Positive Selection Sites Research Articles

Biological Significance of the Genomic Variation and Structural Dynamics of SARS-CoV-2 B.1.617.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants have been emerging and circulating globally since the start of the COVID-19 pandemic, of which B.1.617 lineage that was first reported in India at the end of 2020, soon became predominant. Tracing genomic variations and understanding their impact on the viral properties are the foundations for the vaccine and drug development and for the mitigation measures to be taken or lifted. In this study, 1,051 near-complete genomes and 1,559 spike (S) sequences belonging to the B.1.617 were analyzed. A genome-wide spread of single nucleotide polymorphisms (SNPs) was identified. Of the high frequency mutations identified, 61% (11/18) involved structural proteins, despite two third of the viral genome encoding nonstructural proteins. There were 22 positive selection sites, mostly distributed across the S protein, of which 16 were led by non-C to U transition and should be of a special attention. Haplotype network revealed that a large number of daughter haplotypes were continually derived throughout the pandemic, of which H177, H181 H219 and H286 from the ancestor haplotype H176 of B.1.617.2 were widely prevalent. Besides the well known substitutions of L452R, P681R and deletions of E156 and F157, as well as the potential biological significance, structural analysis in this study still indicated that new amino acid changes in B.1.617, such as E484Q and N501Y, had reshaped the viral bonding network, and increasingly sequenced N501Y mutant with a potential enhanced binding ability was detected in many other countries in the follow-up monitoring. Although we can’t conclude the properties of all the mutants including N501Y thoroughly, it merits focusing on their spread epidemically and biologically.

Genetic Characterizations and Molecular Evolution of the Measles Virus Genotype B3's Hemagglutinin (H) Gene in the Elimination Era.

Measles virus (MeV) genotype B3 is one globally significant circulating genotype. Here, we present a systematic description of long-term evolutionary characterizations of the MeV genotype B3’s hemagglutinin (H) gene in the elimination era. Our results show that the B3 H gene can be divided into two main sub-genotypes, and the highest intra-genotypic diversity was observed in 2004. MeV genotype B3’s H gene diverged in 1976; its overall nucleotide substitution rate is estimated to be 5.697 × 10−4 substitutions/site/year, and is slowing down. The amino acid substitution rate of genotype B3’s H gene is also decreasing, and the mean effective population size has been in a downward trend since 2000. Selection pressure analysis only recognized a few sites under positive selection, and the number of positive selection sites is getting smaller. All of these observations may reveal that genotype B3’s H gene is not under strong selection pressure, and is becoming increasingly conservative. MeV H-gene or whole-genome sequencing should be routine, so as to better elucidate the molecular epidemiology of MeV in the future.

Comparative Chloroplast Genomes of Zosteraceae Species Provide Adaptive Evolution Insights Into Seagrass.

Seagrasses are marine flowering plants found in tropical and sub-tropical areas that live in coastal regions between the sea and land. All seagrass species evolved from terrestrial monocotyledons, providing the opportunity to study plant adaptation to sea environments. Here, we sequenced the chloroplast genomes (cpGenomes) of three Zostera species, then analyzed and compared their cpGenome structures and sequence variations. We also performed a phylogenetic analysis using published seagrass chloroplasts and calculated the selection pressure of 17 species within seagrasses and nine terrestrial monocotyledons, as well as estimated the number of shared genes of eight seagrasses. The cpGenomes of Zosteraceae species ranged in size from 143,877 bp (Zostera marina) to 152,726 bp (Phyllospadix iwatensis), which were conserved and displayed similar structures and gene orders. Additionally, we found 17 variable hotspot regions as candidate DNA barcodes for Zosteraceae species, which will be helpful for studying the phylogenetic relationships and interspecies differences between seagrass species. Interestingly, nine genes had positive selection sites, including two ATP subunit genes (atpA and atpF), two ribosome subunit genes (rps4 and rpl20), two DNA-dependent RNA polymerase genes (rpoC1 and rpoC2), as well as accD, clpP, and ycf2. These gene regions may have played key roles in the seagrass adaptation to diverse environments. The Branch model analysis showed that seagrasses had a higher rate of evolution than terrestrial monocotyledons, suggesting that seagrasses experienced greater environmental pressure. Moreover, a branch-site model identified positively selected sites (PSSs) in ccsA, suggesting their involvement in the adaptation to sea environments. These findings are valuable for further investigations on Zosteraceae cpGenomes and will serve as an excellent resource for future studies on seagrass adaptation to sea environments.

