High Rate Of Nucleotide Substitution Research Articles

Genomic Analyses Uncover Evolutionary Features of Influenza A/H3N2 Viruses in Yunnan Province, China, from 2017 to 2022.

Influenza A viruses evolve at a high rate of nucleotide substitution, thereby requiring continuous monitoring to determine the efficacy of vaccines and antiviral drugs. In the current study, we performed whole-genome sequencing analyses of 253 influenza A/H3N2 strains from Yunnan Province, China, during 2017-2022. The hemagglutinin (HA) segments of Yunnan A/H3N2 strains isolated during 2017-2018 harbored a high genetic diversity due to heterogeneous distribution across branches. The mutation regularity of the predominant antigenic epitopes of HA segments in Yunnan was inconsistent in different years. Some important functional mutations in gene segments associated with viral adaptation and drug tolerance were revealed. The rapid genomic evolution of Yunnan A/H3N2 strains from 2017 to 2022 mainly concentrated on segments, i.e., matrix protein 2 (M2), non-structural protein 1 (NS1), neuraminidase (NA), NS2, and HA, with a high overall non-synonymous/synonymous substitution ratio (dN/dS). Our results highlighted a decline in vaccine efficacy against the A/H3N2 circulating strains, particularly against the Yunnan 2021-2022 A/H3N2 strains. These findings aid our understanding of evolutionary characteristics and epidemiological monitoring of the A/H3N2 viruses and provide in-depth insights into the protective efficacy of influenza vaccines.

Genetic Diversity, Evolutionary Dynamics, and Ongoing Spread of Pedilanthus Leaf Curl Virus.

Pedilanthus leaf curl virus (PeLCV) is a monopartite begomovirus (family Geminiviridae) discovered just a few decades ago. Since then, it has become a widely encountered virus, with reports from ca. 25 plant species across Pakistan and India, indicative of its notable evolutionary success. Viruses mutate at such a swift rate that their ecological and evolutionary behaviors are inextricably linked, and all of these behaviors are imprinted on their genomes as genetic diversity. So, all these imprints can be mapped by computational methods. This study was designed to map the sequence variation dynamics, genetic heterogeneity, regional diversity, phylogeny, and recombination events imprinted on the PeLCV genome. Phylogenetic and network analysis grouped the full-length genome sequences of 52 PeLCV isolates into 7 major clades, displaying some regional delineation but lacking host-specific demarcation. The progenitor of PeLCV was found to have originated in Multan, Pakistan, in 1977, from where it spread concurrently to India and various regions of Pakistan. A high proportion of recombination events, distributed unevenly throughout the genome and involving both inter- and intraspecies recombinants, were inferred. The findings of this study highlight that the PeLCV population is expanding under a high degree of genetic diversity (π = 0.073%), a high rate of mean nucleotide substitution (1.54 × 10-3), demographic selection, and a high rate of recombination. This sets PeLCV apart as a distinctive begomovirus among other begomoviruses. These factors could further exacerbate the PeLCV divergence and adaptation to new hosts. The insights of this study that pinpoint the emergence of PeLCV are outlined.

Buried treasure in a public repository: Mining mitochondrial genes of 32 annelid species from sequence reads deposited in the Sequence Read Archive (SRA).

