Complete Gene Sequences Research Articles

Characterization of two full-length tubuliform silk gene sequences from Neoscona theisi reveals intragenic concerted evolution and multiple copies in genome

Orb-web weaving spiders use a variety of silk types for particular tasks, and each silk type is composed of at least two spider silk proteins (spidroins). In the early stage of divergence, however, the molecular evolutionary processes act on spidroin variants are still unclear because of a lack of knowledge for full-length paralogous and orthologous gene sequences among closely related species. Here, we present two complete gene sequences encoding the tubuliform spidroin TuSp1 variants (TuSp1-v2 and TuSp1-v3) from orb-weaving spider Neoscona theisi. Both N. theisi TuSp1-v2 and TuSp1-v3 genes contain a single enormous exon (14,139 bp for TuSp1-v2 and 13,152 bp for TuSp1-v3) and dozens of tandemly arrayed repeats (25 repeats for TuSp1-v2 and 23 repeats for TuSp1-v3) with extreme intragenic homogenization. The pattern of expression for these two spidroins revealed that the level of TuSp1-v3 mRNA is ~3-fold higher than that of TuSp1-v2 in tubuliform gland. Phylogenetic analyses of spidroins not only show the occurrence of a gene duplication event for TuSp1-v2 and TuSp1-v3 in the common ancestor of the Neoscona and Araneus lineage but reinforce the role of concerted evolution for the extreme homogenization of TuSp1 repeats.

Molecular characterization of NLRC3 and its interaction with other inflammasome components and regulation on the bacterial colonization in Qihe crucian carp Carassius auratus

The NLRC as a very unique subfamily of Nod like receptors (NLRs), is believed to play an important role in the bacterial recognition of animals. However, the molecular characterization and immunological role of NLRC3 in Carassius auratus is little known. In this study, we identified and achieved a complete cDNA sequence of NLRC3 gene in Qihe crucian carp Carassius auratus (named as CaNLRC3). The full-length cDNA sequence of CaNLRC3 was composed of 3823 bp, which contained a 5′-UTR of 251 bp, a 3′-UTR of 158 bp, and an open reading frame (ORF) of 3414 bp encoded 1137 amino acids with a predicted isoelectric point of 8.25 and a molecular mass of 124.1 kDa, characterized with a caspase recruitment domain (CARD) at N-terminus. The mRNA expression of CaNLRC3 was detected to be constitutive in all the examined tissues, with the high expression levels in spleen, skin and intestine. After challenges with bacteria or pathogenic analogue, expression levels of CaNLRC3 gene were strongly induced. Co-localization and co-immunoprecipitation results found that CaNLRC3 can assemble CaASC through CARD domain interaction, then CaASC associated with CaCaspase-1a, presumably to assemble the NLRC3 inflammasome complex. The overexpression of CaNLRC3 could significantly increase the mRNA expression of IL-1β, and promote the bacterial elimination and result in the decrease of bacterial loading in liver, spleen and kidney after bacterial infection. Vice versa, the knockdown of CaNLRC3 could obviously reduce IL-1β expression at mRNA level, and bacterial loading was significantly increased in tissue. Taken together, CaNLRC3 is proved to be a pivotal cytosolic innate immune receptor in this study, which is acted as the potential component of inflammasome to regulate inflammation reaction, and could modify bacterial loading in tissue and restrict bacterial infection in teleost.

Emergence of canine parvovirus type 2c (CPV-2c) of Asian origin in domestic dogs in Myanmar

Canine parvovirus type 2 (CPV-2) causes severe gastroenteric disease in domestic dogs and wild canids. This study aimed to conduct molecular detection and characterization of CPV-2 in domestic dogs in Myanmar from December 2017 to October 2019. Rectal swabs (n = 143) were collected from domestic dogs from shelters and small animal hospitals in Yangon, Myanmar. CPV-2 detection was performed by a PCR assay targeting the VP2 gene. Our result showed that 25.17% (36/143) of swab samples tested positive for CPV-2. CPV-2 strains (n = 15) were selected for complete VP2 gene sequencing. Phylogenetic analysis showed that CPV-2 strains from Myanmar clustered together with Asian CPV-2c from China, Indonesia, Taiwan and Thailand but in separate clusters from CPV-2c from Europe and North America. Characteristic amino acid at residues 267Y and 324I were observed in CPV-2c strains from Myanmar, suggesting the Asian origin. In conclusion, our findings expanded the evidence of the predominance of CPV-2c in Southeast Asia. Thus, the surveillance of CPV-2 in domestic dogs in the countries and regions should be routinely conducted to provide epidemiological information for supporting prevention and control practices.