Molecular evolutionary characteristics of SARS-CoV-2 emerging in the United States.

SARS‐CoV‐2 is a newly discovered beta coronavirus at the end of 2019, which is highly pathogenic and poses a serious threat to human health. In this paper, 1875 SARS‐CoV‐2 whole genome sequences and the sequence coding spike protein (S gene) sampled from the United States were used for bioinformatics analysis to study the molecular evolutionary characteristics of its genome and spike protein. The MCMC method was used to calculate the evolution rate of the whole genome sequence and the nucleotide mutation rate of the S gene. The results showed that the nucleotide mutation rate of the whole genome was 6.677 × 10−4 substitution per site per year, and the nucleotide mutation rate of the S gene was 8.066 × 10−4 substitution per site per year, which was at a medium level compared with other RNA viruses. Our findings confirmed the scientific hypothesis that the rate of evolution of the virus gradually decreases over time. We also found 13 statistically significant positive selection sites in the SARS‐CoV‐2 genome. In addition, the results showed that there were 101 nonsynonymous mutation sites in the amino acid sequence of S protein, including seven putative harmful mutation sites. This paper has preliminarily clarified the evolutionary characteristics of SARS‐CoV‐2 in the United States, providing a scientific basis for future surveillance and prevention of virus variants.

Insights Into the Molecular Evolution of AT-Hook Motif Nuclear Localization Genes in Brassica napus.

AT-hook motif nuclear localization (AHL) proteins belong to a family of transcription factors, and play important roles in plant growth and development and response to various stresses through protein-DNA and protein-protein interactions. To better understand the Brassica napus AHL gene family, AHL genes in B. napus and related species were analyzed. Using Arabidopsis as a reference, 122 AHL gene family members were first identified in B. napus. According to the phylogenetic tree and gene organization, the BnaAHLs were classified into two clades (Clade-A and Clade-B) and three types (Type-I, Type-II, and Type-III). Gene organization and motif distribution analysis suggested that the AHL gene family is relatively conserved during evolution. These BnaAHLs are unevenly distributed on 38 chromosomes and expanded by whole-genome duplication (WGD) or segmental duplication. And large-scale loss events have also occurred in evolution. All types of BnaAHLs are subject to purification or neutral selection, while some positive selection sites are also identified in Type-II and Type-III groups. At the same time, the purification effect of Type-I members are stronger than that of the others. In addition, RNA-seq data and cis-acting element analysis also suggested that the BnaAHLs play important roles in B. napus growth and development, as well as in response to some abiotic and biotic stresses. Protein-protein interaction analysis identified some important BnaAHL-binding proteins, which also play key roles in plant growth and development. This study is helpful to fully understand the origin and evolution of the AHL gene in B. napus, and lays the foundation for their functional studies.

