Complete assembly, annotation of virulence genes and CRISPR editing of the genome of Leishmania amazonensis PH8 strain.

We report the sequencing and assembly of the PH8 strain of Leishmania amazonensis one of the etiological agents of leishmaniasis. After combining data from long Pacbio reads, short Illumina reads and synteny with the Leishmania mexicana genome, the sequence of 34 chromosomes with 8317 annotated genes was generated. Multigene families encoding three virulence factors, A2, amastins and the GP63 metalloproteases, were identified and compared to their annotation in other Leishmania species. As they have been recently recognized as virulence factors essential for disease establishment and progression of the infection, we also identified 14 genes encoding proteins involved in parasite iron and heme metabolism and compared to genes from other Trypanosomatids. To follow these studies with a genetic approach to address the role of virulence factors, we tested two CRISPR-Cas9 protocols to generate L. amazonensis knockout cell lines, using the Miltefosine transporter gene as a proof of concept.

Functional Diversification, Redundancy, and Epistasis among Paralogs of the Drosophila melanogaster Obp50a-d Gene Cluster.

Large multigene families, such as the insect odorant-binding proteins (OBPs), are thought to arise through functional diversification after repeated gene duplications. Whereas many OBPs function in chemoreception, members of this family are also expressed in tissues outside chemosensory organs. Paralogs of the Obp50 gene cluster are expressed in metabolic and male reproductive tissues, but their functions and interrelationships remain unknown. Here, we report the genetic dissection of four members of the Obp50 cluster, which are in close physical proximity without intervening genes. We used CRISPR technology to excise the entire cluster while introducing a PhiC31 reintegration site to reinsert constructs in which different combinations of the constituent Obp genes were either intact or rendered inactive. We performed whole transcriptome sequencing and assessed sexually dimorphic changes in transcript abundances (transcriptional niches) associated with each gene-edited genotype. Using this approach, we were able to estimate redundancy, additivity, diversification, and epistasis among Obp50 paralogs. We analyzed the effects of gene editing of this cluster on organismal phenotypes and found a significant skewing of sex ratios attributable to Obp50a, and sex-specific effects on starvation stress resistance attributable to Obp50d. Thus, there is functional diversification within the Obp50 cluster with Obp50a contributing to development and Obp50d to stress resistance. The deletion-reinsertion approach we applied to the Obp50 cluster provides a general paradigm for the genetic dissection of paralogs of multigene families.

Plasmodium vinckei genomes provide insights into the pan-genome and evolution of rodent malaria parasites.

BACKGROUND: Rodent malaria parasites (RMPs) serve as tractable tools to study malaria parasite biology and host-parasite-vector interactions. Among the four RMPs originally collected from wild thicket rats in sub-Saharan Central Africa and adapted to laboratory mice, Plasmodium vinckei is the most geographically widespread with isolates collected from five separate locations. However, there is a lack of extensive phenotype and genotype data associated with this species, thus hindering its use in experimental studies. RESULTS: We have generated a comprehensive genetic resource for P. vinckei comprising of five reference-quality genomes, one for each of its subspecies, blood-stage RNA sequencing data for five P. vinckei isolates, and genotypes and growth phenotypes for ten isolates. Additionally, we sequenced seven isolates of the RMP species Plasmodium chabaudi and Plasmodium yoelii, thus extending genotypic information for four additional subspecies enabling a re-evaluation of the genotypic diversity and evolutionary history of RMPs. The five subspecies of P. vinckei have diverged widely from their common ancestor and have undergone large-scale genome rearrangements. Comparing P. vinckei genotypes reveals region-specific selection pressures particularly on genes involved in mosquito transmission. Using phylogenetic analyses, we show that RMP multigene families have evolved differently across the vinckei and berghei groups of RMPs and that family-specific expansions in P. chabaudi and P. vinckei occurred in the common vinckei group ancestor prior to speciation. The erythrocyte membrane antigen 1 and fam-c families in particular show considerable expansions among the lowland forest-dwelling P. vinckei parasites. The subspecies from the highland forests of Katanga, P. v. vinckei, has a uniquely smaller genome, a reduced multigene family repertoire and is also amenable to transfection making it an ideal parasite for reverse genetics. We also show that P. vinckei parasites are amenable to genetic crosses. CONCLUSIONS: Plasmodium vinckei isolates display a large degree of phenotypic and genotypic diversity and could serve as a resource to study parasite virulence and immunogenicity. Inclusion of P. vinckei genomes provide new insights into the evolution of RMPs and their multigene families. Amenability to genetic crossing and transfection make them also suitable for classical and functional genetics to study Plasmodium biology.

