticus, and Staphylococcus warneri. In contrast, Af-LEAP2-cys demonstrated weak or no antibacterial activity against the tested bacteria. Af-LEAP2 had a disruptive effect on bacterial cell membrane integrity, whereas Af-LEAP2-cys did not exhibit this effect. Additionally, neither Af-LEAP2 nor Af-LEAP2-cys displayed any observable ability to hydrolyse the genomic DNA of P. aeruginosa.ConclusionsOur study provides clear evidence that linear LEAP2 exhibits better antibacterial activity than oxidised LEAP2, thereby confirming, for the first time, this phenomenon in fish.

Discovery and Derivatization of Tridecaptin Antibiotics with Altered Host Specificity and Enhanced Bioactivity.

The prevalence of multidrug-resistant (MDR) pathogens combined with a decline in antibiotic discovery presents a major challenge for health care. To refill the discovery pipeline, we need to find new ways to uncover new chemical entities. Here, we report the global genome mining-guided discovery of new lipopeptide antibiotics tridecaptin A5 and tridecaptin D, which exhibit unusual bioactivities within their class. The change in the antibacterial spectrum of Oct-TriA5 was explained solely by a Phe to Trp substitution as compared to Oct-TriA1, while Oct-TriD contained 6 substitutions. Metabolomic analysis of producer Paenibacillus sp. JJ-21 validated the predicted amino acid sequence of tridecaptin A5. Screening of tridecaptin analogues substituted at position 9 identified Oct-His9 as a potent congener with exceptional efficacy against Pseudomonas aeruginosa and reduced hemolytic and cytotoxic properties. Our work highlights the promise of tridecaptin analogues to combat MDR pathogens.

Research Note: Characterization and phylodynamic analysis of new infectious bursal disease virus variants circulating in Argentina

Infectious Bursal Disease is a highly contagious disease that affects young chickens and leads to significant economic losses. Its causal agent is a double-stranded RNA virus that, due to its high error rate during the replication process, gives rise to a constant generation of new virus variants. Until 2014, strains of Infectious Bursal Diseases Virus (IBDV) belonging to genogroup 4 predominated in Argentina, but there have been no reports since then regarding the circulating genogroups in poultry. In this study, 11 recent sequences of Argentine from the hypervariable region of VP2 protein (hvVP2) were analyzed to determine their genogroup, origin, evolution, and amino acid sequence. Samples from chickens showing signs of IBDV infection were collected, and the hvVP2 region was amplified using RT-PCR, followed by sequencing. The results indicated that the analyzed strains belong to genogroup 2, with an estimated evolutionary rate of 1.74 × 10-3 substitutions/site/year. It is speculated that the predominant group of sequences began to spread in Argentina around 2014 and had its origins in China. Another sample is related to strains from South Korea and is not closely linked to the main group. Furthermore, the predicted amino acid sequences show similarity to strains that can evade vaccine-induced immunity. These findings underscore the importance of active surveillance in poultry to mitigate losses caused by IBDV.

Differential Expression of Stress Adaptation Genes in a Diatom Ulnaria acus under Different Culture Conditions.