Genome-wide identification and evolutionary analysis of MLO gene family in Rosaceae plants

Mutants lacking wild-type MLO (Mildew resistance Locus O) proteins show broad-spectrum resistance to the powdery mildew fungus, and dysregulated cell death control, with spontaneous cell death in response to developmental or abiotic stimuli. In order to understand the evolution and divergence patterns of the MLO gene family in Rosaceae plants, we analysed systematically genome-wide data from Fragaria vesca, Prunus persica, Prunus mume, Malus domestica, Pyrus bretschneideri and Rubus occidentalis based on bioinformatics methods. Using three phylogenetic methods (the neighbour-joining, maximum likelihood, and Bayesian methods), we identified 117 MLO genes from 6 Rosaceae species. The results of all three phylogenetic analysis methods supported that these genes were divided into six clades. Conserved motif analysis found that only motif 2 was present in all MLO proteins and had 3 nearly invariant amino acid residues. The findings indicated that motif 2 might be shared by the MLO gene family. The structural features of these genes showed large variations in sequence length among different species, although the lengths and the numbers of exons exhibited high degrees of similarity. Selective pressure analysis showed extremely significant differences in all 6 clades, with 2, 1, and 1 site(s) under significant positive selection detected in clades III, IV, and VI, respectively. These positive selection sites were important driving forces for the promotion of the functional differentiation of the MLO genes. Functional divergence analysis showed that the significantly divergent sites were located within the domains of the MLO genes. Functional distance analysis showed that the clade V had more conservative functions and might have retained more original functions during the evolutionary process. However, clade I may have undergone extensive altered functional constraints as a specialised functional role. Moreover, the most original function of the MLO genes in Rosaceae could be related to the evolution of their resistance to powdery mildew, which then gradually evolved into functions such as the regulation of flower development, the control of root morphology, and seed evolution due to the different evolutionary rates after gene duplication. These results provide a theoretical basis for further studies of the molecular evolutionary patterns of the plant MLO gene family.

Insight into the Phylogenetic Relationships among Three Subfamilies within Heptageniidae (Insecta: Ephemeroptera) along with Low-Temperature Selection Pressure Analyses Using Mitogenomes.

Simple SummaryThe Ephemeroptera is an ancient lineage of insects, among which the Heptageniidae is one of the most species-rich families, although its phylogenetic relationships have been controversial. The mitogenomes of Heptageniidae were found gene rearrangements of CR-I-M-Q-M-ND2 and a conserved intergenic gap between trnA and trnR. Thus, 15 complete and two nearly complete mitogenomes of Heptageniidae were used to explore mitogenome structures and clarify the disputes of phylogenetic relationships among Heptageniidae. Additionally, the Heptageniidae samples collected from habitats with significant temperature differences were applied to investigate the adaptive evolution of mitochondrial PCGs under low-temperature stress.We determined 15 complete and two nearly complete mitogenomes of Heptageniidae belonging to three subfamilies (Heptageniinae, Rhithrogeninae, and Ecdyonurinae) and six genera (Afronurus, Epeorus, Leucrocuta, Maccaffertium, Stenacron, and Stenonema). Species of Rhithrogeninae and Ecdyonurinae had the same gene rearrangement of CR-I-M-Q-M-ND2, whereas a novel gene rearrangement of CR-I-M-Q-NCR-ND2 was found in Heptageniinae. Non-coding regions (NCRs) of 25–47 bp located between trnA and trnR were observed in all mayflies of Heptageniidae, which may be a synapomorphy for Heptageniidae. Both the BI and ML phylogenetic analyses supported the monophyly of Heptageniidae and its subfamilies (Heptageniinae, Rhithrogeninae, and Ecdyonurinae). The phylogenetic results combined with gene rearrangements and NCR locations confirmed the relationship of the subfamilies as (Heptageniinae + (Rhithrogeninae + Ecdyonurinae)). To assess the effects of low-temperature stress on Heptageniidae species from Ottawa, Canada, we found 27 positive selection sites in eight protein-coding genes (PCGs) using the branch-site model. The selection pressure analyses suggested that mitochondrial PCGs underwent positive selection to meet the energy requirements under low-temperature stress.

Comparative Genomics Provide Insights Into Function and Evolution of Odorant Binding Proteins in Cydia pomonella.