Identification of Candidate Chemosensory Gene Families by Head Transcriptomes Analysis in the Mexican Fruit Fly, Anastrepha ludens Loew (Diptera: Tephritidae).

Insect chemosensory systems, such as smell and taste, are mediated by chemosensory receptor and non-receptor protein families. In the last decade, many studies have focused on discovering these families in Tephritidae species of agricultural importance. However, to date, there is no information on the Mexican fruit fly Anastrepha ludens Loew, a priority pest of quarantine importance in Mexico and other countries. This work represents the first effort to identify, classify and characterize the six chemosensory gene families by analyzing two head transcriptomes of sexually immature and mature adults of A. ludens from laboratory-reared and wild populations, respectively. We identified 120 chemosensory genes encoding 31 Odorant-Binding Proteins (OBPs), 5 Chemosensory Proteins (CSPs), 2 Sensory Neuron Membrane Proteins (SNMPs), 42 Odorant Receptors (ORs), 17 Ionotropic Receptors (IRs), and 23 Gustatory Receptors (GRs). The 120 described chemosensory proteins of the Mexican fruit fly significantly contribute to the genetic databases of insects, particularly dipterans. Except for some OBPs, this work reports for the first time the repertoire of olfactory proteins for one species of the genus Anastrepha, which provides a further basis for studying the olfactory system in the family Tephritidae, one of the most important for its economic and social impact worldwide.

Understanding the Early Evolutionary Stages of a Tandem Drosophilamelanogaster-Specific Gene Family: A Structural and Functional Population Study.

Gene families underlie genetic innovation and phenotypic diversification. However, our understanding of the early genomic and functional evolution of tandemly arranged gene families remains incomplete as paralog sequence similarity hinders their accurate characterization. The Drosophila melanogaster-specific gene family Sdic is tandemly repeated and impacts sperm competition. We scrutinized Sdic in 20 geographically diverse populations using reference-quality genome assemblies, read-depth methodologies, and qPCR, finding that ∼90% of the individuals harbor 3-7 copies as well as evidence of population differentiation. In strains with reliable gene annotations, copy number variation (CNV) and differential transposable element insertions distinguish one structurally distinct version of the Sdic region per strain. All 31 annotated copies featured protein-coding potential and, based on the protein variant encoded, were categorized into 13 paratypes differing in their 3' ends, with 3-5 paratypes coexisting in any strain examined. Despite widespread gene conversion, the only copy present in all strains has functionally diverged at both coding and regulatory levels under positive selection. Contrary to artificial tandem duplications of the Sdic region that resulted in increased male expression, CNV in cosmopolitan strains did not correlate with expression levels, likely as a result of differential genome modifier composition. Duplicating the region did not enhance sperm competitiveness, suggesting a fitness cost at high expression levels or a plateau effect. Beyond facilitating a minimally optimal expression level, Sdic CNV acts as a catalyst of protein and regulatory diversity, showcasing a possible evolutionary path recently formed tandem multigene families can follow toward long-term consolidation in eukaryotic genomes.

Holosteans contextualize the role of the teleost genome duplication in promoting the rise of evolutionary novelties in the ray-finned fish innate immune system.

