Maximum likelihood estimation and natural pairwise estimating equations are identical for three sequences and a symmetric 2-state substitution model.

Consider the problem of estimating the branch lengths in a symmetric 2-state substitution model with a known topology and a general, clock-like or star-shaped tree with three leaves. We show that the maximum likelihood estimates are analytically tractable and can be obtained from pairwise sequence comparisons. Furthermore, we demonstrate that this property does not generalize to larger state spaces, more complex models or larger trees. Our arguments are based on an enumeration of the free parameters of the model and the dimension of the minimal sufficient data vector. Our interest in this problem arose from discussions with our former colleague Freddy Bugge Christiansen.

Robustness of Felsenstein's Versus Transfer Bootstrap Supports With Respect to Taxon Sampling.

The bootstrap method is based on resampling sequence alignments and re-estimating trees. Felsenstein's bootstrap proportions (FBP) are the most common approach to assess the reliability and robustness of sequence-based phylogenies. However, when increasing taxon sampling (i.e., the number of sequences) to hundreds or thousands of taxa, FBP tend to return low support for deep branches. The transfer bootstrap expectation (TBE) has been recently suggested as an alternative to FBP. TBE is measured using a continuous transfer index in [0,1] for each bootstrap tree, instead of the binary {0,1} index used in FBP to measure the presence/absence of the branch of interest. TBE has been shown to yield higher and more informative supports while inducing a very low number of falsely supported branches. Nonetheless, it has been argued that TBE must be used with care due to sampling issues, especially in datasets with a high number of closely related taxa. In this study, we conduct multiple experiments by varying taxon sampling and comparing FBP and TBE support values on different phylogenetic depths, using empirical datasets. Our results show that the main critique of TBE stands in extreme cases with shallow branches and highly unbalanced sampling among clades, but that TBE is still robust in most cases, while FBP is inescapably negatively impacted by high taxon sampling. We suggest guidelines and good practices in TBE (and FBP) computing and interpretation.

Maximum Likelihood Estimation for Unrooted 3-Leaf Trees: An Analytic Solution for the CFN Model.

Maximum likelihood estimation is among the most widely-used methods for inferring phylogenetic trees from sequence data. This paper solves the problem of computing solutions to the maximum likelihood problem for 3-leaf trees under the 2-state symmetric mutation model (CFN model). Our main result is a closed-form solution to the maximum likelihood problem for unrooted 3-leaf trees, given generic data; this result characterizes all of the ways that a maximum likelihood estimate can fail to exist for generic data and provides theoretical validation for predictions made in Parks and Goldman (Syst Biol 63(5):798-811, 2014). Our proof makes use of both classical tools for studying group-based phylogenetic models such as Hadamard conjugation and reparameterization in terms of Fourier coordinates, as well as more recent results concerning the semi-algebraic constraints of the CFN model. To be able to put these into practice, we also give a complete characterization to test genericity.

NewickTreeModifier: A simple web tool to prune and modify Newick trees.

Large-scale selection analyses of protein-coding sequences and phylogenetic tree reconstructions require suitable trees in Newick format. We developed the NewickTreeModifier (NTM), a simple web-based tool to trim and modify Newick trees for such analyses. The users can choose provided master trees or upload a tree to prune it to selected species available in FASTA, NEXUS, or PHYLIP sequence format with an internal converter, a simple species list, or directly determined from a checklist interface of the master trees. Plant, insect, and vertebrate master trees comprise the maximum number of species in an up-to-date phylogenetic order directly transferable to the pruned Newick outfile. NTM is available at https://retrogenomics.uni-muenster.de/tools/ntm.

Genomic and phylogenomic analysis of Fusobacteriaceae family and proposal to reclassify Fusobacterium naviforme Jungano 1909 into a novel genus as Zandiella naviformis gen. nov., comb. nov. and reclassification of Fusobacterium necrophorum subsp. funduliforme as later heterotypic synonym of Fusobacterium necrophorum subsp. necrophorum and Fusobacterium equinum as later heterotypic synonym of Fusobacterium gonidiaformans.