BaZFP1, a C2H2 Subfamily Gene in Desiccation-Tolerant Moss Bryum argenteum, Positively Regulates Growth and Development in Arabidopsis and Mosses.

C2H2 zinc finger protein (C2H2-ZFP) plays an important role in regulating plant growth, development, and response to abiotic stress. To date, there have been no analyses of the C2H2-ZFP family in desiccation-tolerant moss. In this study, we identified 57 BaZFP transcripts across the Bryum argenteum (B. argenteum) transcriptome. The BaZFP proteins were phylogenetically divided into four groups (I-IV). Additionally, we studied the BaZFP1 gene, which is a nuclear C2H2-ZFP and acts as a positive regulator of growth and development in both moss and Arabidopsis thaliana. The complete coding sequence of the BaZFP1 gene was isolated from B. argenteum cDNA, which showed a high expression level in a dehydration-rehydration treatment process. The overexpression of the BaZFP1 gene in the Physcomitrium patens and B. argenteum promoted differentiation and growth of gametophytes. Heterologous expression in Arabidopsis regulated the whole growth and development cycle. In addition, we quantitatively analyzed the genes related to growth and development in transgenic moss and Arabidopsis, including HLS1, HY5, ANT, LFY, FT, EIN3, MUS, APB4, SEC6, and STM1, and found that their expression levels changed significantly. This study may pave the way for substantial insights into the role of C2H2-ZFPs in plants as well as suggest appropriate candidate genes for crop breeding.

Detection and Genome Sequence Analysis of Avian Metapneumovirus Subtype A Viruses Circulating in Commercial Chicken Flocks in Mexico.

Simple SummaryThe use of next-generation sequencing to identify pathogens in clinical samples is continually improving, providing genomic data that can be used to: (1) identify what viruses are present in particular samples, (2) determine the viral genotype, pathotype, or lineage, (3) predict which viruses are likely to cause severe disease, and (4) help guide what vaccines to use for control efforts. As part of a project to improve NGS for diagnostics, samples were collected from commercial chicken farms in Mexico and transported on FTA cards. One of the commonly identified viruses was avian metapneumovirus, an important upper respiratory avian pathogen consisting of subtypes A-D. Complete genomes of seven subtype-A isolates were identified, which are distinct from other previously reported subtype-A strains. Although subtype-A viruses were previously reported in Mexico, this is the first comprehensive complete genome sequence analysis of Mexican subtype-A viruses. These data provide a much clearer picture of viruses circulating in Mexico and demonstrate the value of next-generation sequencing to identify pathogens where surveillance is not routinely performed. Because subtype-A is not found in the U.S., a modified real-time reverse-transcriptase polymerase chain reaction test was evaluated that can quickly identify the virus in poultry samples.Avian metapneumoviruses (aMPV subtypes A-D) are respiratory and reproductive pathogens of poultry. Since aMPV-A was initially reported in Mexico in 2014, there have been no additional reports of its detection in the country. Using nontargeted next-generation sequencing (NGS) of FTA card-spotted respiratory samples from commercial chickens in Mexico, seven full genome sequences of aMPV-A (lengths of 13,288–13,381 nucleotides) were de novo assembled. Additionally, complete coding sequences of genes N (n = 2), P and M (n = 7 each), F and L (n = 1 each), M2 (n = 6), SH (n = 5) and G (n = 2) were reference-based assembled from another seven samples. The Mexican isolates phylogenetically group with, but in a distinct clade separate from, other aMPV-A strains. The genome and G-gene nt sequences of the Mexican aMPVs are closest to strain UK/8544/06 (97.22–97.47% and 95.07–95.83%, respectively). Various amino acid variations distinguish the Mexican isolates from each other, and other aMPV-A strains, most of which are in the G (n = 38), F (n = 12), and L (n = 19) proteins. Using our sequence data and publicly available aMPV-A data, we revised a previously published rRT-PCR test, which resulted in different cycling and amplification conditions for aMPV-A to make it more compatible with other commonly used rRT-PCR diagnostic cycling conditions. This is the first comprehensive sequence analysis of aMPVs in Mexico and demonstrates the value of nontargeted NGS to identify pathogens where targeted virus surveillance is likely not routinely performed.