Over 99% of ray-finned fishes (Actinopterygii) are teleosts, a clade that comprises half of all living vertebrate species that have diversified across virtually all fresh and saltwater ecosystems. This ecological breadth raises the question of how the immunogenetic diversity required to persist under heterogeneous pathogen pressures evolved. The teleost genome duplication (TGD) has been hypothesized as the evolutionary event that provided the substrate for rapid genomic evolution and innovation. However, studies of putative teleost-specific innate immune receptors have been largely limited to comparisons either among teleosts or between teleosts and distantly related vertebrate clades such as tetrapods. Here we describe and characterize the receptor diversity of two clustered innate immune gene families in the teleost sister lineage: Holostei (bowfin and gars). Using genomic and transcriptomic data, we provide a detailed investigation of the phylogenetic history and conserved synteny of gene clusters encoding diverse immunoglobulin domain-containing proteins (DICPs) and novel immune-type receptors (NITRs). These data demonstrate an ancient linkage of DICPs to the major histocompatibility complex (MHC) and reveal an evolutionary origin of NITR variable-joining (VJ) exons that predate the TGD by at least 50 million years. Further characterizing the receptor diversity of Holostean DICPs and NITRs illuminates a sequence diversity that rivals the diversity of these innate immune receptor families in many teleosts. Taken together, our findings provide important historical context for the evolution of these gene families that challenge prevailing expectations concerning the consequences of the TGD during actinopterygiian evolution.

Evolutionary history of the human multigene families reveals widespread gene duplications throughout the history of animals.

BACKGROUND: The hypothesis that vertebrates have experienced two ancient, whole genome duplications (WGDs) is of central interest to evolutionary biology and has been implicated in evolution of developmental complexity. Three-way and Four-way paralogy regions in human and other vertebrate genomes are considered as vital evidence to support this hypothesis. Alternatively, it has been proposed that such paralogy regions are created by small-scale duplications that occurred at different intervals over the evolution of life. RESULTS: To address this debate, the present study investigates the evolutionary history of multigene families with at least three-fold representation on human chromosomes 1, 2, 8 and 20. Phylogenetic analysis and the tree topology comparisons classified the members of 36 multigene families into four distinct co-duplicated groups. Gene families falling within the same co-duplicated group might have duplicated together, whereas genes belong to different co-duplicated groups might have distinct evolutionary origins. CONCLUSION: Taken together with previous investigations, the current study yielded no proof in favor of WGDs hypothesis. Rather, it appears that the vertebrate genome evolved as a result of small-scale duplication events, that cover the entire span of the animals' history.

Chromosome Mapping of H1 and H4 Histones in Parodontidae (Actinopterygii: Characiformes): Dispersed and/or Co-Opted Transposable Elements?

The karyotypes of the family Parodontidae consist of 2n = 54 chromosomes. The main chromosomal evolutionary changes of its species are attributed to chromosome rearrangements in repetitive DNA regions in their genomes. Physical mapping of the H1 and H4 histones was performed in 7 Parodontidae species to analyze the chromosome rearrangements involved in karyotype diversification in the group. In parallel, the observation of a partial sequence of an endogenous retrovirus (ERV) retrotransposon in the H1 histone sequence was evaluated to verify molecular co-option of the transposable elements (TEs) and to assess paralogous sequence dispersion in the karyotypes. Six of the studied species had an interstitial histone gene cluster in the short arm of the autosomal pair 13. Besides this interstitial cluster, in Apareiodon davisi, a probable further site was detected in the terminal region of the long arm in the same chromosome pair. The H1/H4 clusters in Parodon cf. pongoensis were located in the smallest chromosomes (pair 20). In addition, scattered H1 signals were observed on the chromosomes in all species. The H1 sequence showed an ERV in the open reading frame (ORF), and the scattered H1 signals on the chromosomes were attributed to the ERV's location. The H4 sequence had no similarity to the TEs and displayed no dispersed signals. Furthermore, the degeneration of the inner ERV in the H1 sequence (which overlapped a stretch of the H1 ORF) was discussed regarding the likelihood of molecular co-option of this retroelement in histone gene function in Parodontidae.

Multigene Family of Pore-Forming Toxins from Sea Anemone Heteractis crispa.

Sea anemones produce pore-forming toxins, actinoporins, which are interesting as tools for cytoplasmic membranes study, as well as being potential therapeutic agents for cancer therapy. This investigation is devoted to structural and functional study of the Heteractis crispa actinoporins diversity. Here, we described a multigene family consisting of 47 representatives expressed in the sea anemone tentacles as prepropeptide-coding transcripts. The phylogenetic analysis revealed that actinoporin clustering is consistent with the division of sea anemones into superfamilies and families. The transcriptomes of both H. crispa and Heteractis magnifica appear to contain a large repertoire of similar genes representing a rapid expansion of the actinoporin family due to gene duplication and sequence divergence. The presence of the most abundant specific group of actinoporins in H. crispa is the major difference between these species. The functional analysis of six recombinant actinoporins revealed that H. crispa actinoporin grouping was consistent with the different hemolytic activity of their representatives. According to molecular modeling data, we assume that the direction of the N-terminal dipole moment tightly reflects the actinoporins' ability to possess hemolytic activity.