Diatoms are a group of unicellular eukaryotes that are essential primary producers in aquatic ecosystems. The dynamic nature of their habitat necessitates a quick and specific response to various stresses. However, the molecular mechanisms of their physiological adaptations are still underexplored. In this work, we study the response of the cosmopolitan freshwater diatom Ulnaria acus (Bacillariophyceae, Fragilariophycidae, Licmophorales, Ulnariaceae, Ulnaria) in relation to a range of stress factors, namely silica deficiency, prolonged cultivation, and interaction with an algicidal bacterium. Fluorescent staining and light microscopy were used to determine the physiological state of cells under these stresses. To explore molecular reactions, we studied the genes involved in the stress response-type III metacaspase (MC), metacaspase-like proteases (MCP), death-specific protein (DSP), delta-1-pyrroline-5-carboxylate dehydrogenase (ALDH12), and glutathione synthetase (GSHS). We have described the structure of these genes, analyzed the predicted amino acid sequences, and measured their expression dynamics in vitro using qRT-PCR. We demonstrated that the expression of UaMC1, UaMC3, and UaDSP increased during the first five days of silicon starvation. On the seventh day, it was replaced with the expression of UaMC2, UaGSHS, and UaALDH. After 45 days of culture, cells stopped growing, and the expression of UaMC1, UaMC2, UaGSHS, and UaDSP increased. Exposure to an algicidal bacterial filtrate induced a higher expression of UaMC1 and UaGSHS. Thus, we can conclude that these proteins are involved in diatoms' adaptions to environmental changes. Further, these data show that the molecular adaptation mechanisms in diatoms depend on the nature and exposure duration of a stress factor.

Characterization and function analysis of cathepsin C in Marsupenaeusjaponicus

Cathepsin C is a cysteine protease widely found in invertebrates and vertebrates, and has the important physiological role participating in proteolysis in vivo and activating various functional proteases in immune/inflammatory cells in the animals. In order to study the role of cathepsin C in the disease resistance of shrimp, we cloned cathepsin C gene (MjcathC) from Marsupenaeus japonicus, analyzed its expression patterns in various tissues, performed MjcathC-knockdown, and finally challenged experimental shrimps with Vibrio alginolyticus and WSSV. The results have shown the full length of MjcathC is 1782 bp, containing an open reading frame of 1350 bp encoding 449 amino acids. Homology analysis revealed that the predicted amino acid sequence of MjcathC shared respectively 88.42 %, 87.36 % and 87.58 % similarity with Penaeus monodon, Fenneropenaeus penicillatus and Litopenaeus vannamei. The expression levels of MjcathC in various tissues of healthy M. japonicus are the highest in the liver, followed by the gills and heart, and the lowest in the stomach. The expression levels of MjcathC were significantly up-regulated in all examined tissues of shrimp challenged with WSSV or V. alginolyticus. After knockdown-MjcathC using RNAi technology in M. japonicus, the expression levels of lectin and heat shock protein 70 in MjcathC-knockdown shrimp were significantly down-regulated, and the mortality of MjcathC-knockdown shrimp challenged by WSSV and V. alginolyticus significantly increased. Knockdown of the MjcathC reduced the resistance of M. japonicus to WSSV and V. alginolyticus. The above results have indicated that cathepsin C may play an important role in the antibacterial and antiviral innate immunity of M. japonicus.

Identification of new nucleotide sequences of the Glu-B1-1 gene encoding x-type glutenins in bread wheat.

Studies of the genetic base and polymorphism of bread wheat cultivars aimed at identifying alleles of genes associated with high baking and other economically valuable traits seem to be relevant, since bread wheat, along with all representatives of the Triticeae tribe, has a huge genetic potential for creating cultivars with high technological and rheological properties of grain flour. The aim of this study was sequencing and analysis of the nucleotide sequences of the Glu-B1-1 gene, and analysis of the predicted amino acid sequences of its protein product in three cultivars of bread wheat. Thus, in the course of genotyping cultivars and lines of bread wheat for the Glu-B1-1 gene, in the cultivars 'Avesta', 'Leningradka krupnozernaya' and line C-75094, previously undescribed changes in the size of amplifiable regions of the Glu-B1-1 gene for high-molecular weight glutenins were found. Comparative analysis of the nucleotide sequences of these genes with known sequences showed the presence of two deletions in 'Avesta' and C-75094 and the presence of seven single-nucleotide substitutions in 'Leningradka krupnozernaya'. Alignment of the predicted Glu-B1 amino acid sequences of the studied accessions and the standard cultivar carrying the Glu-B1-a allele showed that deletions in the amino acid sequences of 'Avesta' and C-75094 accessions are localized in the central domain of the protein and affect the amount of tri-, hexa-, and nonapeptides, and in 'Leningradka krupnozernaya', a decrease in GQQ and PGQGQQ by one unit was revealed. In addition, substitutions of five amino acids were found in 'Leningradka krupnozernaya'. Thus, we have found previously undescribed deletions and substitutions in the nucleotide sequences of the Glu-B1-1 gene for high-molecular-weight glutenins, which lead to changes in amino acid sequences in functionally important regions, namely, in the central domains of protein molecules. The identified mutations can be used for genotyping bread wheat cultivars.