The family Fusobacteriaceae is a large family within the phylum Fusobacteriota. The reclassification of F. naviforme as Zandiella naviformis gen. nov., comb. nov. is proposed because of the separate and distinct phylogenetic situation on the basis of the results of 16S rRNA gene sequence analysis, the genetic and genomic differences from all other species and subspecies in the Fusobacteriaceae family. The type strain is ATCC 25832; CCUG 50052; NCTC 13121. In phylogenetic trees drawn using complete genome sequences and 16S rRNA gene sequences, F. necrophorum subsp. funduliforme and F. equinum were clades together with F. necrophorum subsp. necrophorum and F. gonidiaformans, respectively. The average nucleotide identity, average amino acid identity, and digital DNA-DNA hybridization values between themes exceeded the cut-off values for species delineation. Based on these results, F. necrophorum subsp. funduliforme and F. equinum should be reclassified as later heterotypic synonyms of F. necrophorum subsp. necrophorum and F. gonidiaformans, respectively.

Molecular Characterization and Pathogenicity of an Infectious cDNA Clone of Youcai Mosaic Virus on Solanum nigrum.

Virus infections cause devastative economic losses for various plant species, and early diagnosis and prevention are the most effective strategies to avoid the losses. Exploring virus genomic evolution and constructing virus infectious cDNA clones is essential to achieve a deeper understanding of the interaction between host plant and virus. Therefore, this work aims to guide people to better prevent, control, and utilize the youcai mosaic virus (YoMV). Here, the YoMV was found to infect the Solanum nigrum under natural conditions. Then, an infectious cDNA clone of YoMV was successfully constructed using triple-shuttling vector-based yeast recombination. Furthermore, we established phylogenetic trees based on the complete genomic sequences, the replicase gene, movement protein gene, and coat protein gene using the corresponding deposited sequences in NCBI. Simultaneously, the evolutionary relationship of the YoMV discovered on S. nigrum to others was determined and analyzed. Moreover, the constructed cDNA infectious clone of YoMV from S. nigrum could systematically infect the Nicotiana benthamiana and S. nigrum by agrobacterium-mediated infiltration. Our investigation supplied a reverse genetic tool for YoMV study, which will also contribute to in-depth study and profound understanding of the interaction between YoMV and host plant.

The whole-genome sequencing of prevalent DENV-1 strains during the largest dengue virus outbreak in Xishuangbanna Dai autonomous prefecture in 2019.

In 2019, a serious dengue virus (DENV) infection broke out in the Xishuangbanna Dai Autonomous Prefecture, China. Therefore, we conducted a molecular epidemiological analysis in people that contracted DENV serotype 1 (DENV-1) during this year. We analyzed the molecular epidemiology of six DENV-1 epidemic strains in 2019 by full-length genome sequencing, amino acid mutation site analysis, evolutionary tree analysis, and recombination site comparison analysis. Through the analysis of amino acid mutation sites, it was found that DENV-1 strain (MW386867) was different from the other five epidemic DENV-1 strains in Xishuangbanna in 2019. MW386867 had unique mutation sites at six loci. The six epidemic DENV-1 strains in Xishuangbanna in 2019 were divided into two clusters. MW386867 was highly similar to the MG679800 (Myanmar 2017), MG679801 (Myanmar 2017), and KC172834 (Laos 2008), and the other five strains were highly similar to JQ045660 (Vietnam 2011), FJ176780 (GuangDong 2006). Genetic recombination analysis revealed that there was no recombination signal in the six epidemic DENV-1 strains in Xishuangbanna in 2019. We speculate that the DENV-1 epidemic in 2019 has a co-epidemic of local strains and cross-border strains.

Topological analysis of vertebrate phylogenetic trees utilizing Horton's first law.

Phylogenetic trees visually represent evolution and diversification. While many studies have focused on the number and length of edges (branches), topological properties, such as edge connection patterns, are also important. In this study, the topological properties of phylogenetic trees were quantified, focusing on edge connection patterns. Horton's first law was applied to quantify the overall, rather than local, topological properties of phylogenetic trees. The topological properties of vertebrate phylogenetic trees for spiny-rayed fishes, Amphibians, turtles, Squamata, Aves, and placental mammals were analyzed. The topological features discussed herein include the number of first-order edges, maximum order, and bifurcation ratio. The average bifurcation ratio of all trees was approximately 3, suggesting that phylogenetic trees for different taxa have a common mechanism of evolution. Vertebrate phylogenetic trees were compared with artificial branching objects created from neutral stochastic branching model simulations. The topological properties of the actual vertebrate phylogenetic trees agreed with those of the artificial branching objects. Our study suggests that evolutionary events do not change the overall topological properties of actual phylogenetic trees, even if the number and length of the edges change. Specifically, non-neutral events (e.g., environmental changes and mass extinction) are not main factors associated with topological properties. The results instead demonstrate a relationship between the bifurcation ratio and symmetricity in the context of temporal changes of topological properties. When the number of first-order edges increased and the maximum order remained constant, the bifurcation ratio increased and symmetricity decreased. When the number of first-order edges increased and the maximum order increased by one, the bifurcation ratio decreased and symmetricity increased.