Metallothionein Gene Family in the Sea Urchin Paracentrotus lividus: Gene Structure, Differential Expression and Phylogenetic Analysis.

Metallothioneins (MT) are small and cysteine-rich proteins that bind metal ions such as zinc, copper, cadmium, and nickel. In order to shed some light on MT gene structure and evolution, we cloned seven Paracentrotus lividus MT genes, comparing them to Echinodermata and Chordata genes. Moreover, we performed a phylogenetic analysis of 32 MTs from different classes of echinoderms and 13 MTs from the most ancient chordates, highlighting the relationships between them. Since MTs have multiple roles in the cells, we performed RT-qPCR and in situ hybridization experiments to understand better MT functions in sea urchin embryos. Results showed that the expression of MTs is regulated throughout development in a cell type-specific manner and in response to various metals. The MT7 transcript is expressed in all tissues, especially in the stomach and in the intestine of the larva, but it is less metal-responsive. In contrast, MT8 is ectodermic and rises only at relatively high metal doses. MT5 and MT6 expression is highly stimulated by metals in the mesenchyme cells. Our results suggest that the P. lividus MT family originated after the speciation events by gene duplications, evolving developmental and environmental sub-functionalization.

Retracing the path of planar cell polarity.

BACKGROUND: The Planar Cell Polarity pathway (PCP) has been described as the main feature involved in patterning cell orientation in bilaterian tissues. Recently, a similar phenomenon was revealed in cnidarians, in which the inhibition of this pathway results in the absence of cilia orientation in larvae, consequently proving the functional conservation of PCP signaling between Cnidaria and Bilateria. Nevertheless, despite the growing accumulation of databases concerning basal lineages of metazoans, very few information concerning the existence of PCP components have been gathered outside of Bilateria and Cnidaria. Thus, the origin of this module or its prevalence in early emerging metazoans has yet to be elucidated. RESULTS: The present study addresses this question by investigating the genomes and transcriptomes from all poriferan lineages in addition to Trichoplax (Placozoa) and Mnemiopsis (Ctenophora) genomes for the presence of the core components of this pathway. Our results confirm that several PCP components are metazoan innovations. In addition, we show that all members of the PCP pathway, including a bona fide Strabismus ortholog (Van gogh), are retrieved only in one sponge lineage (Homoscleromorpha) out of four. This highly suggests that the full PCP pathway dates back at least to the emergence of homoscleromorph sponges. Consequently, several secondary gene losses would have occurred in the three other poriferan lineages including Amphimedon queenslandica (Demospongiae). Several proteins were not retrieved either in placozoans or ctenophores leading us to discuss the difficulties to predict orthologous proteins in basally branching animals. Finally, we reveal how the study of multigene families may be helpful to unravel the relationships at the base of the metazoan tree. CONCLUSION: The PCP pathway antedates the radiation of Porifera and may have arisen in the last common ancestor of animals. Oscarella species now appear as key organisms to understand the ancestral function of PCP signaling and its potential links with Wnt pathways.

Targeted modification of storage protein content resulting in improved amino acid composition of barley grain.

C-hordein in barley and ω-gliadins in wheat are members of the prolamins protein families. Prolamins are the major component of cereal storage proteins and composed of non-essential amino acids (AA) such as proline and glutamine therefore have low nutritional value. Using double stranded RNAi silencing technology directed towards C-hordein we obtained transgenic barley lines with up to 94.7% reduction in the levels of C-hordein protein relative to the parental line. The composition of the prolamin fraction of the barley parental line cv. Golden Promise was resolved using SDS-PAGE electrophoresis, the protein band were excised and the proteins identified by quadrupole-time-of-flight mass spectrometry. Subsequent SDS-PAGE separation and analysis of the prolamin fraction of the transgenic lines revealed a reduction in the amounts of C-hordeins and increases in the content of other hordein family members. Analysis of the AA composition of the transgenic lines showed that the level of essential amino acids increased with a concomitant reduction in proline and glutamine. Both the barley C-hordein and wheat ω-gliadin genes proved successful for RNAi-gene mediated suppression of barley C-hordein level. All transgenic lines that exhibited a reduction for C-hordein showed off-target effects: the lines exhibited increased level of B/γ-hordein while D-hordein level was reduced. Furthermore, the multicopy insertions correlated negatively with silencing.