Insect olfaction is vital for foraging, mating, host-seeking, and avoidance of predators/pathogens. In insects, odorant binding proteins (OBPs) are involved in transporting hydrophobic odor molecules from the external environment to receptor neurons. The codling moth, Cydia pomonella, one of the most destructive insect fruit pests, causes enormous economic losses. However, little is known about the number, variety, gains and losses, and evolution of OBP genes in C. pomonella. Here we report the identification of 40 OBPs in C. pomonella, most (75%) of which are classic OBPs, using genomic and transcriptomic analyses. Two OBP genes were lost in C. pomonella relative to possible distant ancestor in Lepidoptera lineage based on an analysis of gene gains and losses. The phylogenetic tree and chromosome location showed that the expansion of OBP genes mainly resulted from tandem duplications, as the CpomGOBP2 gene was duplicated twice along with loss of CpomPBPB. Two positive selection sites of the CpomGOBP1 gene were identified while other OBP genes evolved under purifying selection. Our results provide fundamental knowledge of OBP genes allowing further study of their function in C. pomonella.

Polymorphism and varying selection within the MHC class I of four Anas species.

Ducks (Anatidae) are often vectors for the spread of pathogens because of their long-distance migrations. These migrations also expose ducks to a wide variety of pathogens in their wintering and breeding grounds, and, as a consequence, we might expect strong selection on their immune genes. Here, we studied exons 2 and 3 of the MHC class I in four species of Anas ducks (A. platyrhynchos, A. poecilorhyncha, A. formosa, and A. querquedula) using Illumina-sequencing. Both exons 2 and 3 code for the peptide-binding region of class I molecules; however, most previous studies of birds have only focused on exon 3. Here, we found stronger positive selection on exon 2 than exon 3, as indicated by more species with dN/dS > 1 and higher Wu-Kabat values. There was little evidence that divergence time influenced polymorphism, the numbers of identical alleles (partial α1 or α2 regions) among four Anas, or selection, suggesting that these widespread species might share similar levels of selection from pathogens. The high similarity of allele numbers, positively selected sites (PSS), conserved motifs, and variable protein sites (VPS) supported the persistence of trans-species polymorphism in Anas for at least 10 million years. Our study revealed exon 2 as a relatively unexplored source of variation in avian MHC class I, which should be considered in future studies.

Evolution of the GII.3[P12] Norovirus from 2010 to 2019 in Jiangsu, China

ObjectivesNorovirus genotype GII.3[P12] strains have been an important pathogen for sporadic gastroenteritis infection. In previous studies of GII.3[P12], the number of specimens and time span are relatively small, which is difficult to truly reflect the infection and evolution of this type of norovirus. Here we report a molecular epidemiological study of the NoVs prevalent in Jiangsu between 2010 and 2019 to investigate the evolution of the GII.3[P12] strains in China.MethodsIn this study 60 GII.3[P12] norovirus strains were sequenced and analyzed for evolution, recombination, and selection pressure using bioanalysis software.ResultsThe GII.3[P12] strains were continuously detected during the study period, which showed a high constituent ratio in males, in winter and among children aged 0–11 months, respectively. A time-scaled evolutionary tree showed that both GII.P12 RdRp and GII.3 VP1 sequences were grouped into three major clusters (Cluster I–III). Most GII.3[P12] strains were mainly located in sub-cluster (SC) II of Cluster III. A SimPlot analysis identified GII.3[P12] strain to be as an ORF1-intragenic recombinant of GII.4[P12] and GII.3[P21]. The RdRp genes of the GII.3[P12] showed a higher mean substitution rate than those of all GII.P12, while the VP1 genes of the GII.3[P12] showed a lower mean substitution rate than those of all GII.3. Alignment of the GII.3 capsid sequences revealed that three HBGA binding sites of all known GII.3 strains remained conserved, while several amino acid mutations in the predicted antibody binding sites were detected. The mutation at 385 was within predicted antibody binding regions, close to host attachment factor binding sites. Positive and negative selection sites were estimated. Two common positively selected sites (sites 385 and 406) were located on the surface of the protruding domain. Moreover, an amino acid substitution (aa204) was estimated to be near the active site of the RdRp protein.ConclusionsWe conducted a comprehensive analysis on the epidemic and evolution of GII.3[P12] noroviruses and the results suggested that evolution was possibly driven by intergenic recombination and mutations in some key amino acid sites.