The mitochondrial genomes (mitogenomes) of metazoans generally include the same set of protein-coding genes, which ensures the homology of mitochondrial genes between species. The mitochondrial genes are often used as reference data for species identification based on genetic data (DNA barcoding). The need for such reference data has been increasing due to the application of environmental DNA (eDNA) analysis for environmental assessments. Recently, the number of publicly available sequence reads obtained with next-generation sequencing (NGS) has been increasing in the public database (the NCBI Sequence Read Archive, SRA). Such freely available NGS reads would be promising sources for assembling mitochondrial protein-coding genes (mPCGs) of organisms whose mitochondrial genes are not available in GenBank. The present study aimed to assemble annelid mPCGs from raw data deposited in the SRA. The recent progress in the classification of Annelida was briefly introduced. In the present study, the mPCGs of 32 annelid species of 19 families in clitellates and allies in Sedentaria (echiurans and polychaetes) were newly assembled from the reads deposited in the SRA. Assembly was performed with a recently published pipeline mitoRNA, which includes cycles of Bowtie2 mapping and Trinity assembly. Assembled mPCGs were deposited in GenBank as Third Party Data (TPA) data. A phylogenetic tree was reconstructed with maximum likelihood (ML) analysis, together with other mPCGs deposited in GenBank. mPCG assembly was largely successful except for Travisia forbesii; only four genes were detected from the assembled contigs of the species probably due to the reads targeting its parasite. Most genes were largely successfully obtained, whereas atp8, nad2, and nad4l were only successful in 22-24 species. The high nucleotide substitution rates of these genes might be relevant to the failure in the assembly although nad6, which showed a similarly high substitution rate, was successfully assembled. Although the phylogenetic positions of several lineages were not resolved in the present study, the phylogenetic relationships of some polychaetes and leeches that were not inferred by transcriptomes were well resolved probably due to a more dense taxon sampling than previous phylogenetic analyses based on transcriptomes. Although NGS data are generally better sources for resolving phylogenetic relationships of both higher and lower classifications, there are ensuring needs for specific loci of the mitochondrial genes for analyses that do not require high resolutions, such as DNA barcoding, eDNA, and phylogenetic analysis among lower taxa. Assembly from publicly available NGS reads would help design specific primers for the mitochondrial gene sequences of species, whose mitochondrial genes are hard to amplify by Sanger sequencing using universal primers.

Natural recombination of the torque teno canis virus within the ORF1, -2, and -3 genes and the untranslated region

The torque teno canis virus (TTCaV) was first reported in 2001 and it shares similarities with the known Torque teno virus (TTV) in terms of genomic organization and putative transcriptional features. It is a single-stranded DNA virus characterized by high rates of recombination and nucleotide substitution, like RNA viruses. Studies reported recombination events in torque teno virus; however, there is limited reporting of TTCaV reorganization events. This study screened fecal samples from domestic dogs in Henan Province. There was a positivity rate of 16.5% (19/115) for TTCaV. Four nearly complete TTCaV genomes, namely Canine/HeNan/4, 5, 6, and 13/2019, were obtained from the 19 positive fecal samples, whose genome sequence was obtained using gap-filling PCR. Sequence analysis revealed two unique amino acid mutation sites in the TTCaV strains, K278Q (compared with the first isolate Cf-TTV10 in Japan) and V/L268I (compared with the TTCaV strain from southern China). Subsequently, 17 near full-length TTCaV genome sequences were subjected to phylogenetic and recombination detection program analyzes. We obtained evidence supporting recombination events in the Chinese TTCaV strains. These findings suggest that mutation and recombination occurred in the three individual gene segments (ORF1, ORF2, ORF3) and the untranslated region, an area of major recombination in the Chinese TTCaV strain GX265 genome. Interestingly, the TTCaV strain (Canine/HeNan/6/2019) was a major parent involved in the genetic recombination of the GX265 strain. This study provides insights into the genetic variability and evolution of TTCaV.

Genetic diversity and molecular evolution of Seoul virus in Hebei province, China.

Seoul virus (SEOV) is a major pathogen which causes hemorrhagic fever with renal syndrome (HFRS), and is present all over the world. However, there are currently few long-term systematic studies of SEOV's phylogenetic and evolutionary mechanisms in epidemic areas. Thus, in this study, we used RT-PCR combined with NGS to obtain the genomes of six SEOV viruses from 1993, as well as 56 Hebei province-specific tissue samples from 1999 to 2022. Phylogenetic analysis showed that the SEOV samples could be divided into seven groups and showed geographic clustering. The geographic region may be the main factor affecting the genetic diversity of SEOV. We also found that SEOV was subject to strong overall purifying selection and positive selection at certain sites during evolution. Recombination events and high nucleotide substitution rates were also shown to accelerate SEOV's evolution. Evolutionary feature of the L segment is more representative of complete genome. Our detailed analysis provides a deeper understanding of the genetic diversity and evolutionary drivers of SEOV within its primary epidemic areas. It will be important to further monitor epidemiological trends and drivers of variation to help increase our understanding of the pathogenicity of SEOV infections.