High similarity of U2 snDNA sequence between A and B chromosomes in the grasshopper Abracris flavolineata.

B chromosomes are frequently enriched for a wide variety of repetitive DNAs. Among grasshoppers in the species Abracris flavolineata (Ommatolampidinae) the B chromosomes are submetacentric, C-negative and harbor repetitive DNAs such as, U2 snDNA, C 0 t-1 DNA, two Mariner-like elements and some microsatellites. Here, we provide evidence showing the intragenome similarity between the B chromosome and the A complement in A. flavolineata, combining analysis of microdissection and chromosome painting and B chromosome-specific amplification through polymerase chain reaction (PCR) of U2 snDNA. Chromosome painting revealed signals spread through the C-negative regions, including the A and B chromosomes. Moreover, significant clustered signals forming bands were observed in some A chromosomes, and for the B chromosome, significant signals were located on both arms, which could be caused by accumulation of repetitive DNA sequences. The C-positive regions did not reveal any signals. Sequence comparison of U2 snDNA between that obtained from a genome without the B chromosome and that from µB-DNA revealed high similarity with the occurrence of four shared haplotypes, one of them (i.e., Hap1) being highly prevalent and putatively ancestral. The highest divergence from Hap1 was observed for Hap3, which was caused by only six mutational steps. These data support an intraspecific origin of the B chromosome in A. flavolineata that is highly similar with the A complement, and the low U2 snDNA sequence diversity observed in the B chromosome could be related to its recent origin, besides intrachromosomal concerted evolution for U2 snDNA repeats in the B chromosome.

Partial sequence homogenization in the 5S multigene families may generate sequence chimeras and spurious results in phylogenetic reconstructions.

Multigene families have provided opportunities for evolutionary biologists to assess molecular evolution processes and phylogenetic reconstructions at deep and shallow systematic levels. However, the use of these markers is not free of technical and analytical challenges. Many evolutionary studies that used the nuclear 5S rDNA gene family rarely used contiguous 5S coding sequences due to the routine use of head-to-tail polymerase chain reaction primers that are anchored to the coding region. Moreover, the 5S coding sequences have been concatenated with independent, adjacent gene units in many studies, creating simulated chimeric genes as the raw data for evolutionary analysis. This practice is based on the tacitly assumed, but rarely tested, hypothesis that strict intra-locus concerted evolution processes are operating in 5S rDNA genes, without any empirical evidence as to whether it holds for the recovered data. The potential pitfalls of analysing the patterns of molecular evolution and reconstructing phylogenies based on these chimeric genes have not been assessed to date. Here, we compared the sequence integrity and phylogenetic behavior of entire versus concatenated 5S coding regions from a real data set obtained from closely related plant species (Medicago, Fabaceae). Our results suggest that within arrays sequence homogenization is partially operating in the 5S coding region, which is traditionally assumed to be highly conserved. Consequently, concatenating 5S genes increases haplotype diversity, generating novel chimeric genotypes that most likely do not exist within the genome. In addition, the patterns of gene evolution are distorted, leading to incorrect haplotype relationships in some evolutionary reconstructions.

Template-specific optimization of NGS genotyping pipelines reveals allele-specific variation in MHC gene expression.