Evolutionary conservation of motifs within vanA and vanB of vancomycin-resistant enterococci.

Global Health is threatened by the rapid emergence of multidrug-resistant bacteria. Antibiotic resistomes rapidly evolve, yet conserved motifs elucidated in our study have the potential for future drug targets for precision medicine. This study aimed to identify conserved genetic sequences and their evolutionary pathways among vancomycin-resistant Enterococcus species such as Enterococcus faecium and Enterococcus faecalis. We retrieved a total of 26 complete amino acid and nucleotide sequences of resistance determinant genes against vancomycin (vanA and vanB), streptomycin (aac-aah), and penicillin (pbp5) from the publicly available genetic sequence database, GenBank. The sequences were comprised of bacteria classified under the genera of Enterococcus, Staphylococcus, Amycolatopsis, Ruminococcus, and Clostridium. Sequences were aligned with Clustal Omega Multiple Sequence Alignment program and Percent Identity Matrices were derived. Phylogenetic analyses to elucidate evolutionary relationships between sequences were conducted with the neighbor-end joining method through the Molecular Evolutionary Genetics Analysis (MEGAX) software, developed by the Institute of Molecular Evolutionary Genetics at Pennsylvania State University. Subsequent network analyses of the resistance gene, vanB, within E. faecium were derived from ScanProsite and InterPro. We observed the highest nucleotide sequence similarity of vanA regions within strains of E. faecium (100%) and E. faecalis (100%). Between Enterococcus genera, we continued to observe high sequence conservation for vanA and vanB, up to 99.9% similarity. Phylogenetic tree analyses suggest rapid acquisition of these determinants between strains within vanA and vanB, particularly between strains of Enterococcus genera, which may be indicative of horizontal gene transfer. Within E. faecium, Adenosine 5'-Triphosphate (ATP)-Grasp and D-ala-D-ala ligase (Ddl) were found as conserved domains of vanA and vanB. We additionally found that there is notable sequence conservation, up to 66.67%, between resistomes against vancomycin and streptomycin among E. faecium. Resistance genes against vancomycin have highly conserved sequences between strains of Enterococcus bacteria. These conserved sequences within vanA and vanB encode for ATP-Grasp and Ddl motifs, which have functional properties for maintaining cell wall integrity. High sequence conservation is also observed among resistance genes against penicillin and streptomycin, which can inform future drug targets for broader spectrum therapies.

S5.1d Mechanisms of azole antifungal resistance in clinical isolates of Candida tropicalis

S5.1 Antifungal resistance, September 22, 2022, 3:00 PM - 4:30 PMObjectivesIn tropical countries, the azole resistance in Candida tropicalis is on the rise. There are limited studies available regarding the azole resistance mechanisms in C. tropicalis. This study was designed to understand the molecular mechanisms of azole resistance in C. tropicalis by using genetic and bioinformatics approaches.MethodsA total of 32 azole-resistant (R) and 10 azole-susceptible (S) clinical isolates of C. tropicalis were included in this study. All the isolates were subjected to complete gene sequencing of azole target genes including ERG11 to analyze the mutations which could lead the azole resistance. Four fragments were amplified, sequenced, and aligned to get full-length ERG11 gene. Inducible expression analysis of 17 other genes potentially associated with azole resistance was also evaluated. Homology modeling and molecular docking analysis were performed to study the effect of amino acid alterations in mediating azole resistance.ResultsOf the 32 resistant isolates, 12 (37.5%) showed A395T and C461T mutations in the ERG11 gene. The mean overexpression of CDR1, CDR3, TAC1, ERG1, ERG2, ERG3, ERG11, UPC2, and MKC1 in resistant isolates without mutation (R-WTM) was significantly higher (P <.05) than those with mutation (R-WM) and the sensitive isolates (3.2-11 vs. 0.2-2.5, and 0.3-2.2 folds, respectively). Although the R-WTM and R-WM had higher (P <.05) CDR2 and MRR1 expression compared to S isolates, noticeable variation was not seen among the other genes. Protein homology modeling and molecular docking revealed that the mutations in the ERG11 gene were responsible for structural alteration and low binding efficiency between ERG11p and ligands. Isolates with ERG11 mutations also presented A220C in ERG1 and together T503C, G751A mutations in UPC2.ConclusionsNonsynonymous mutations in the ERG11 gene and coordinated overexpression of various genes including different transporters, ergosterol biosynthesis pathway, transcription factors, and stress-responsive genes are associated with azole resistance in clinical isolates of C. tropicalis.