The FDA-approved drug nitazoxanide is a potent inhibitor of human seasonal coronaviruses acting at postentry level: effect on the viral spike glycoprotein.

Coronaviridae is recognized as one of the most rapidly evolving virus family as a consequence of the high genomic nucleotide substitution rates and recombination. The family comprises a large number of enveloped, positive-sense single-stranded RNA viruses, causing an array of diseases of varying severity in animals and humans. To date, seven human coronaviruses (HCoV) have been identified, namely HCoV-229E, HCoV-NL63, HCoV-OC43 and HCoV-HKU1, which are globally circulating in the human population (seasonal HCoV, sHCoV), and the highly pathogenic SARS-CoV, MERS-CoV and SARS-CoV-2. Seasonal HCoV are estimated to contribute to 15-30% of common cold cases in humans; although diseases are generally self-limiting, sHCoV can sometimes cause severe lower respiratory infections and life-threatening diseases in a subset of patients. No specific treatment is presently available for sHCoV infections. Herein we show that the anti-infective drug nitazoxanide has a potent antiviral activity against three human endemic coronaviruses, the Alpha-coronaviruses HCoV-229E and HCoV-NL63, and the Beta-coronavirus HCoV-OC43 in cell culture with IC50 ranging between 0.05 and 0.15 μg/mL and high selectivity indexes. We found that nitazoxanide does not affect HCoV adsorption, entry or uncoating, but acts at postentry level and interferes with the spike glycoprotein maturation, hampering its terminal glycosylation at an endoglycosidase H-sensitive stage. Altogether the results indicate that nitazoxanide, due to its broad-spectrum anti-coronavirus activity, may represent a readily available useful tool in the treatment of seasonal coronavirus infections.

Kinetoplast Genome of Leishmania spp. Is under Strong Purifying Selection.

Instability is an intriguing characteristic of many protist genomes, and trypanosomatids are not an exception in this respect. Some regions of trypanosomatid genomes evolve fast. For instance, the trypanosomatid mitochondrial (kinetoplast) genome consists of fairly conserved maxicircle and minicircle molecules that can, nevertheless, possess high nucleotide substitution rates between closely related strains. Recent experiments have demonstrated that rapid laboratory evolution can result in the non-functionality of multiple genes of kinetoplast genomes due to the accumulation of mutations or loss of critical genomic components. An example of a loss of critical components is the reported loss of entire minicircle classes in Leishmania tarentolae during laboratory cultivation, which results in an inability to generate some correctly encoded genes. In the current work, we estimated the evolutionary rates of mitochondrial and nuclear genome regions of multiple natural Leishmania spp. We analyzed synonymous and non-synonymous substitutions and, rather unexpectedly, found that the coding regions of kinetoplast maxicircles are among the most variable regions of both genomes. In addition, we demonstrate that synonymous substitutions greatly predominate among maxicircle coding regions and that most maxicircle genes show signs of purifying selection. These results imply that maxicircles in natural Leishmania populations remain functional despite their high mutation rate.

Molecular Identification and Phylogenetic Diversity of Native Entomopathogenic Nematodes, and Their Bacterial Endosymbionts, Isolated from Banana and Plantain Crops in Western Colombia

With the increasing negative impacts on worldwide food production caused by pests, the recovery of native entomopathogenic nematodes (EPNs) is relevant, since they are adapted to local environments, entomofauna, and significant virulence. Therefore, the present study was designed to recover and understand the phylogenetic diversity of EPNs and their associated bacterial endosymbionts, from banana and plantain crops, as alternatives for the control of weevil species. An extensive sampling of western Colombia covered 325 ha, yielding the recovery of three EPNs’ isolates (0.49% of the samples). The molecular characterization included four mitochondrial and nuclear loci, which, after merging with the sequences of 48 species, confirmed the presence of Steinernema carpocapsae, the first report of S. costaricense in South America, and monophyly in most of the Steinernema clades. The tree topologies were consistent for the nuclear loci but not for mitochondrial, probably due to the high nucleotide substitution rate, deficit in the number of species available for these loci, and incomplete lineage sorting. The endosymbiotic bacteria associated with S. carpocapsae were identified as Xenorhabdus nematophila. However, the S. costaricense bacterial symbiont presented a genetic similarity to X. koppenhoeferi and X. khoisanae, which are still uncertain in their classification. The identification of S. costaricense in South America indicates the wide range distribution of this species in the Americas and its ability to persist in different soil types. For the first time, EPN isolation and phylogenetic characterization are directed to plantain and banana crops. Leveraging EPNs’ diversity promises novel applications for crop protection, while the genetic resources from the bacterial endosymbionts may provide metabolites with a wide spectrum of uses, either for agricultural or medicinal purposes.