First Report of the Occurrence of Diaporthe nobilis Causing Postharvest Fruit Rot on 'Shine Muscat' Grape in Yuncheng, China.

In recent years, 'Shine Muscat' (Vitis labruscana × Vitis vinifera) has been the most popular table grape among consumers in Asian countries (Choi et al., 2021). In November 2020, fruit rot was observed in packing house 2 months after cold-stored in Yuncheng (35°16'N, 111°08'E), Shanxi Province, China. Of 38 boxes (5 kg per box) grape were selected randomly from the packing house with a stock of 7,000 kg. The incidence of grape rot was 71.5%, observed in 143 of the 200 clusters sampled. The diseased fruit showed symptoms including pedicel browning, and extending lesions and softening of berries. Samples (1 mm2) cut from the border between diseased and healthy tissue were surface sterilized with 70% ethanol for 1 min, then rinsed with sterile distilled water thrice, and placed on potato dextrose agar (PDA) plate 25 ° C in the dark for 5 days. After subculturing for three times, 26 isolates with a similar type of fungal colony were obtained. Colonies on PDA was initially white fluffy aerial, but gradually turned grey. Pycnidia were black and globes, producing α and ß conidia. α conidia abundant, aseptate, hyaline, ellipsoid, both ends obtuse, biguttulate, 6.52~9.05 µm×2.19~3.25 µm (n=50). ß conidia abundant, hyaline, aseptate, filiform, flexuous to J-shaped, 22.79~36.75 µm×1.24~1.92 µm (n=50). For further identification, phylogenetic analysis of combined internal transcribed spacer (ITS), ß-tubulin (TUB), translation elongation factor (EF1-α) and calmodulin (CAL) datasets were carried out. The representative isolate SS1 and SS2 were selected for identification. Primers ITS1 and ITS4 (White et al. 1990) were used to amplify the ITS region. The gene fragments of TUB, CAL and EF1-α were amplified by the primers Bt2a/Bt2b (Glass and Donaldson 1995), CAL228F/CAL737R and EF1-728F/EF1-986R, respectively (Carbone and Kohn 1999). The sequences were deposited in GenBank (SS1: MW644526 for ITS, OQ718912 - 4 for CAL, TEF-1α, and TUB, respectively; SS2: MW644527 for ITS, OQ718915 -7 for CAL, TEF-1α, and TUB, respectively). BLASTn analysis of the NCBI database indicated that the ITS, TUB, EF1-α, and CAL sequences had very high nucleotide homology (98 to 100%) to ex-type sequences for D. nobilis (CBS 116953, Gomes et al., 2013) in GenBank (ITS: KC343147, TUB: KC344115, EF1-α: KC343873, and CAL: KC343389). Phylogenetic tree based on the combined sequences revealed that the two isolates clustered well with D. nobilis. To confirm its pathogenicity, 30 fresh-harvested 'Shine Muscat' grape fruits were used to mock inoculate. Wounds (5 mm wide by 2 mm deep) were created uniformly at the equator of the grape fruit with a sterile puncher and inoculated with mycelial plugs (5 mm in diameter) prepared from 7-day-old fungal cultures grown on PDA (Wild 1994). The control fruits were inoculated with agar disks without mycelium. Inoculated fruits were placed in a chamber with the 80% relative humidity at 25°C for 8 days. After inoculation, all treated fruits turned brown, with significant necrotic lesions; fruits in control showed no symptoms. Fungal colonies reisolated from inoculated fruits had similar morphological characteristics as D. nobilis. Diaporthe spp. are responsible for diseases on a wide range of plants hosts, causing root and fruit rots, dieback, cankers, leaf spots, blights, decay and wilt (Gomes et al., 2013). D. nobilis causing postharvest grape rot can seriously restrict the development of local grape industry.

The Connection of the Generalized Robinson-Foulds Metric with Partial Wiener Indices.

In this work we propose the partial Wiener index as one possible measure of branching in phylogenetic evolutionary trees. We establish the connection between the generalized Robinson-Foulds (RF) metric for measuring the similarity of phylogenetic trees and partial Wiener indices by expressing the number of conflicting pairs of edges in the generalized RF metric in terms of partial Wiener indices. To do so we compute the minimum and maximum value of the partial Wiener index [Formula: see text], where [Formula: see text] is a binary rooted tree with root [Formula: see text] and [Formula: see text] leaves. Moreover, under the Yule probabilistic model, we show how to compute the expected value of [Formula: see text]. As a direct consequence, we give exact formulas for the upper bound and the expected number of conflicting pairs. By doing so we provide a better theoretical understanding of the computational complexity of the generalized RF metric.