Codon usage patterns and evolution of HSP60 in birds

Heat shock protein 60 (HSP60) is highly conserved from prokaryotic to eukaryotic organisms, acting as molecular chaperone and other vital biological functions. In spite of increasing knowledge of HSP60, its evolutionary mechanism on functional adaption is still far from completely understood. Moreover, analysis of codon usage bias (CUB) is a powerful tool to understand evolutionary association studies. However, so far, as we know, no scientific work on CUB of HSP60 in birds has been reported. In this study, we provide a comprehensive analysis on the codon usage and molecular evolution of HSP60 across birds. The results indicated that HSP60 had a weak codon usage bias with high ENC values (range from 52.66 to 61), low RSCU, and A/T-ending codons were mostly preferred. Meanwhile, it was considered that mutation, natural selection, and genetic drift combined to shape codon usage patterns with different strength proportions among various birds for HSP60. Then, the LRT tests suggested that different lineages of birds might be under similar selective pressures. Besides, the two positive selection sites (151 and 131) were detected and might undergo radical changes. These findings would contribute to understand function diversity and molecular evolution of HSP60 in birds.

Genotyping and Molecular Characterization of Classical Swine Fever Virus Isolated in China during 2016–2018

Classical swine fever (CSF) is a highly contagious disease of swine caused by classical swine fever virus (CSFV). For decades the disease has been controlled in China by a modified live vaccine (C-strain) of genotype 1. The emergent genotype 2 strains have become predominant in China in the past years that are genetically distant from the vaccine strain. Here, we aimed to evaluate the current infectious status of CSF, and for this purpose 24 isolates of CSFV were identified from different areas of China during 2016–2018. Phylogenetic analysis of NS5B, E2 and full genome revealed that the new isolates were clustered into subgenotype 2.1d and 2.1b, while subgenotype 2.1d was predominant. Moreover, E2 and Erns displayed multiple variations in neutralizing epitope regions. Furthermore, the new isolates exhibited capacity to escape C-strain-derived antibody neutralization compared with the Shimen strain (genotype 1). Potential positive selection sites were identified in antigenic regions of E2 and Erns, which are related with antibody binding affinity. Recombination events were predicted in the new isolates with vaccine strains in the E2 gene region. In conclusion, the new isolates showed molecular variations and antigenic alterations, which provide evidence for the emergence of vaccine-escaping mutants and emphasize the need of updated strategies for CSF control.

Genetic variability of human papillomavirus type 39 based on E6, E7 and L1 genes in Southwest China