Prevalence and genetic characterization of bovine coronavirus identified from diarrheic pre-weaned native Korean calves from 2019 to 2021.

Bovine coronavirus (BCoV) is associated with severe diarrhea in calves, winter dysentery in adult cattle, and respiratory diseases in cattle. However, there is currently limited information regarding its molecular characterization in the Republic of Korea (KOR). Therefore, this study investigated the prevalence of BCoV in diarrheic pre-weaned calves (aged ≤60days) and compared BCoV genome sequences identified globally. A total of 846 fecal samples were collected from calves with diarrhea across 100 beef farms in the KOR. The samples were divided into three groups based on age as follows: 1-10days (n=490), 11-30days (n=277), and 31-60days (n=79). BCoV infection was detected in 50 calves by real-time RT-PCR analysis. The results showed that the prevalence of BCoV was associated with calf age (P=0.028) and was significantly higher in calves aged 31-60days (odds ratio: 2.69, 95% confidence interval: 1.24-5.85; P=0.012) than in those aged 1-10days. Our findings show that BCoV is an important etiological agent of diarrhea in calves aged 31-60days. Fifteen full genome sequences (2019-2021 variants) of the spike, hemagglutinin/esterase, and nucleocapsid were obtained from the 50 BCoV-positive samples. Phylogenetic analysis of each gene revealed that BCoVs circulating worldwide might have no boundary between enteric and respiratory tropisms, demonstrating the presence of three BCoVs groups: the classical, Asia/USA, and European. Initially, Korean BCoVs were originated from the USA, but diverged since the 1980s and rapidly evolved independently, unlike in other Asian countries. In this study, Korean BCoVs are more recent BCoVs and present relatively high nucleotide substitution rates in all genes compared with other BCoVs. Our results showed that the 2019-2021 variants undergo continuous genetic evolution and that there are genetic differences among globally distributed BCoVs.

Abstract GS3-09: Loss of ASXL1 tumor suppressor promotes resistance to CDK4/6 inhibitors in ER+ breast cancer