An Unsupervised Classifier for Whole-Genome Phylogenies, the Maxwell© Tool.

The development of phylogenetic trees based on RNA or DNA sequences generally requires a precise and limited choice of important RNAs, e.g., messenger RNAs of essential proteins or ribosomal RNAs (like 16S), but rarely complete genomes, making it possible to explain evolution and speciation. In this article, we propose revisiting a classic phylogeny of archaea from only the information on the succession of nucleotides of their entire genome. For this purpose, we use a new tool, the unsupervised classifier Maxwell, whose principle lies in the Burrows-Wheeler compression transform, and we show its efficiency in clustering whole archaeal genomes.

Phylogenetic Signal, Congruence, and Uncertainty across Bacteria and Archaea.

Reconstruction of the Tree of Life is a central goal in biology. Although numerous novel phyla of bacteria and archaea have recently been discovered, inconsistent phylogenetic relationships are routinely reported, and many inter-phylum and inter-domain evolutionary relationships remain unclear. Here, we benchmark different marker genes often used in constructing multidomain phylogenetic trees of bacteria and archaea and present a set of marker genes that perform best for multidomain trees constructed from concatenated alignments. We use recently-developed Tree Certainty metrics to assess the confidence of our results and to obviate the complications of traditional bootstrap-based metrics. Given the vastly disparate number of genomes available for different phyla of bacteria and archaea, we also assessed the impact of taxon sampling on multidomain tree construction. Our results demonstrate that biases between the representation of different taxonomic groups can dramatically impact the topology of resulting trees. Inspection of our highest-quality tree supports the division of most bacteria into Terrabacteria and Gracilicutes, with Thermatogota and Synergistota branching earlier from these superphyla. This tree also supports the inclusion of the Patescibacteria within the Terrabacteria as a sister group to the Chloroflexota instead of as a basal-branching lineage. For the Archaea, our tree supports three monophyletic lineages (DPANN, Euryarchaeota, and TACK/Asgard), although we note the basal placement of the DPANN may still represent an artifact caused by biased sequence composition. Our findings provide a robust and standardized framework for multidomain phylogenetic reconstruction that can be used to evaluate inter-phylum relationships and assess uncertainty in conflicting topologies of the Tree of Life.

Coevolutionary and Phylogenetic Analysis of Mimiviral Replication Machinery Suggest the Cellular Origin of Mimiviruses.

Mimivirus is one of the most complex and largest viruses known. The origin and evolution of Mimivirus and other giant viruses have been a subject of intense study in the last two decades. The two prevailing hypotheses on the origin of Mimivirus and other viruses are the reduction hypothesis, which posits that viruses emerged from modern unicellular organisms; whereas the virus-first hypothesis proposes viruses as relics of precellular forms of life. In this study, to gain insights into the origin of Mimivirus, we have carried out extensive phylogenetic, correlation, and multidimensional scaling analyses of the putative proteins involved in the replication of its 1.2-Mb large genome. Correlation analysis and multidimensional scaling methods were validated using bacteriophage, bacteria, archaea, and eukaryotic replication proteins before applying to Mimivirus. We show that a large fraction of mimiviral replication proteins, including polymerase B, clamp, and clamp loaders are of eukaryotic origin and are coevolving. Although phylogenetic analysis places some components along the lineages of phage and bacteria, we show that all the replication-related genes have been homogenized and are under purifying selection. Collectively our analysis supports the idea that Mimivirus originated from a complex cellular ancestor. We hypothesize that Mimivirus has largely retained complex replication machinery reminiscent of its progenitor while losing most of the other genes related to processes such as metabolism and translation.

Conserved C-terminal motifs in odorant receptors instruct their cell surface expression and cAMP signaling.