BackgroundHuman papillomavirus type 39 associated with genital intraepithelial neoplasia and invasive cancers, has a high prevalence in Southwest China. HPV E6, E7 are two main papillomavirus oncoproteins, closely relate to the function of HPV immortalization, cell transformation, and carcinogenesis. L1 is the major capsid protein, can reflect the replication status of the virus in cells and the progression of cervical lesions. The purpose of this study is to reveal the prevalence of HPV 39 and the genetic polymorphisms of HPV39 based on E6, E7 and L1 gene in southwest China.MethodsCell samples were collected by cervical scraped for HPV detecting and typing, and HPV39 positive samples were selected out. Important E6, E7 and L1 genes of HPV39 were sequenced and analyzed for the study of HPV39 genetic polymorphisms. Phylogenetic trees were constructed by Maximum-likelihood and Kimura 2-parameters methods in Molecular Evolutionary Genetics Analysis version 6.0. The selection pressures of E6, E7 and L1 genes were estimated by Datamonkey web server. The secondary and three-dimensional structure of HPV39 E6, E7 proteins were created by sopma server and SWISS-MODEL software.Results344 HPV39 positive samples were selected from 5718 HPV positive cell samples. Among HPV39 E6-E7 sequences, 20 single nucleotide mutations were detected, including 10 non-synonymous and 10 synonymous mutations; 26 single nucleotide mutations were detected in HPV39 L1 sequences, including 7 non-synonymous and 19 synonymous mutations respectively. 11 novel variants of HPV39 E6-E7 (5 in E6 and 6 in E7) and 14 novel variants of HPV39 L1 were identified in this study. A-branch was the most frequent HPV39 lineage in southwest China during our investigation. Selective pressure analysis showed that codon sites 26, 87, 151 in E6 and 75, 180, 222, 272, 284, 346, 356 in L1 were positively selected sites, as well as codon sites 45, 138, 309, 381 were negative selection sites in L1 gene, E7 has neither positive selection sites nor negative selection sites. A certain degree of secondary and three-dimensional structure dislocation was existed due to the non-synonymous mutations.ConclusionsAmino acid substitution affected the secondary and three-dimensional structure of HPV39, and resulting in the differences of carcinogenic potential and biological functions as well as the immune response due to the antigen epitopes difference, the antigen epitopes with stronger adaptability in Southwest will be screened out based on the above research results for the later vaccine development. And gene polymorphism of HPV39 in Southwest China may improve the effectiveness of clinical test and vaccine design, specifically for women in Southwest China.

The highly conserved rps12 gene in ferns provides strong evidence for decreased substitution rates in the inverted repeat region

The typical chloroplast genome consists of two inverted repeat (IR) and two single-copy (SC) regions. Each region has its own pattern of molecular evolution. Many studies have shown that the IR region has a low substitution rate. The plastid rps12 gene encodes ribosomal protein small subunit 12, which is composed of 5′-rps12 (exon 1) and 3′-rps12 (exon 2–3); 3′-rps12 is near the IR region and enters or leaves the IR region as it expands and contracts, enabling study of the variation in its substitution rate. We analyzed the evolutionary rate and adaptive evolution of the rps12 genes of 118 ferns and 2 lycophytes in a phylogenetic context using the maximum likelihood method. We found that the substitution rate of exon 2–3 was significantly lower in the IR region than in the LSC (1–11 times higher in the LSC), and also lower than that of exon 1 in the LSC region (1.9–6.7 times higher for exon 1). In addition, there was more GC content at the third codon position, and the rps12 gene encoded 123 amino acids, with 107 negative selection sites and 4 positive selection sites. These results prove that the substitution rate decreases after the sequence fragments enter the IR region and demonstrate the highly conserved nature of the rps12 gene.

The char of migration inhibitory factor and Cathepsin B from Asian swamp eel (Monopterus albus) and their response to challenge with Aeromonas hydrophila

AbstractAeromonas hydrophila causes sepsis and is responsible for significant economic losses in Asian finless eel (Monopterus albus) aquaculture. Previous studies have found that macrophage migration inhibitory factor (MIF) and Cathepsin B (CTSB) may be expressed in many vertebrate tissues and organs. However, there have been no reports discussing the importance of MIF and CTSB in Asian swamp eel. The complete ORF sequences of MAMIF and MACTSB were found to be 348 and 993 bp, respectively. The adaptive analysis showed that the sequences of MAMIF and MACTSB were highly conserved, and only two positive selection sites were identified in MAMIF. Quantitative real time polymerase chain reaction (qRT‐PCR) demonstrated that MAMIF and MACTSB genes were expressed in all tissues evaluated with the highest expression occurring in the head kidney, spleen and liver. Challenge with Aeromonas hydrophila resulted in a significant increase in the expression of MAMIF and MACTSB in skin, liver, head kidney and spleen that peaked at 12‐h post‐infection. The results show that MAMIF and MACTSB play important roles in the immune function of Monopterus albus.