Abstract Background: CDK4/6 inhibitors (CDK4/6i) in combination with antiestrogens have prolonged survival of patients with ER+ metastatic breast cancer. However, this combination is not curative mainly due to acquired drug resistance. Knowledge about mechanisms of such resistance remains quite incomplete. We report herein a forward-genetics screen to discover a broad spectrum of novel somatic mutations causal to CDK4/6i resistance. Methods: We used CRISPR/Cas9 to delete the DNA mismatch repair (MMR) gene MSH2 in MCF7 and T47D ER+ breast cancer cells. Deficiency of DNA MMR proteins such as MSH2 results in a high nucleotide substitution rate which, in turn, predisposes cells to acquire drug resistance-associated mutations. MSH2-/- MCF7 and T47D cells were infected with a lentiviral barcode library containing ~1000 unique DNA barcodes. MSH2-/- barcoded cells were expanded for ~25 doublings to allow the accumulation of random mutations. Clones resistant to CDK4/6i were selected in the presence of IC90 of palbociclib (200 nM) or abemaciclib (500 nM) for 4-6 weeks. CDK4/6i resistant clones with unique barcode IDs were subjected to whole exome sequencing (WES). Results: Following drug selection, ~73 uniquely barcoded resistant colonies emerged from MCF7 and T47D MSH2-/- clonal lines. As expected, MCF7 and T47D MSH2-/- clones harbored a high mutation burden compared to parental cells. Candidate variants were distilled based on (a) functionality prediction and (b) mutation frequency in Project GENIE. We observed RB1 (5/73 clones; 6.8%) mutations in CDK4/6i resistant clones, providing proof-of-principle that this approach can identify clinically-relevant drug resistant alterations. Overall, we identified non-synonymous alterations in 2,206 genes in T47D palbociclib-resistant, 2,195 genes in T47D abemaciclib-resistant, and 1,312 genes in MCF7 palbociclib-resistant lines. A secondary screen of the 10 genes recurrently mutated in all three CDK4/6i resistant groups identified loss of ASXL1 as top hit. ASXL1 encodes a polycomb repressive complex protein that regulates chromatin accessibility. Loss of ASXL1 has been implicated in myeloid transformation through epigenetic reprogramming. WES of 76 CDK4/6i resistant tumor biopsies (DFCI/MBCproject cohort) identified ASXL1 alterations in two and four patients with acquired and primary resistance, respectively (6/76=7.9%). One of the tumors that progressed after an initial response to palbociclib had acquired the same ASXL1 R549C mutation that was identified in our screen. Among 1,769 tumors from patients treated with CDK4/6i (TEMPUS database), 37 exhibited ASXL1 alterations (4 frameshift, 6 truncating, 3 in-frame del, 24 missense mutations). DNAseq of patient-derived organoids established from post-CDK4/6i metastases identified ASXL1 mutations in 2/7 organoids (29%). ASXL1-/- MCF7 and T47D cells were cross-resistant to fulvestrant. GSEA analysis of RNA-seq data showed upregulation of E2F targets in palbociclib-treated cells stably transduced with ASXL1 shRNA but not control shRNA (Enrichment score=0.75, q=1.00E-09). This was associated with maintenance of RB phosphorylation in the presence of CDK4/6i, markedly higher levels of CDK2, CDK6, cyclins E and A, and downregulation of p21 and p27. Finally, siRNAs targeting CDK2 or cyclin A reduced the viability of ASXL1-deficient T47D cells by 50% and 90%, respectively. Conclusions: An accelerated mutagenesis approach using MMR-deficient ER+ breast cancer cells identified loss of ASXL1 as a novel mechanism of resistance to CDK4/6i. ASXL1 alterations were found in ~8% of tumors from patients with de novo or acquired resistance to CDK4/6i. Knockdown of CDK2 and cyclin A restored sensitivity to CDK4/6i and reduced viability of ASXL1 deficient cells, suggesting CDK2 inhibitors are a treatment approach against these drug-resistant tumors. Citation Format: Dhivya R. Sudhan, Sumanta Chatterjee, Jiwoong Kim, Yunguan Wang, Vishal Kandagatla, Dan Ye, Chang-Ching Lin, Jorge Gomez Tejeda Zanudo, Esha Jain, Arnaldo Marin, Alberto Servetto, Kyung-min Lee, Juan Manuel Povedano, David McFadden, Alex Barrett, Nikhil Wagle, Ariella B. Hanker, Carlos L. Arteaga. Loss of ASXL1 tumor suppressor promotes resistance to CDK4/6 inhibitors in ER+ breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr GS3-09.

Coronavirus: History, Genome Structure and Pathogenesis

Background: The positive sense and inordinate large RNA genome enclosed by helical nucleocapsids along with an outermost layer belongs to the family Coronaviridae. The phylogenetic tree of this family has been classified into Class1 as alpha, Class 2 as beta, Class 3 as gamma, and Class 4 as delta CoV. The mammalian respiratory and gastrointestinal tracts are the main target organs of this enveloped virus with misperceived mechanisms. The relevance of this virus family has considerably increased by the recent emergence of the Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS), which are caused by viruses that belong to the beta-CoV group. Aim: Aforesaid illustrations of the emergence of coronavirus diseases over the past two decades, SARS (2002; 2003) and MERS (2012 to present) - the ongoing COVID-19 outbreak has pressurized the WHO to take innovative measures for public health, research and medical communities. The aim of the present review is to have proficiency in the coronavirus replication and transcription process which is still in its infancy. Conclusion: As an outcome of epidemics, it is being recognized as one of the most advancing viruses by the virtue of high genomic nucleotide substitution rates and recombination. The hallmark of coronavirus replication is discontinuous transcription resulting in the production of multiple subgenomic mRNAs having sequences complementary to both ends of the genome. Therefore, the complete genome sequence of coronavirus will be used as a frame of reference for comprehending this classical phenomenon of the RNA replication process. Finally, research on the pathogenesis of coronaviruses and the host immunopathological response will aid in designing vaccines and minimizing the mortality rate.