The highly individual plasma membrane expression and cAMP signaling of odorant receptors have hampered their ligand assignment and functional characterization in test cell systems. Chaperones have been identified to support the cell surface expression of only a portion of odorant receptors, with mechanisms remaining unclear. The presence of amino acid motifs that might be responsible for odorant receptors' individual intracellular retention or cell surface expression, and thus, for cAMP signaling, is under debate: so far, no such protein motifs have been suggested. Here, we demonstrate the existence of highly conserved C-terminal amino acid motifs, which discriminate at least between class-I and class-II odorant receptors, with their numbers of motifs increasing during evolution, by comparing C-terminal protein sequences from 4808 receptors across eight species. Truncation experiments and mutation analysis of C-terminal motifs, largely overlapping with helix 8, revealed single amino acids and their combinations to have differential impact on the cell surface expression and on stimulus-dependent cAMP signaling of odorant receptors in NxG 108CC15 cells. Our results demonstrate class-specific and individual C-terminal motif equipment of odorant receptors, which instruct their functional expression in a test cell system, and in situ may regulate their individual cell surface expression and intracellular cAMP signaling.

Phylogenomic discordance suggests polytomies along the backbone of the large genus Solanum.

PREMISE: Evolutionary studies require solid phylogenetic frameworks, but increased volumes of phylogenomic data have revealed incongruent topologies among gene trees in many organisms both between and within genomes. Some of these incongruences indicate polytomies that may remain impossible to resolve. Here we investigate the degree of gene-tree discordance in Solanum, one of the largest flowering plant genera that includes the cultivated potato, tomato, and eggplant, as well as 24 minor crop plants. METHODS: A densely sampled species-level phylogeny of Solanum is built using unpublished and publicly available Sanger sequences comprising 60% of all accepted species (742 spp.) and nine regions (ITS, waxy, and seven plastid markers). The robustness of this topology is tested by examining a full plastome dataset with 140 species and a nuclear target-capture dataset with 39 species of Solanum (Angiosperms353 probe set). RESULTS: While the taxonomic framework of Solanum remained stable, gene tree conflicts and discordance between phylogenetic trees generated from the target-capture and plastome datasets were observed. The latter correspond to regions with short internodal branches, and network analysis and polytomy tests suggest the backbone is composed of three polytomies found at different evolutionary depths. The strongest area of discordance, near the crown node of Solanum, could potentially represent a hard polytomy. CONCLUSIONS: We argue that incomplete lineage sorting due to rapid diversification is the most likely cause for these polytomies, and that embracing the uncertainty that underlies them is crucial to understand the evolution of large and rapidly radiating lineages.

Maximum parsimony reconciliation in the DTLOR model.

BACKGROUND: Analyses of microbial evolution often use reconciliation methods. However, the standard duplication-transfer-loss (DTL) model does not account for the fact that species trees are often not fully sampled and thus, from the perspective of reconciliation, a gene family may enter the species tree from the outside. Moreover, within the genome, genes are often rearranged, causing them to move to new syntenic regions. RESULTS: We extend the DTL model to account for two events that commonly arise in the evolution of microbes: origin of a gene from outside the sampled species tree and rearrangement of gene syntenic regions. We describe an efficient algorithm for maximum parsimony reconciliation in this new DTLOR model and then show how it can be extended to account for non-binary gene trees to handle uncertainty in gene tree topologies. Finally, we describe preliminary experimental results from the integration of our algorithm into the existing xenoGI tool for reconstructing the histories of genomic islands in closely related bacteria. CONCLUSIONS: Reconciliation in the DTLOR model can offer new insights into the evolution of microbes that is not currently possible under the DTL model.

Discordant evolution of organellar genomes in peas (Pisum L.).

Plastids and mitochondria have their own small genomes, which do not undergo meiotic recombination and may have evolutionary fates different from each other and that of the nuclear genome. For the first time, we sequenced mitochondrial genomes of pea (Pisum L.) from 42 accessions mostly representing diverse wild germplasm from throughout the wild pea geographical range. Six structural types of the pea mitochondrial genome were revealed. From the same accessions, plastid genomes were sequenced. Phylogenetic trees based on the plastid and mitochondrial genomes were compared. The topologies of these trees were highly discordant, implying not less than six events of hybridisation between diverged wild peas in the past, with plastids and mitochondria differently inherited by the descendants. Such discordant inheritance of organelles could have been driven by plastid-nuclear incompatibility, which is known to be widespread in crosses involving wild peas and affects organellar inheritance. The topology of the phylogenetic tree based on nucleotide sequences of a nuclear gene, His5, encoding a histone H1 subtype, corresponded to the current taxonomy and resembled that based on the plastid genome. Wild peas (Pisum sativum subsp. elatius s.l.) inhabiting Southern Europe were shown to be of hybrid origin, resulting from crosses of peas related to those presently inhabiting the eastern Mediterranean in a broad sense. These results highlight the roles of hybridisation and cytonuclear conflict in shaping plant microevolution.