Contribution of the intron retained in the &lt;i&gt;Nxf1&lt;/i&gt; gene transcript to the phylogeny of the order &lt;i&gt;Chiroptera&lt;/i&gt;

This work is devoted to the possibilities of using a specific intron-containing block of the Nxf1 gene in phylogenetic studies.&#x0D; Our attention was drawn to the conservative intron of the Nxf1 (nuclear export factor) gene. This intron is a part of an evolutionarily conserved block formed with flanking 110 bp and 37 bp exons, similar in representatives of various taxonomic groups. This evolutionary conservative block in our previous works was designated cassette intron. The Nxf1 genes are found in all representatives of Opisthokonta, and may be a convenient object for phylogenetic studies.&#x0D; The Nxf1 gene sequences of seventeen representatives of the order Chiroptera obtained from publicly available databases (ensembl, ncbi). Alignment algorithm: MUSCLE. Programs: MEGA-X version 10.1.7, IQTree, Mesquite, MrBayes, and FigTree.v1.4.4. Estimation methods: Maximum Likelihood and Bayes Inference.&#x0D; The use of Nxf1 gene sequences that include only exons or only introns leads to unequal loss of accuracy in establishing evolutionary relationships in comparison with the model based on the complete gene sequence. Sequences involving all exons plus a cassette intron give the same result as the complete Nxf1 gene sequence.&#x0D; The obtained results indicate the importance of the cassette intron in the evolution of the Nxf1 gene of Chiroptera.

Genomic sequence of the non-pathogen Neisseria sp. strain MA1-1 with antibiotic resistance and virulence factors isolated from a head and neck cancer patient.

Recent research has claimed virulence factors or antimicrobial resistance in commensal or non-pathogenic Neisseria spp. This study aimed to isolate and analyze commensal microorganisms related to the genus Neisseria from the oral cavity of a patient with head and neck cancer. We successfully isolated strain MA1-1 and identified its functional gene contents. Although strain MA1-1 was related to Neisseria flava based on 16S rRNA gene sequence similarity, genomic relatedness analysis revealed that strain MA1-1 was closely related to Neisseria mucosa, reported as a commensal Neisseria species. The strain MA1-1 genome harbored genes for microaerobic respiration and the complete core metabolic pathway with few transporters for nutrients. A number of genes have been associated with virulence factors and resistance to various antibiotics. In addition, the comparative genomic analysis showed that most genes identified in the strain MA1-1 were shared with other Neisseria spp. including two well-known pathogens, Neisseria gonorrhoeae and Neisseria meningitidis. This indicates that the gene content of intra-members of the genus Neisseria has been evolutionarily conserved and is stable, with no gene recombination with other microbes in the host. Finally, this study provides more fundamental interpretations for the complete gene sequence of commensal Neisseria spp. and will contribute to advancing public health knowledge.

The first identified invertebrate LGP2-like homolog gene in the Pacific oyster Crassostrea gigas