The Bayesian reconstruction and the evolutionary history of Salivirus type 1 and type 2: the worldwide spreading.

Salivirus (SalV) represents an emerging problem in public health especially during the recent years. In this study, the Bayesian evolutionary history and the spread of the virus through the different countries have been reported. a database of 81 sequences of SalV structural VP1 fragment were downloaded from GenBank, aligned and manually edited by Bioedit Software. ModelTest v. 3.7 software was used to estimate the simplest evolutionary model fitting the sequence dataset. A Maximum-Likelihood tree has been generated using MEGA-X to test the "clockliness" signal using TempEst 1.5.1. The Bayesian phylogenetic tree was built by BEAST. Homology modelling was performed by SWISS-Model and protein variability evaluated by ConSurf server. the phylogenetic tree showed a clade of SalV A2 and three main clades of SalV A1, revealing several infections in humans in South Korea, India, Tunisia, China, Nigeria, Ethiopia and USA. The Bayesian maximum clade credibility tree and the time of the most common recent ancestor dated back the root of the tree to the year 1788 with the probable origin in USA. Selective pressure analysis revealed two positive selection sites, His at 100th and Leu at 116th positions that at the homology modelling resulted important to guarantee protein stability and variability. This could contribute to the development of new mutations modifying the clinical features of this evolving virus. Bayesian phylogenetic and phylodynamic represented a useful tool to follow the transmission dynamic of SalV and to prevent new epidemics worldwide.

Complete genome characterization of the 2018 dengue outbreak in Hunan, an inland province in central South China

In 2018, a small-scale dengue epidemic broke out in Hunan Province, an inland province in central South China, with 172 people infected. To verify the causative agent, complete genome information was obtained by PCR and sequencing based on the viral RNAs extracted from patient serum samples. Mutation and evolutionary analysis were performed by MEGA7.0 software. The online softwares "Predict protein" and "Mfold" were used to predict the secondary structure of proteins and untranslated regions, respectively. Phylogenetic analysis showed that all five isolates in this study were DENV type 2, which is most closely related to the Zhejiang strain (2017-MH110588). Compared with the DENV-2 standard strain, 773 nucleotide mutations occurred in the isolated strain, of which 666 were nonsense mutations. Of the 80 mutated amino acids, 22 occurred in the structural protein region (2 in C region, 8 in PrM/M region, 12 in E region), and 58 in the non-structural (NS) protein region (9 in NS1 region, 10 in NS2 region, 12 in NS3 region, 7 in NS4 region, 20 in NS5 region). The prM/M region had the highest AA mutation rate, while NS4B was conservative. Three amino acid mutations (E: N390thS, and NS5: S676thN, K800thT) may important for the replication and virulence of the DENV. Secondary structure prediction observed 28 changes in polynucleotide binding regions and 110 changes in protein binding sites of coding sequence. 2 and 4 base substitutions resulted in 2 and 6 significant changes in the RNA secondary structure of 5′ UTR and 3′ UTR, respectively. Two significant positive selection sites were observed in NS5. To our knowledge, this research is the first complete genome analysis of the epidemic strain of the 2018 dengue outbreak in Hunan and will benefit further research for virus traceability and vaccine development.