Hundreds of nuclear and plastid loci yield novel insights into orchid relationships.

PREMISE: The inference of evolutionary relationships in the species-rich family Orchidaceae has hitherto relied heavily on plastid DNA sequences and limited taxon sampling. Previous studies have provided a robust plastid phylogenetic framework, which was used to classify orchids and investigate the drivers of orchid diversification. However, the extent to which phylogenetic inference based on the plastid genome is congruent with the nuclear genome has been only poorly assessed. METHODS: We inferred higher-level phylogenetic relationships of orchids based on likelihood and ASTRAL analyses of 294 low-copy nuclear genes sequenced using the Angiosperms353 universal probe set for 75 species (representing 69 genera, 16 tribes, 24 subtribes) and a concatenated analysis of 78 plastid genes for 264 species (117 genera, 18 tribes, 28 subtribes). We compared phylogenetic informativeness and support for the nuclear and plastid phylogenetic hypotheses. RESULTS: Phylogenetic inference using nuclear data sets provides well-supported orchid relationships that are highly congruent between analyses. Comparisons of nuclear gene trees and a plastid supermatrix tree showed that the trees are mostly congruent, but revealed instances of strongly supported phylogenetic incongruence in both shallow and deep time. The phylogenetic informativeness of individual Angiosperms353 genes is in general better than that of most plastid genes. CONCLUSIONS: Our study provides the first robust nuclear phylogenomic framework for Orchidaceae and an assessment of intragenomic nuclear discordance, plastid-nuclear tree incongruence, and phylogenetic informativeness across the family. Our results also demonstrate what has long been known but rarely thoroughly documented: nuclear and plastid phylogenetic trees can contain strongly supported discordances, and this incongruence must be reconciled prior to interpretation in evolutionary studies, such as taxonomy, biogeography, and character evolution.

An extended model for phylogenetic maximum likelihood based on discrete morphological characters.

Maximum likelihood is a common method of estimating a phylogenetic tree based on a set of genetic data. However, models of evolution for certain types of genetic data are highly flawed in their specification, and this misspecification can have an adverse impact on phylogenetic inference. Our attention here is focused on extending an existing class of models for estimating phylogenetic trees from discrete morphological characters. The main advance of this work is a model that allows unequal equilibrium frequencies in the estimation of phylogenetic trees from discrete morphological character data using likelihood methods. Possible extensions of the proposed model will also be discussed.

Molecular characterization of the viral structural protein genes in the first outbreak of dengue virus type 2 in Hunan Province, inland China in 2018.

BACKGROUND: An unexpected dengue outbreak occurred in Hunan Province in 2018. This was the first dengue outbreak in this area of inland China, and 172 cases were reported. METHODS: To verify the causative agent of this outbreak and characterise the viral genes, the genes encoding the structural proteins C/prM/E of viruses isolated from local residents were sequenced followed by mutation and phylogenetic analysis. Recombination, selection pressure, potential secondary structure and three-dimensional structure analyses were also performed. RESULTS: Phylogenetic analysis revealed that all epidemic strains were of the cosmopolitan DENV-2 genotype and were most closely related to the Zhejiang strain (MH010629, 2017) and then the Malaysia strain (KJ806803, 2013). Compared with the sequence of DENV-2SS, 151 base substitutions were found in the sequences of 89 isolates; these substitutions resulted in 20 non-synonymous mutations, of which 17 mutations existed in all samples (two in the capsid protein, six in the prM/M proteins, and nine in the envelope proteins). Moreover, amino acid substitutions at the 602nd (E322:Q → H) and 670th (E390: N → S) amino acids may have enhanced the virulence of the epidemic strains. One new DNA binding site and five new protein binding sites were observed. Two polynucleotide binding sites and seven protein binding sites were lost in the epidemic strains compared with DENV-2SS. Meanwhile, five changes were found in helical regions. Minor changes were observed in helical transmembrane and disordered regions. The 429th amino acid of the E protein switched from a histamine (positively charged) to an asparagine (neutral) in all 89 isolated strains. No recombination events or positive selection pressure sites were observed. To our knowledge, this study is the first to analyse the genetic characteristics of epidemic strains in the first dengue outbreak in Hunan Province in inland China. CONCLUSIONS: The causative agent is likely to come from Zhejiang Province, a neighbouring province where dengue fever broke out in 2017. This study may help clarify the intrinsic geographical relatedness of DENV-2 and contribute to further research on pathogenicity and vaccine development.