Analysis of Predicted Amino Acid Sequences of Diatom Microtubule Center Components.

Diatoms synthesize species-specific exoskeletons inside cells under the control of the cytoskeleton and microtubule center. Previous studies have been conducted with the visualization of the microtubule center; however, its composition has not been studied and reliably established. In the present study, several components of MTOC in diatoms, GCP (gamma complex proteins), Aurora A, and centrins have been identified. Analysis of the predicted amino acid sequences of these proteins revealed structural features typical for diatoms. We analyzed the conserved amino acids and the motives necessary for the functioning of proteins. Phylogenetic analysis of GCP showed that all major groups of diatoms are distributed over phylogenetic trees according to their systematic position. This work is a theoretical study; however, it allows drawing some conclusions about the functioning of the studied components and possible ways to regulate them.

Multilocus Sequence Analysis and Detection of Copper Ion Resistance of Xanthomonas phaseoli pv. manihotis Causing Bacterial Blight in Cassava.

Cassava (Manihot esculenta Crantz) is an important tropical tuber crop around the world. Cassava bacterial blight, caused by Xanthomonas phaseoli pv. manihotis, is a key disease that influences cassava production worldwide. Between 2008 and 2020, 50 X. phaseoli pv. manihotis strains were isolated from diseased plant samples or acquired from China, Uganda, Cambodia, Colombia, Malaysia, and Micronesia. Using multilocus sequence analysis, the genetic diversity of X. phaseoli pv. manihotis strains was evaluated. A neighbor-joining phylogenetic dendrogram was constructed based on partial sequences of five housekeeping genes (atpD-dnaK-gyrB-efp-rpoD). The strains clustered into three groups whose clusters were consistent with atpD and RpoD gene sequences. Group I contained 46 strains from China, Uganda, Cambodia, and Micronesia, and the other two groups were comprised of strains from Colombia and Malaysia, respectively. The resistance of all these strains to copper ion (Cu2+) was determined, the minimal inhibitory concentration was between 1.3 and 1.7 mM, and there was no significant difference between strains from different geographic region. During genome annotation of the X. phaseoli pv. manihotis strain CHN01, homologous gene clusters of copLAB and xmeRSA were identified. The predicted amino acid sequences of two gene clusters were highly homologous with the copper-resistant protein from Xanthomonas strains. CopLAB and xmeRSA were amplified from all these strains, suggesting that the regulation of copper resistance is associated with two distinct metabolic pathways. CopLAB and xmeRSA were highly conserved among strains from different geographic regions, possibly associated with other conserved function.

Generative design of de novo proteins based on secondary-structure constraints using an attention-based diffusion model

We report two generative deep learning models that predict amino acid sequences and 3D protein structures based on secondary structure design objectives via either overall content or per-residue structure. Both models are robust regarding imperfect inputs and offer de novo design capacity as they can discover new protein sequences not yet discovered from natural mechanisms or systems. The residue-level secondary structure design model generally yields higher accuracy and more diverse sequences. These findings suggest unexplored opportunities for protein designs and functional outcomes within the vast amino acid sequences beyond known proteins. Our models, based on an attention-based diffusion model and trained on a dataset extracted from experimentally known 3D protein structures, offer numerous downstream applications in conditional generative design of various biological or engineering systems. Future work may include additional conditioning, and an exploration of other functional properties of the generated proteins for various properties beyond structural objectives.