Complete genome analysis identifies recombinant events and positive selection sites of hepatitis C virus from mainland China during 2010–2019

Identification of new recombinant HCV strains and positive selection sites are crucially important for the formulation of virus intervention measures. However, little is known about the recombinant variant information and positive selection sites of circulating HCV strains in mainland China. In this study, we systematically identified recombinant variants and positive selection sites of HCV in mainland China during the 2010–2019. Phylogenetic analysis results indicated that HCV-6 was one of the dominant genotypes in mainland China during 2010–2019, whereas genotypes 7 and 8 were not detected. Recombinant analysis based on 102 full-length genome sequences of Chinese epidemic strains of HCV identified four intra-genotypic recombinants (strains WYHCV286, GB28, GZ2983, and HCV156) and one inter-genotypic recombinant (strain HH075). Specifically, two breakpoints in the 5′ UTR of two recombinants, the strains HH075 and WYHCV286, are rather unusual and has not been described before. Further, selection pressure analyses revealed five positive selective sites, which were located in the core, E2, and NS5B protein. Notably, positive selective sites in NS5B and core protein may be partially responsible for the drug resistance and immune evasion. To the best of our knowledge, this study firstly reported five specific intertypic and intratypic recombinants of Chinese epidemic strains of HCV, which highlight their significance for anti−HCV treatment and vaccine development.

Transcriptome-wide identification and characterization of toll-like receptors response to Vibrio anguillarum infection in Manila clam (Ruditapes philippinarum)

The Manila clam (Ruditapes philippinarum), one of the major marine aquaculture species in China, is susceptible to infection with the pathogen Vibrio, which results in massive mortality and economic losses. Toll-like receptors (TLRs) are significant pattern recognition receptors (PRRs) of innate immunity that are involved in immune regulation against pathogenic invasion. Molecular characterization of Manila clam TLRs and investigations of their immune functions are essential to prevent and control Vibrio infection. In the present research, eight cDNA sequences of R. philippinarum TLRs (RpTLRs) were identified from previous transcriptome libraries and then classified into four groups, namely, P-TLR (one sequence), V-TLR (one sequence), Ls-TLR (two sequences) and sP-TLR (four sequences), based on the corresponding LRR domain arrangement of their protein structures within the typical TLR motifs. A selective pressure test firstly suggested that the molluscan P-TLR, V-TLR, Ls-TLR and sP-TLR families underwent positive selection, and different numbers of positive selection sites (PSSs) were identified in different domains of the four types of RpTLRs, as determined by PAML and analysis of website data. These findings indicated that the evolution of RpTLRs may be associated with their immune recognition and function. Furthermore, tissue-specific expression analysis showed that all RpTLRs were ubiquitously expressed in all test tissues and were dominant in hemocytes. Quantitative real-time PCR revealed that the cDNA expression of all eight RpTLRs was upregulated after injection with Vibrio anguillarum (P < 0.01) in R. philippinarum hemocytes, revealing that these RpTLRs play important roles in responding to pathogenic stimulation. In summary, these findings provide a foundation for future investigations of the molecular classification and evolutionary patterns of Toll-like receptors in invertebrates, and the innate immune responses of TLR signaling pathways in Mollusca.

Analysis of Adaptive Evolution and Coevolution of rbcL Gene in the Genus Galdieria (Rhodophyta).

To research the adaptive evolution and coevolution of the rbcL gene in the genus Galdieria, 36 sequences were selected. The bioinformatics of proteins encoded by rbcL genes of Galdieria were analyzed, and phylogenetic trees were constructed by the maximum-likelihood method. Then, adaptive evolution and coevolution were analyzed. The phylogenetic tree showed that the inner groups were clustered into four branches, in which the sequences of Galdieria maxima were divided into two small branches, and the posterior probability of each branch is above 94.9%. Eleven reliable positive selection sites were detected in the branched-site model, indicating that the rbcL protein-coding gene of Galdieria underwent adaptive evolution to adapt to extreme environments. Site 269 F is located in the loop 6 domain, while sites 272 D and 273 W are located in the 6-helix structure. Many coevolution pairs were detected, which were closely related to the hydrophobic and molecular weight correlation values of amino acids. The results are helpful to research the evolution process of freshwater red algae, to explore the changes of its essential genes and protein functions to adapt to different environmental pressures, and to understand the close relationship between amino acids in proteins and the molecular mechanism of evolution.