Canning Processes Reduce the DNA-Based Traceability of Commercial Tropical Tunas.

Canned tuna is one of the most widely traded seafood products internationally and is of growing demand. There is an increasing concern over the vulnerability of canned tuna supply chains to species mislabelling and fraud. Extensive processing conditions in canning operations can lead to the degradation and fragmentation of DNA, complicating product traceability. We here employed a forensically validated DNA barcoding tool (cytochrome b partial sequences) to assess the effects of canning processes on DNA degradation and the identification of four tropical tuna species (yellowfin, bigeye, skipjack and longtail tuna) collected on a global scale, along their commercial chains. Each species was studied under five different canning processes i.e., freezing, defrosting, cooking, and canning in oil and brine, in order to investigate how these affect DNA-based species identification and traceability. The highest percentage of nucleotide substitutions were observed after brine-canning operations and were greatest for yellowfin and skipjack tuna. Overall, we found that DNA degradation significantly increased along the tuna canning process for most specimens. Consequently, most of the specimens canned in oil or brine were misidentified due to the high rate of nucleotide substitution in diagnostic sequences.

Molecular and phylogenetic analysis of Vietnamese canine parvovirus 2C originated from dogs reveals a new Asia-IV clade.

Canine parvovirus type 2 (CPV-2) is a small, single-stranded DNA virus causing fatal haemorrhagic enteritis in dogs. Currently, CPV-2 is classified into CPV-2a, CPV-2b and CPV-2c based on genetic variation in the VP2 gene. The CPV-2c variant has become ubiquitous worldwide and gained attention for monitoring parvoviral evolution. In this study, we characterized the full-length genome sequences of CPV-2c strains obtained from 59 dogs in Vietnam. Molecular analysis revealed that Vietnamese CPV-2c shared a common evolutionary pattern with the Asian CPV-2 clade, which is marked by genetic signature patterns in the structural and nonstructural proteins. In addition, these Vietnamese CPV-2c strains exhibited unique Thr112Ile and Ile447Met mutations in the VP1 and VP2 sequence, respectively. Interestingly, phylogenetic analysis indicated that the mutations of amino acid residues in both the structural and nonstructural genes have contributed to the emergence of a new clade, designated here as the Asia-IV clade. The substitution rates, estimated from a dataset containing 199 sequences over the last 42years, confirmed that CPV-2 showed a high rate of nucleotide substitution, at about 2.49×10-4 nucleotide substitutions per site per year (nt/s/y), with VP1/2 and NS1/2 estimates of 3.06×10-4 and 3.16×10-4 nt/s/y, respectively. Even though no evidence of genetic recombination in these Vietnamese CPV-2c strains was established, potential positive selection sites were observed in both the structural and nonstructural genes, suggesting the viral evolutionary process has occurred in both the structural and nonstructural proteins. Genetic and evolutionary analysis of the full-length genome sequence is necessary to gain evolutionary insight of CPV-2.

Exceptional Enlargement of the Mitochondrial Genome Results from Distinct Causes in Different Rain Frogs (Anura: Brevicipitidae: Breviceps).