Host defence peptide LEAP2 contributes to antimicrobial activity in a mustache toad (Leptobrachium liui)

BackgroundThe liver-expressed antimicrobial peptide 2 (LEAP2) is essential in host immunity against harmful pathogens and is only known to act as an extracellular modulator to regulate embryonic development in amphibians. However, there is a dearth of information on the antimicrobial function of amphibian LEAP2. Hence, a LEAP2 homologue from Leptobrachium liui was identified, characterized, and chemically synthesized, and its antibacterial activities and mechanisms were determined.ResultsIn this study, LEAP2 gene (Ll-LEAP2) cDNA was cloned and sequenced from the Chong’an Moustache Toad (Leptobrachium liui). The predicted amino acid sequence of Ll-LEAP2 comprises a signal peptide, a mature peptide, and a prodomain. From sequence analysis, it was revealed that Ll-LEAP2 belongs to the cluster of amphibian LEAP2 and displays high similarity to the Tropical Clawed Frog (Xenopus tropicalis)‘s LEAP2. Our study revealed that LEAP2 protein was found in different tissues, with the highest concentration in the kidney and liver of L. liui; and Ll-LEAP2 mRNA transcripts were expressed in various tissues with the kidney having the highest mRNA expression level. As a result of Aeromonas hydrophila infection, Ll-LEAP2 underwent a noticeable up-regulation in the skin while it was down-regulated in the intestines. The chemically synthesized Ll-LEAP2 mature peptide was selective in its antimicrobial activity against several in vitro bacteria including both gram-positive and negative bacteria. Additionally, Ll-LEAP2 can kill specific bacteria by disrupting bacterial membrane and hydrolyzing bacterial gDNA.ConclusionsThis study is the first report on the antibacterial activity and mechanism of amphibian LEAP2. With more to uncover, the immunomodulatory functions and wound-healing activities of Ll-LEAP2 holds great potential for future research.

Isolation and characterization of full-length coding sequences of gibberellic acid biosynthetic genes in coconut (Cocos nucifera L.) and their expression analysis

Gibberellic acid (GA) is intricately associated with plant growth and developmental pathways. To elucidate GA metabolism in coconut, we cloned full-length coding regions of the seven GA biosynthetic pathway genes (CPS, KS, KO, KAO, GA20ox, GA3ox and GA2ox) using a comparative genomics approach, using sequences from oil palm and date palm. The predicted amino acid sequences of the seven coconut GA biosynthetic genes shared high identities with oil palm and date palm GA biosynthetic genes. In silico structural and functional analyses were performed, and secondary and tertiary structures were predicted. Compared to the other two palms, the evolutionary analysis of the seven genes in coconut revealed that these genes may not be evolving rapidly enough to keep pace with occurring mutations. Additionally, we performed expression profiling of these seven genes during germination of mature zygotic embryos under in vitro conditions in two phenotypically contrasting coconut accessions (West Coast Tall and Chowghat Green Dwarf) and in response to exogenously supplemented GA3 and paclobutrazol (PBZ) in the embryo culture medium. While PBZ had an antagonistic effect on the GA biosynthesis pathway, GA3 enhanced the overall GA production. These results provide the first insights into GA biosynthesis in coconut, laying the foundation for future studies to decipher the genetics of dwarfism in coconut.