Using high-throughput sequencing for precise genotyping of multi-locus gene families, such as the major histocompatibility complex (MHC), remains challenging, due to the complexity of the data and difficulties in distinguishing genuine from erroneous variants. Several dedicated genotyping pipelines for data from high-throughput sequencing, such as next-generation sequencing (NGS), have been developed to tackle the ensuing risk of artificially inflated diversity. Here, we thoroughly assess three such multi-locus genotyping pipelines for NGS data, the DOC method, AmpliSAS and ACACIA, using MHC class IIß data sets of three-spined stickleback gDNA, cDNA and "artificial" plasmid samples with known allelic diversity. We show that genotyping of gDNA and plasmid samples at optimal pipeline parameters was highly accurate and reproducible across methods. However, for cDNA data, the gDNA-optimal parameter configuration yielded decreased overall genotyping precision and consistency between pipelines. Further adjustments of key clustering parameters were required tο account for higher error rates and larger variation in sequencing depth per allele, highlighting the importance of template-specific pipeline optimization for reliable genotyping of multi-locus gene families. Through accurate paired gDNA-cDNA typing and MHC-II haplotype inference, we show that MHC-II allele-specific expression levels correlate negatively with allele number across haplotypes. Lastly, sibship-assisted cDNA-typing of MHC-I revealed novel variants linked in haplotype blocks, and a higher-than-previously-reported individual MHC-I allelic diversity. In conclusion, we provide novel genotyping protocols for the three-spined stickleback MHC-I and -II genes, and evaluate the performance of popular NGS-genotyping pipelines. We also show that fine-tuned genotyping of paired gDNA-cDNA samples facilitates amplification bias-corrected MHC allele expression analysis.

Whole genome sequence and diversity in multigene families of Babesia ovis.

Ovine babesiosis, caused by Babesia ovis, is an acute, lethal, and endemic disease worldwide and causes a huge economic loss to animal industry. Pathogen genome sequences can be utilized for selecting diagnostic markers, drug targets, and antigens for vaccine development; however, those for B. ovis have not been available so far. In this study, we obtained a draft genome sequence for B. ovis isolated from an infected sheep in Turkey. The genome size was 7.81 Mbp with 3,419 protein-coding genes. It consisted of 41 contigs, and the N50 was 526 Kbp. There were 259 orthologs identified among eight Babesia spp., Plasmodium falciparum, and Toxoplasma gondii. A phylogeny was estimated on the basis of the orthologs, which showed B. ovis to be closest to B. bovis. There were 43 ves genes identified using hmm model as well. They formed a discriminating cluster to other ves multigene family of Babesia spp. but showed certain similarities to those of B. bovis, B. caballi, and Babesia sp. Xinjiang, which is consistent with the phylogeny. Comparative genomics among B. ovis and B. bovis elucidated uniquely evolved genes in these species, which may account for the adaptation.

Identification of African swine fever virus MGF505-2R as a potent inhibitor of innate immunity in vitro.

African swine fever (ASF) is etiologically an acute, highly contagious and hemorrhagic disease caused by African swine fever virus (ASFV). Due to its genetic variation and phenotypic diversity, until now, no efficient commercial vaccines or therapeutic options are available. The ASFV genome contains a conserved middle region and two flexible ends that code for five multigene families (MGFs), while the biological functions of the MGFs are not fully characterized. Here, ASFV MGF505-2R-deficient mutant ASFV-Δ2R was constructed based on a highly virulent genotype II field isolate ASFV CN/GS/2018 currently circulating in China. Transcriptomic profiling demonstrated that ASFV-Δ2R was capable of inducing a larger number of differentially expressed genes (DEGs) compared with ASFV CN/GS/2018. Hierarchical clustering of up-regulated DEGs revealed that ASFV-Δ2R induced the most dramatic expression of interferon-related genes and inflammatory and innate immune genes, as further validated by RT-qPCR. The GO and KEGG pathway analysis identified significantly enriched pathways involved in pathogen recognition and innate antiviral immunity. Conversely, pharmacological activation of those antiviral immune responses by exogenous cytokines, including type I/II IFNs, TNF-α and IL-1ß, exerted combinatory effects and synergized in antiviral capacity against ASFV replication. Collectively, MGF505-2R is a newly identified inhibitor of innate immunity potentially implicated in immune evasion.

Comparative genomics reveals unique features of two Babesia motasi subspecies: Babesia motasi lintanensis and Babesia motasi hebeiensis.