The LGP2 (Laboratory of Genetics and Physiology 2) protein is a member of the retinoic acid-inducible gene I (RIG-I)-like receptor (RLRs) family, which is a class of antiviral pattern recognition receptors located in the cytoplasm. However, few studies have investigated the function of LGP2 in invertebrates. In this study, the complete coding sequence of the LGP2 gene of the Pacific oyster, Crassostrea gigas, was obtained and named CgLGP2-like. Sequence analysis revealed that CgLGP2-like encodes 803 amino acids, and the encoded protein contains a DEXDc, HELICc, and C-terminal regulatory domains. Multiple sequence alignment demonstrated that the sequences of these key protein functional domains were relatively conserved. Phylogenetic analysis revealed that CgLGP2-like was a new member of the animal LGP2 family. Quantitative real-time PCR results showed that CgLGP2-like mRNA was expressed in all tested oyster tissues, with the highest expression observed in the labial palpus and digestive glands. CgLGP2-like expression in gill tissues was significantly induced after the poly(I:C) challenge. Furthermore, multiple IRF and NF-κB binding sites were identified in the CgLGP2-like promoter region, which may be one of the reasons why CgLGP2-like responds to poly(I:C) stimulation. Finally, the results of dual-luciferase reporter gene assays revealed that overexpression of CgLGP2-like may have a regulatory effect on the human IFN, AP-1, and oyster CgIL-17 genes in HEK293T cells. Overall, our results preliminarily elucidate the immune functions of invertebrate LGP2 protein and provide valuable information for the development of comparative immunology.

Serological Investigation and Genetic Characteristics of Pseudorabies Virus between 2019 and 2021 in Henan Province of China

In late 2011, severe pseudorabies (PR) outbreaks occurred among swine herds vaccinated with the Bartha-K61 vaccine in many provinces of China, causing enormous economic losses for the pork industry. To understand the epidemic profile and genetic characteristics of the pseudorabies virus (PRV), a total of 35,796 serum samples were collected from 1090 pig farms of different breeding scales between 2019 and 2021 in the Henan province where swine had been immunized with the Bartha-K61 vaccine, and PRV glycoprotein E (gE)-specific antibodies were detected using an enzyme-linked immunosorbent assay (ELISA). The results reveal that the overall positive rate for PRV gE antibodies was 20.33% (7276/35,796), which decreased from 25.00% (2596/10,385) in 2019 to 16.69% (2222/13,315) in 2021, demonstrating that PR still existed widely in pig herds in the Henan province but displayed a decreasing trend. Further analysis suggested that the PRV-seropositive rate may be associated with farm size, farm category, quarter, region and the cross-regional transportation of livestock. Moreover, the gE gene complete sequences of 18 PRV isolates were obtained, and they shared a high identity (97.1–100.0%) with reference strains at the nucleotide level. Interestingly, the phylogenetic analysis based on the gE complete sequences found that there were both classical strains and variant strains in pig herds. The deduced amino acid sequence analysis of the gE gene showed that there were unique amino acids in the classical strains, the variant strains and genotype Ⅱ strains. This study provides epidemiological data that could be useful in the prevention of pseudorabies in Henan, China, and this finding contributed to our understanding of the epidemiology and evolution of PRV.

The Diversity and Spatiotemporally Evolutionary Dynamic of Atypical Porcine Pestivirus in China.

The presence of congenital tremor (CT) type A-II in newborn piglets, caused by atypical porcine pestivirus (APPV), has been a focus since 2016. However, the source, evolutionary history, and transmission pattern of APPV in China remain poorly understood. In this study, we undertook phylogenetic analyses based on available complete E2 gene sequences along with 98 newly sequenced E2 genes between 2016 and 2020 in China within the context of global genetic diversity. The phylogenies revealed four distinct lineages of APPV, and interestingly, all lineages could be detected in China with the greatest diversity. Bayesian phylogenetic analyses showed that the E2 gene evolves at a mean rate of 1.22 × 10−3 (8.54 × 10−4-1.60 × 10−3) substitutions/site/year. The most recent common ancestor for APPVs is dated to 1886 (1837–1924) CE, somewhat earlier than the documented emergence of CT (1922 CE). Our phylogeographic analyses suggested that the APPV population possibly originated in the Netherlands, a country with developed livestock husbandry, and was introduced into China during the period 1837–2010. Guangdong, as a primary seeding population together with Central and Southwest China as epidemic linkers, was responsible for the dispersal of APPVs in China. The transmission pattern of “China lineages” (lineage 3 and lineage 4) presented a “south to north” movement tendency, which was likely associated with the implementation of strict environmental policy in China since 2000. Reconstruction of demographic history showed that APPV population size experienced multiple changes, which correlated well with the dynamic of the number of pigs in the past decades in China. Besides, positively selected pressure and geography-driven adaptation were supposed to be key factors for the diversification of APPV lineages. Our findings provide comprehensive insights into the diversity and spatiotemporal dynamic of APPV in China.