The mitochondrial (mt) genome of the bushveld rain frog (Breviceps adspersus, Brevicipitidae, Afrobatrachia) is the largest (28.8 kbp) among the vertebrates investigated to date. The major cause of genome size enlargement in this species is the duplication of multiple genomic regions. To investigate the evolutionary lineage, timing, and process of mt genome enlargement, we sequenced the complete mtDNAs of two congeneric rain frogs, B. mossambicus and B. poweri. The mt genomic organization, gene content, and gene arrangements of these two rain frogs are very similar to each other but differ from those of B. adspersus. The B. mossambicus mt genome (22.5 kbp) does not differ significantly from that of most other afrobatrachians. In contrast, the B. poweri mtDNA (28.1 kbp) is considerably larger: currently the second largest among vertebrates, after B. adspersus. The main causes of genome enlargement differ among Breviceps species. Unusual elongation (12.5 kbp) of the control region (CR), a single major noncoding region of the vertebrate mt genome, is responsible for the extremely large mt genome in B. poweri. Based on the current Breviceps phylogeny and estimated divergence age, it can be concluded that the genome enlargements occurred independently in each species lineage within relatively short periods. Furthermore, a high nucleotide substitution rate and relaxation of selective pressures, which are considered to be involved in changes in genome size, were also detected in afrobatrachian lineages. Our results suggest that these factors were not direct causes but may have indirectly affected mt genome enlargements in Breviceps.

Genetic Characterization and Molecular Evolution of Urban Seoul Virus in Southern China

Seoul virus (SEOV), which causes hemorrhagic fever with renal syndrome (HFRS) in humans, has spread all over the world, especially in mainland China. Understanding basic mechanisms of SEOV evolution is essential to better combat and prevent viral diseases. Here, we examined SEOV prevalence and evolution in the residential area of four districts in Guangzhou city, China. The carriage of SEOV was observed in 33.33% of the sampled rodents, with 35.96% of the sampled Rattus norvegicus and 13.33% of R. tanezumi. Based on the comprehensive analyses of large (L), medium (M), and small (S) segments, our study first demonstrated that the genetic characterization of urban SEOV was shaped by high nucleotide substitution rates, purifying selection, and recombination. Additionally, we detected mutational saturation in the S segment of SEOV, which may lead to the biases of genetic divergence and substitution rates in our study. Importantly, we have filled the gap of SEOV evolution in the urban area. The genetic variation of SEOV may highlight the risk of HFRS, which merits further investigation.

Molecular evolution of mammalian genes with epistatic interactions in fertilization

BackgroundGenes that encode proteins associated with sperm competition, fertilization, and sexual conflicts of interest are often among the most rapidly evolving parts of animal genomes. One family of sperm-expressed genes (Zp3r, C4bpa) in the mammalian gene cluster called the regulator of complement activation (RCA) encodes proteins that bind eggs and mediate reproductive success, and are therefore expected to show high relative rates of nonsynonymous nucleotide substitution in response to sexual selection in comparison to other genes not involved in gamete binding at fertilization. We tested that working hypothesis by using phylogenetic models of codon evolution to identify episodes of diversifying positive selection. We used a comparative approach to quantify the evidence for episodic diversifying selection acting on RCA genes with known functions in fertilization (and sensitivity to sexual selection), and contrast them with other RCA genes in the same gene family that function in innate immunity (and are not sensitive to sexual selection).ResultsWe expected but did not find evidence for more episodes of positive selection on Zp3r in Glires (the rodents and lagomorphs) or on C4BPA in Primates, in comparison to other paralogous RCA genes in the same taxon, or in comparison to the same orthologous RCA gene in the other taxon. That result was not unique to RCA genes: we also found little evidence for more episodes of diversifying selection on genes that encode selective sperm-binding molecules in the egg coat or zona pellucida (Zp2, Zp3) in comparison to members of the same gene family that encode structural elements of the egg coat (Zp1, Zp4). Similarly, we found little evidence for episodic diversifying selection acting on two other recently discovered genes (Juno, Izumo1) that encode essential molecules for sperm–egg fusion.ConclusionsThese negative results help to illustrate the importance of a comparative context for this type of codon model analysis. The results may also point to other phylogenetic contexts in which the effects of selection acting on these fertilization proteins might be more readily discovered and documented in mammals and other taxa.

Feline coronavirus isolates from a part of Brazil: insights into molecular epidemiology and phylogeny inferred from the 7b gene.