Parasites of the Babesia genus are prevalent worldwide and infect a wide diversity of domestic animals and humans. Herein, using Oxford Nanopore Technology and Illumina sequencing technologies, we sequenced two Babesia subspecies, Babesia motasi lintanensis and Babesia motasi hebeiensis. We identified 3,815 one-to-one ortholog genes that are specific to ovine Babesia spp. Phylogenetic analysis reveals that the two B. motasi subspecies form a distinct clade from other piroplasmas. Consistent with their phylogenetic position, comparative genomic analysis reveals that these two ovine Babesia spp. share higher colinearity with Babesia bovis than with Babesia microti. Concerning the speciation date, B. m. lintanensis split from B. m. hebeiensis approximately 17 million years ago. Genes correlated to transcription, translation, protein modification and degradation, as well as differential/specialized gene family expansions in these two subspecies may favor adaptation to vertebrate and tick hosts. The close relationship between B. m. lintanensis and B. m. hebeiensis is underlined by a high degree of genomic synteny. Compositions of most invasion, virulence, development, and gene transcript regulation-related multigene families, including spherical body protein, variant erythrocyte surface antigen, glycosylphosphatidylinositol anchored proteins, and transcription factor Apetala 2 genes, is largely conserved, but in contrast to this conserved situation, we observe major differences in species-specific genes that may be involved in multiple functions in parasite biology. For the first time in Babesia spp., we find abundant fragments of long terminal repeat-retrotransposons in these two species. We provide fundamental information to characterize the genomes of B. m. lintanensis and B. m. hebeiensis, providing insights into the evolution of B. motasi group parasites.

Differential Trafficking and Expression of PIR Proteins in Acute and Chronic Plasmodium Infections.

Plasmodium multigene families are thought to play important roles in the pathogenesis of malaria. Plasmodium interspersed repeat (pir) genes comprise the largest multigene family in many Plasmodium species. However, their expression pattern and localisation remain to be elucidated. Understanding protein subcellular localisation is fundamental to reveal the functional importance and cell-cell interactions of the PIR proteins. Here, we use the rodent malaria parasite, Plasmodium chabaudi chabaudi, as a model to investigate the localisation pattern of this gene family. We found that most PIR proteins are co-expressed in clusters during acute and chronic infection; members of the S7 clade are predominantly expressed during the acute-phase, whereas members of the L1 clade dominate the chronic-phase of infection. Using peptide antisera specific for S7 or L1 PIRS, we show that these PIRs have different localisations within the infected red blood cells. S7 PIRs are exported into the infected red blood cell cytoplasm where they are co-localised with parasite-induced host cell modifications termed Maurer's clefts, whereas L1 PIRs are localised on or close to the parasitophorous vacuolar membrane. This localisation pattern changes following mosquito transmission and during progression from acute- to chronic-phase of infection. The presence of PIRs in Maurer's clefts, as seen for Plasmodium falciparum RIFIN and STEVOR proteins, might suggest trafficking of the PIRs on the surface of the infected erythrocytes. However, neither S7 nor L1 PIR proteins detected by the peptide antisera are localised on the surface of infected red blood cells, suggesting that they are unlikely to be targets of surface variant-specific antibodies or to be directly involved in adhesion of infected red blood cells to host cells, as described for Plasmodium falciparum VAR proteins. The differences in subcellular localisation of the two major clades of Plasmodium chabaudi PIRs across the blood cycle, and the apparent lack of expression on the red cell surface strongly suggest that the function(s) of this gene family may differ from those of other multigene families of Plasmodium, such as the var genes of Plasmodium falciparum.

Complete genome analysis of the African swine fever virus isolated from a wild boar responsible for the first viral outbreak in Korea, 2019.

African swine fever (ASF), a highly contagious and severe hemorrhagic viral disease in swine, is emerging as a major threat not only in Korea but also worldwide. The first confirmed case of ASF in Korea was reported in 2019. Despite the occurrence of ASF in Korea, only a few studies have genetically characterized the causative ASF virus (ASFV). In this study, we aimed to genetically characterize the ASFV responsible for the 2019 outbreak in Korea. The genome of the ASFV isolated during the first outbreak in Korea was analyzed. The Korea/YC1/2019 strain has 188,950 base pairs, with a GC content of 38.4%. The complete genome sequence was compared with other ASFV genomes annotated in the NCBI database. The Korea/YC1/2019 strain shared the highest similarity with Georgia 2007, Belgium 2018/1, and ASFV-wbBS01 strains. This study expands our knowledge of the genetic diversity of ASFV, providing valuable information for epidemiology, diagnostics, therapies, and vaccine development.