Loss of anthocyanidin synthase gene is associated with white flowers of Salvia miltiorrhiza Bge. f. alba, a natural variant of S. miltiorrhiza.

SmANS deletion leads to white flower mutation in Salvia miltiorrhiza. SmANS deletion leads to white flower mutation in Salvia miltiorrhiza. Abstract Salvia miltiorrhiza is an essential traditional Chinese medicine (TCM) with purple flowers, and S. miltiorrhiza Bge. f. alba is a unique intraspecific variation with white flowers. The molecular mechanism of flower color formation in S. miltiorrhiza will provide vital information for the variation and evolution. Here, we performed HPLC, transcriptomic, and re-sequencing analyses of purple-flowered S. miltiorrhiza line 'Zihua105' (ZH105) and white-flowered S. miltiorrhiza Bge. f. alba line 'Baihua18' (BH18). Delphinidin was the most anthocyanidin in ZH105, which become the main different between ZH105 vs. BH18 flowers. Transcriptome analysis revealed 299 differentially expressed genes (DEGs). SmANS, the anthocyanidin synthase gene in the down-stream anthocyanin biosynthesis pathway, was significantly expressed in ZH105 corollas, suggesting it might play a key role in white petal formation. Whole-genome re-sequencing revealed that a 6.75kb segment located on chromosome 5, which contains the complete sequence of the SmANS genes, was lost in BH18 and another S. miltiorrhiza Bge. f. alba line. In contrast, the other five purple-flowered S. miltiorrhiza lines both possessed this segment. Further molecular marker identification also confirmed that wild S. miltiorrhiza Bge. f. alba lines lost regions that contained a complete or important part of SmANS sequences. Subsequently, the research showed that the deletion mutant of SmANS genes resulted in the natural white flower color variant of S. miltiorrhiza.

Comparing the structural characteristics and expression of GA2ox gene in dwarf banana and its wild type

It is necessary to understand the molecular mechanism of banana dwarfing mutation in order to improve the high stem character of banana varieties and breed the new excellent dwarf lines. To elucidate the molecular-level regulation of banana dwarf mutations and identify the relevant genes, the complete cDNA sequence of the gibberellin 2-oxidase gene (GA2ox) in the dwarf banana and its wild type was cloned by RT-PCR and the encoded amino acid sequences bioinformatically was analyzed. Meanwhile, the expression levels of banana GA2ox gene in different tissues of dwarfed banana and its wild type were analyzed by qRT-PCR. Between the GA2ox gene sequences of the dwarf banana and its wildtype, there were 5 base pairs of variation, leading to the predicted GA2ox proteins having different molecular weights, isoelectric points, fat indices, total numbers of positive and negative charges, and hydrophilicity. In addition, the expression of GA2ox in the leaves, stalks, and fruits of dwarf banana plants was higher than that in the wild type in the early and middle stages of banana development. The results showed that the GA2ox gene may have important regulatory effects on banana stem dwarfing, the knowledge of which can help our understanding and manipulation of this important plant.

Scallop RIG-I-like receptor 1 responses to polyinosinic:polycytidylic acid challenge and its interactions with the mitochondrial antiviral signaling protein

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are a class of pattern recognition receptors located in the cytoplasm that play a key role in antiviral innate immunity in animals. However, few studies have been conducted on the function of RLR proteins in invertebrates. In this study, the complete coding sequence of the RLR gene of the Zhikong scallop, Chlamys farreri, was obtained and named CfRLR1 with an aim to study the response of CfRLR1 to polyinosinic:polycytidylic acid [poly (I:C)] stimulation and the interaction between the CfRLR1 and C. farreri mitochondrial antiviral signaling (MAVS) protein. Sequence analysis revealed that CfRLR1 encodes 1161 amino acids, and the encoded protein covers two tandem caspase activation and recruitment domains (CARDs), a helicase domain, and a C-terminal regulatory domain. Phylogenetic analysis revealed that CfRLR1 belongs to the RLR family of mollusks. Quantitative real-time polymerase chain reaction showed that CfRLR1 mRNA was expressed in all tested tissues, with its highest expression observed in feet and gill tissues. Furthermore, CfRLR1 expression in the gill tissues was significantly induced after the poly (I:C) challenge. Finally, the results of co-immunoprecipitation and yeast two-hybrid assays revealed that CfRLR1 can bind to the CfMAVS protein via CARD–CARD interactions. Overall, our results elucidate the immune function of invertebrate RLR proteins and provide valuable information on viral disease control for scallop farming.