Characterization, expression analysis and RNAi-mediated knockdown of two aquaporin genes in the cotton leafworm, Spodoptera littoralis (Lepidoptera: Noctuidae)

Aquaporins (AQPs) are integral membrane proteins that can transport water and small molecules across cell membranes in organisms. In a previous report, we identified an AQP (SlAQP1) gene of the cotton leafworm, Spodoptera littoralis. In the current study, we identify and characterize two more SlAQP genes and their developmental and tissues expression. Predicted amino acid sequence and phylogenetic analysis revealed that SlAQP2 is a glycerol channel belonging to the insect specific Eglp subfamily, whereas SlAQP3 is a water specific channel belonging to the Prip subfamily. SlAQP2 expression was detected mainly in the first four larval instars and adult males, whereas SlAQP3 was detected in all developmental stages. SlAQP2 gene expression was mainly detected in larval midgut and Malpighian tubules and adult male testes, whereas SlAQP3 was detected in all the tissues tested. RNAi mediated knockdown of each gene separately resulted in deleterious effects including larval and pupal mortality, deformed pupae and adults and prolonged development. Results were confirmed using qRT-PCR, which revealed downregulation of both genes after injection of larvae with gene specific dsRNA. Our results confirm the presence of Prip and Eglp AQPs in S. littoralis and suggest that Eglp has a role in male reproductive ability.

Molecular Features and Antimicrobial Susceptibilities of Streptococcus equi ssp. equi Isolates from Strangles Cases in Indonesia

Strangles, caused by Streptococcus equi ssp. equi (S. equi equi), is a highly infectious and frequent disease of equines worldwide. No data are available regarding the molecular epidemiology of strangles in Indonesia. This study aimed to characterize S. equi equi isolates obtained from suspected strangles cases in Indonesia in 2018. Isolates originated from seven diseased horses on four different farms located in three provinces of Indonesia. Whole genome sequences of these isolates were determined and used for seM typing, multilocus sequence typing (MLST), and core genome MLS typing (cgMLST). Genomes were also screened for known antimicrobial resistance genes and genes encoding for the recombinant antigens used in the commercial Strangvac® subunit vaccine. All seven S. equi equi isolates from Indonesia belonged to ST179 and carried seM allele 166. Isolates differed from each other by only 2 to 14 cgSNPs and built an exclusive sub-cluster within the Bayesian Analysis of Population Structure (BAPS) cluster 2 (BAPS-2) of the S. equi equi cgMLST scheme. All isolates revealed predicted amino acid sequence identity to seven and high similarity to one of the eight antigen fragments contained in Strangvac®. Furthermore, all isolates were susceptible to beta-lactam antibiotics penicillin G, ampicillin, and ceftiofur. Our data suggest that the horses from this study were affected by strains of the same novel sublineage within globally distributed BAPS-2 of S. equi equi. Nevertheless, penicillin G can be used as a first-choice antibiotic against these strains and Strangvac® may also be protective against Indonesian strains.

On the lookout for influenza viruses in Italy during the 2021-2022 season: Along came A(H3N2) viruses with a new phylogenetic makeup of their hemagglutinin

AimsTo assess influenza viruses (IVs) circulation and to evaluate A(H3N2) molecular evolution during the 2021-2022 season in Italy. Materials and methods12,393 respiratory specimens (nasopharyngeal swabs or broncho-alveolar lavages) collected from in/outpatients with influenza illness in the period spanning from January 1, 2022 (week 2022-01) to May 31, 2022 (week 2022-22) were analysed to identify IV genome and were molecularly characterized by 12 laboratories throughout Italy. A(H3N2) evolution was studied by conducting an in-depth phylogenetic analysis of the hemagglutinin (HA) gene sequences. The predicted vaccine efficacy (pVE) of vaccine strain against circulating A(H3N2) viruses was estimated using the sequence-based Pepitope model. ResultsThe overall IV-positive rate was 7.2% (894/12,393), all were type A IVs. Almost all influenza A viruses (846/894; 94.6%) were H3N2 that circulated in Italy with a clear epidemic trend, with 10% positivity rate threshold crossed for six consecutive weeks from week 2022-11 to week 2022-16. According to the phylogenetic analysis of a subset of A(H3N2) strains (n=161), the study HA sequences were distributed into five different genetic clusters, all of them belonging to the clade 3C.2a, sub-clade 3C.2a1 and the genetic subgroup 3C.2a1b.2a.2. The selective pressure analysis of A(H3N2) sequences showed evidence of diversifying selection particularly in the amino acid position 156. The comparison between the predicted amino acid sequence of the 2021-2022 vaccine strain (A/Cambodia/e0826360/2020) and the study strains revealed 65 mutations in 59 HA amino acid positions, including the substitution H156S and Y159N in antigenic site B, within major antigenic sites adjacent to the receptor-binding site, suggesting the presence of drifted strains. According to the sequence-based Pepitope model, antigenic site B was the dominant antigenic site and the p(VE) against circulating A(H3N2) viruses was estimated to be -28.9%. Discussion and conclusionAfter a long period of very low IV activity since public health control measures have been introduced to face COVID-19 pandemic, along came A(H3N2) with a new phylogenetic makeup. Although the delayed 2021-2022 influenza season in Italy was characterized by a significant reduction of the width of the epidemic curve and in the intensity of the influenza activity compared to historical data, a marked genetic diversity of the HA of circulating A(H3N2) strains was observed. The identification of the H156S and Y159N substitutions within the main antigenic sites of most HA sequences also suggested the circulation of drifted variants with respect to the 2021-2022 vaccine strain. Molecular surveillance plays a critical role in the influenza surveillance architecture and it has to be strengthened also at local level to timely assess vaccine effectiveness and detect novel strains with potential impact on public health.

Meta-Analysis of the Public RNA-Seq Data of the Western Honeybee Apis mellifera to Construct Reference Transcriptome Data.

Simple SummaryThe honeybee is an economically and ecologically essential insect species and a critical social behavior model. It is vital to honey production and pollination. Therefore, it has been studied extensively in entomological research. The Western honeybee (Apis mellifera) is a representative species of honeybees. Reference transcriptome data are required to perform the meta-analyses using public RNA-Seq data and transcriptome analysis using newly sequenced RNA-Seq data. The reference transcriptome sequences were constructed, which consisted of 149,685 transcripts, and 194,174 predicted amino acid sequences. Half the predicted protein sequences were functionally annotated using protein sequence datasets for several model and insect species. We performed meta-analysis to search for novel immune-response-related transcripts. We identified 3–20 transcripts that were upregulated or downregulated in response to both viral and bacterial infections. However, the immune-related transcripts were not included among them. Autophagy-related protein 3 was downregulated by viral infections. The results suggested that autophagy was implicated in the viral immune responses, and autophagy was not regulated by the canonical immune pathways in A. mellifera. The results of the meta-analysis suggested that the reference transcriptome data could be used for transcriptome analysis in A. mellifera.The Western honeybee (Apis mellifera) is valuable in biological research and agriculture. Its genome sequence was published before those for other insect species. RNA-Seq data for A. mellifera have been applied in several recently published studies. Nevertheless, these data have not been prepared for use in subsequent meta-analyses. To promote A. mellifera transcriptome analysis, we constructed reference transcriptome data using the reference genome sequence and RNA-Seq data curated from about 1,000 runs of public databases. The new reference transcriptome data construct comprised 149,685 transcripts, and 194,174 protein sequences were predicted. Approximately 50–60% of the predicted protein sequences were functionally annotated using the protein sequence data for several model and insect species. Novel candidate immune-related transcripts were searched by meta-analysis using immune-response-related RNA-Seq and reference transcriptome data. Three to twenty candidate transcripts including autophagy-related protein 3 were upregulated or downregulated in response to both viral and bacterial infections. The constructed reference transcriptome data may facilitate future transcriptome analyses of A. mellifera.

Survey of porcine circovirus type 2 and parvovirus in swine breeding herds of Colombia.