Bayesian Phylodynamic Analysis Reveals the Dispersal Patterns of African Swine Fever Virus.

The evolutionary and demographic history of African swine fever virus (ASFV) is potentially quite valuable for developing efficient and sustainable management strategies. In this study, we performed phylogenetic, phylodynamic, and phylogeographic analyses of worldwide ASFV based on complete ASFV genomes, B646L gene, and E183L gene sequences obtained from NCBI to understand the epidemiology of ASFV. Bayesian phylodynamic analysis and phylogenetic analysis showed highly similar results of group clustering between E183L and the complete genome. The evidence of migration and the demographic history of ASFV were also revealed by the Bayesian phylodynamic analysis. The evolutionary rate was estimated to be 1.14 × 10−5 substitution/site/year. The large out-migration from the viral population in South Africa played a crucial role in spreading the virus worldwide. Our study not only provides resources for the better utilization of genomic data but also reveals the comprehensive worldwide evolutionary history of ASFV with a broad sampling window across ~70 years. The characteristics of the virus spatiotemporal transmission are also elucidated, which could be of great importance for devising strategies to control the virus.

Molecular insights into the population structure and haplotype network of Theileria annulata based on the small-subunit ribosomal RNA (18S rRNA) gene.

The present study was conducted to elucidate the population genetic diversity and haplotype network of Theileria annulata based on all available nearly complete 18S rRNA gene sequences in the GenBank™. In total, 52 sequences of the nuclear 18S rRNA gene used to assess the relationship of T. annulata with their country of origin identified 34 haplotypes. Haplotype 4 was widespread, occurring in India, China, Turkey and Iran, while the remaining haplotypes were singleton and unique to one country. Haplotype 4 displayed numerous single haplotypes around it and the stellate shape of the network suggested a rapid population expansion. India exhibited the largest number of haplotypes (h=25) followed by Turkey (h=6), China (h=4), and Iran and Italy (h=1). No geographical clustering of haplotypes was recorded. Nucleotide diversity was the highest in the Turkish followed by the Indian and Chinese populations. Similarly, haplotype diversity was the highest in China followed by Turkey, and the lowest in India. The overall dataset exhibited a low nucleotide diversity (0.00253±0.00035), but high haplotype diversity (0.917±0.034). It suggested the presence of only minor differences (01-11 nucleotide) between haplotypes which was also evident from the haplotype network. A high level of genetic diversity was documented within the Indian, Chinese and Turkish populations of T. annulata, whereas little genetic differentiation was noticed among these populations with a very high level of gene flow. Analysis of molecular variance (AMOVA) of T. annulata sequences revealed higher genetic variation within countries (83.58%) as compared to the variation among countries (16.42%). Neutrality indices, viz., Tajima's D, Fu and Li's F, Fu's Fs, and R2, along with the unimodal mismatch distributions demonstrated a recent population expansion of T. annulata in India and the overall dataset. However, the non-significant values of Tajima's D, Fu and Li's F, and Fu's Fs for the Chinese population along with a bimodal mismatch distribution signified a constant population size. For the Turkish population, the neutrality and mismatch distribution tests either indicated a constant or a slight increase in population size. The present study provides novel insights into the population genetics and haplotype network of T. annulata based on the 18S rRNA gene for the first time.

Genetic Analysis of the Complete S1 Gene in Japanese Infectious Bronchitis Virus Strains.