One of the consequences of the presentation of reproductive failures in sows is the economic losses in production because it alters the estimated values of the volume of production, decreasing the productivity of the farm. Porcine circovirosis by porcine circovirus 2 (PCV2) has been associated with reproductive disorders, and porcine parvovirus (PVP) is one of the pathological agents most related to the presentation of reproductive failure in pigs. In Colombia, there are reports of the presence of PCV2 through molecular techniques, and PVP through serum tests; however, in the department of Tolima, the prevalence of these two viruses is unknown. In this study, the aim was to establish a report of the prevalence of viruses in five municipalities of the department of Tolima-Colombia. Blood samples from 150 breeding sows of five municipalities in Tolima, Colombia, were obtained. Quantitative polymerase chain reaction (qPCR) was used to detect the PCV2 and PVP virus in the blood samples followed by PCR and sequencing of 16 PCR products of the amplification of the cap gene of PCV2. A phylogenetic tree was constructed to identify the genotype of the PCV2 virus. The presence of PCV2d in sows was detected in 135 samples (90%), as well as the identification of PVP in 2.6% of the samples. In addition, the phylogenetic analysis showed that 16 isolates were the PCV2d2 genotype. PCV2d and PVP were found to coinfect the females, and the identification of variability in regions in the predicted amino acid sequence of the PCV2 capsid may be associated with virus pathogenicity.

The Gallus gallus RJF reference genome reveals an MHCY haplotype organized in gene blocks that contain 107 loci including 45 specialized, polymorphic MHC class I loci, 41 C-type lectin-like loci, and other loci amid hundreds of transposable elements.

MHCY is a second major histocompatibility complex-like gene region in chickens originally identified by the presence of major histocompatibility complex class I-like and class II-like gene sequences. Up to now, the MHCY gene region has been poorly represented in genomic sequence data. A high density of repetitive sequence and multiple members of several gene families prevented the accurate assembly of short-read sequence data for MHCY. Identified here by single-molecule real-time sequencing sequencing of BAC clones for the Gallus gallus Red Jungle Fowl reference genome are 107 MHCY region genes (45 major histocompatibility complex class I-like, 41 c-type-lectin-like, 8 major histocompatibility complex class IIβ, 8 LENG9-like, 4 zinc finger protein loci, and a single only zinc finger-like locus) located amid hundreds of retroelements within 4 contigs representing the region. Sequences obtained for nearby ribosomal RNA genes have allowed MHCY to be precisely mapped with respect to the nucleolar organizer region. Gene sequences provide insights into the unusual structure of the MHCY class I molecules. The MHCY class I loci are polymorphic and group into 22 types based on predicted amino acid sequences. Some MHCY class I loci are full-length major histocompatibility complex class I genes. Others with altered gene structure are considered gene candidates. The amino acid side chains at many of the polymorphic positions in MHCY class I are directed away rather than into the antigen-binding groove as is typical of peptide-binding major histocompatibility complex class I molecules. Identical and nearly identical blocks of genomic sequence contribute to the observed multiplicity of identical MHCY genes and the large size (>639 kb) of the Red Jungle Fowl MHCY haplotype. Multiple points of hybridization observed in fluorescence in situ hybridization suggest that the Red Jungle Fowl MHCY haplotype is made up of linked, but physically separated genomic segments. The unusual gene content, the evidence of highly similar duplicated segments, and additional evidence of variation in haplotype size distinguish polymorphic MHCY from classical polymorphic major histocompatibility complex regions.

Molecular characterization of a novel victorivirus isolated from Botryosphaeria dothidea, the causal agent of longan leaf spot disease.