The complete nucleotide sequence of the S1 glycoprotein gene of the Japanese infectious bronchitis virus (IBV) strains was determined and genetically analyzed. A total of 61 Japanese IBV strains were classified into seven genotypes, namely GI-1, 3, 7, 13, 18, 19, and GVI-1 using the classification scheme that was proposed by Valastro et al, with three exceptions. These genotypes practically corresponded to those defined in Japan, namely Mass, Gray, JP-II, 4/91, JP-I, JP-III, and JP-IV, which have been identified through their partial nucleotide sequences containing hypervariable regions 1 and 2. In addition, three exceptive strains were considered to be derived from recombination within the S1 gene of IBV strains G1-13 and GI-19. By analyzing the amino acid polymorphism of the S1 glycoprotein among Japanese genotypes, a diversity was observed based on the genotype-specific amino acid residue, the proteolytic cleavage motif at the S1/S2 cleavage site, and the position of the potential N-glycosylation sites.

Widely used, short 16S rRNA mitochondrial gene fragments yield poor and erratic results in phylogenetic estimation and species delimitation of amphibians

BackgroundThe 16S mitochondrial rRNA gene is the most widely sequenced molecular marker in amphibian systematic studies, making it comparable to the universal CO1 barcode that is more commonly used in other animal groups. However, studies employ different primer combinations that target different lengths/regions of the 16S gene ranging from complete gene sequences (~ 1500 bp) to short fragments (~ 500 bp), the latter of which is the most ubiquitously used. Sequences of different lengths are often concatenated, compared, and/or jointly analyzed to infer phylogenetic relationships, estimate genetic divergence (p-distances), and justify the recognition of new species (species delimitation), making the 16S gene region, by far, the most influential molecular marker in amphibian systematics. Despite their ubiquitous and multifarious use, no studies have ever been conducted to evaluate the congruence and performance among the different fragment lengths.ResultsUsing empirical data derived from both Sanger-based and genomic approaches, we show that full-length 16S sequences recover the most accurate phylogenetic relationships, highest branch support, lowest variation in genetic distances (pairwise p-distances), and best-scoring species delimitation partitions. In contrast, widely used short fragments produce inaccurate phylogenetic reconstructions, lower and more variable branch support, erratic genetic distances, and low-scoring species delimitation partitions, the numbers of which are vastly overestimated. The relatively poor performance of short 16S fragments is likely due to insufficient phylogenetic information content.ConclusionsTaken together, our results demonstrate that short 16S fragments are unable to match the efficacy achieved by full-length sequences in terms of topological accuracy, heuristic branch support, genetic divergences, and species delimitation partitions, and thus, phylogenetic and taxonomic inferences that are predicated on short 16S fragments should be interpreted with caution. However, short 16S fragments can still be useful for species identification, rapid assessments, or definitively coupling complex life stages in natural history studies and faunal inventories. While the full 16S sequence performs best, it requires the use of several primer pairs that increases cost, time, and effort. As a compromise, our results demonstrate that practitioners should utilize medium-length primers in favor of the short-fragment primers because they have the potential to markedly improve phylogenetic inference and species delimitation without additional cost.

Connecting materials, performance and evolution: a case study of the glue of moth-catching spiders (Cyrtarachninae).

Morphological structures and extended phenotypes are made possible by materials that are encoded by the genome. Nearly all biomaterials are viscoelastic, which means that to understand performance, one must understand the strain rate-dependent properties of these materials in relevant ecological interactions, as the behavior of a material can vary dramatically and rapidly. Spider silks are an example of materials whose properties vary substantially intra- and inter-specifically. Here, we focus on aggregate silk, which functions as a biological adhesive. As a case study to understand how a material manifests from genome through organism to ecology, we highlight moth-specialist spiders, the Cyrtarachninae, and their glues as an ideal experimental system to investigate the relationship between genomics and ecologically variable performance of a biological material. There is a clear eco-evolutionary innovation that Cyrtarachne akirai and related species have evolved, a unique trait not found in other spiders, a glue which overcomes the scales of moths. By examining traditional orb-weavers, C. akirai and other subfamily members using biomechanical testing and genomic analysis, we argue that we can track the evolution of this novel bioadhesive and comment on the selection pressures influencing prey specialization. The importance of the ecological context of materials testing is exemplified by the poor performance of C. akirai glue on glass and the exceptional spreading ability and adhesive strength on moths. The genetic basis for these performance properties is experimentally tractable because spider silk genes are minimally pleiotropic and advances in genomic technologies now make possible the discovery of complete silk gene sequences.