Mycoviruses are widespread in all major taxonomic groups of filamentous fungi. Previous research has indicated that mycoviruses are associated with the phytopathogenic fungus Botryosphaeria dothidea. In this study, three distinct double-stranded RNA viruses were detected in B. dothidea strain YCLYY11 isolated from a leaf spot of longan (Dimocarpus longana). The results of BLAST analysis revealed that the predicted amino acid sequences of those viruses were similar to those of Botryosphaeria dothidea chrysovirus 1, Botryosphaeria dothidea partitivirus 1, and an apparent novel victorivirus. Sequencing and analysis of the complete genome of the novel victorivirus indicated it is 5218 bp in length and contains two open reading frames (ORFs) that overlap at the tetranucleotide AUGA. BLASTp analysis of the proteins encoded by ORF1 and ORF2 showed that they were most similar to the coat protein and RNA-dependent RNA polymerase of Sphaeropsis sapinea RNA virus 2 (81.37% and 74.09% identical, respectively). A phylogenetic tree showed that the novel virus clustered together with victoriviruses and was separate from members of the other four genera of the family Totiviridae. Based on its genome structure and the results of phylogenetic analysis, we propose that this novel victorivirus should be named "Botryosphaeria dothidea victorivirus 3". This is also the first report of these three mycoviruses coinfecting a strain of B. dothidea.

Brown planthopper E78 regulates moulting and ovarian development by interacting with E93

The brown planthopper (Nilaparvata lugens) is the main migratory pest in many rice growing areas in Asia. E78 is a member of the nuclear hormone receptor superfamily which plays an important role in egg development and maternal regulation of early embryogenesis. In this study, brown planthopper E78 (NlE78) was cloned, and the predicted amino acid sequence showed that it contains two conserved domains: NR-LBD and DBD. qRT-PCR showed that the expression of NlE78 is high in the fifth instar nymphs and the ovaries of females. After downregulation of NlE78, the rate of moulting failure (33.2%) increased significantly, and ovarian development was delayed. However, when NlE78 was downregulated together with NlE93, the emergence rate increased significantly (78.79%), and ovarian development was similar to that when NlE78 was downregulated but not delayed. A co-immunoprecipitation experiment showed that NlE78 interacts with NlE93, a crucial downstream transcription factor of the ecdysone signalling pathway. Cellular localization by immunofluorescence revealed that NlE78 and NlE93 are expressed in the nucleus. This study indicates that NlE78 regulates ovarian development and moulting, possibly through its interaction with NlE93. This study is of great significance for the development of new pesticides and control methods based on newly discovered targets.

Novel KEL allele associated with loss of Kpb identified in a white blood donor.

The importance of identifying variant alleles among blood donors is significant to the safety of transfusion for recipients. Molecular methods have become more prominent in the routine process of antigen typing donor units. Some variant antigens cannot be detected using only serologic methods. Molecular testing allows the determination of nucleotide sequences that are used to predict a phenotype. Antigens of the Kell blood group system are known for being highly immunogenic and causing adverse reactions upon antibody formation. A female white blood donor who typed Kp(b-) using serologic methods on multiple donations since 2005 was the subject of a typing discrepancy investigation. Routine genotyping using a commercial genotyping kit (HemoID DQS Panel; Agena Bioscience, San Diego, CA) predicted the donor to type Kp(a+b+). Investigation of the discrepancy between these two results identified a rare single nucleotide variant in the KEL gene at nucleotide position c.948G>T that alters amino acid residue 316 from tryptophan (Trp) to cysteine (Cys). After discovery of the novel allele, adsorption and elution studies were performed to see if there was weakened Kpb expression. The elution studies yielded negative results, which indicated that Kpb is not expressed. The KEL transcripts expressed by the donor were determined using cDNA analysis, and the predicted amino acid sequence of the novel allele was modeled to investigate the impact of the amino acid sequence on the structure of the KEL polypeptide. Both SWISS-MODEL and Robetta software were used to evaluate the impact of the p.Trp316Cys on the three-dimensional protein structure. There was no conformational change noted with SWISS-MODEL, whereas the Robetta software showed a significant conformational change compared with the normal Kp(b+) reference sequence. Because the donor is homozygous for variants associated with k and Jsb expression, it was not possible to determine whether the novel allele is associated with loss of Kpb only or loss of all Kell antigens.