The Feline coronavirus (FCoV) can lead to Feline infectious peritonitis (FIP), which the precise cause is still unknown. The theory of internal mutation suggests that a less virulent biotype of FCoV (FECV) would lead to another more pathogenic biotype (FIPV) capable of causing FIP. In this work, the 7b gene was amplified from 51 domestic cat plasma samples by semi-nested PCR and tested through phylogenetic and phylogeographical approaches. The 7b gene of Brazilian isolates displayed high conservation, a strong correlation between the geographic origin of the viral isolates and their genealogy, and its evolution was possibly shaped by a combination of high rates of nucleotide substitution and purifying selection.

Possible risks posed by single-stranded DNA viruses of pigs associated with xenotransplantation.

Routine large‐scale xenotransplantation from pigs to humans is getting closer to clinical reality owing to several state‐of‐the‐art technologies, especially the ability to rapidly engineer genetically defined pigs. However, using pig organs in humans poses risks including unwanted cross‐species transfer of viruses and adaption of these pig viruses to the human organ recipient. Recent developments in the field of virology, including the advent of metagenomic techniques to characterize entire viromes, have led to the identification of a plethora of viruses in many niches. Single‐stranded DNA (ssDNA) viruses are the largest group prevalent in virome studies in mammals. Specifically, the ssDNA viral genomes are characterized by a high rate of nucleotide substitution, which confers a proclivity to adapt to new hosts and cross‐species barriers. Pig‐associated ssDNA viruses include torque teno sus viruses (TTSuV) in the Anelloviridae family, porcine parvoviruses (PPV), and porcine bocaviruses (PBoV) both in the family of Parvoviridae, and porcine circoviruses (PCV) in the Circoviridae family, some of which have been confirmed to be pathogenic to pigs. The risks of these viruses for the human recipient during xenotransplantation procedures are relatively unknown. Based on the scant knowledge available on the prevalence, predilection, and pathogenicity of pig‐associated ssDNA viruses, careful screening and monitoring are required. In the case of positive identification, risk assessments and strategies to eliminate these viruses in xenotransplantation pig stock may be needed.

Madagascar ground gecko genome analysis characterizes asymmetric fates of duplicated genes

BackgroundConventionally, comparison among amniotes – birds, mammals, and reptiles – has often been approached through analyses of mammals and, for comparison, birds. However, birds are morphologically and physiologically derived and, moreover, some parts of their genomes are recognized as difficult to sequence and/or assemble and are thus missing in genome assemblies. Therefore, sequencing the genomes of reptiles would aid comparative studies on amniotes by providing more comprehensive coverage to help understand the molecular mechanisms underpinning evolutionary changes.ResultsHerein, we present the whole genome sequences of the Madagascar ground gecko (Paroedura picta), a promising study system especially in developmental biology, and used it to identify changes in gene repertoire across amniotes. The genome-wide analysis of the Madagascar ground gecko allowed us to reconstruct a comprehensive set of gene phylogenies comprising 13,043 ortholog groups from diverse amniotes. Our study revealed 469 genes retained by some reptiles but absent from available genome-wide sequence data of both mammals and birds. Importantly, these genes, herein collectively designated as ‘elusive’ genes, exhibited high nucleotide substitution rates and uneven intra-genomic distribution. Furthermore, the genomic regions flanking these elusive genes exhibited distinct characteristics that tended to be associated with increased gene density, repeat element density, and GC content.ConclusionThis highly continuous and nearly complete genome assembly of the Madagascar ground gecko will facilitate the use of this species as an experimental animal in diverse fields of biology. Gene repertoire comparisons across amniotes further demonstrated that the fate of a duplicated gene can be affected by the intrinsic properties of its genomic location, which can persist for hundreds of millions of years.

Genome wide analysis of the evolution of Senecavirus A from swine clinical material and assembly yard environmental samples.

Senecavirus A (SVA), previously known as Seneca Valley virus, was first isolated in the United States in 2002. SVA was associated with porcine idiopathic vesicular disease in Canada and the USA in 2007 and 2012, respectively. Recent increase in SVA outbreaks resulting in neonatal mortality of piglets and/or vesicular lesions in sows in Brazil, the USA and Canada point to the necessity to study the pathogenicity and molecular epidemiology of the virus. Here, we report the analysis of the complete coding sequences of SVA from 2 clinical cases and 9 assembly yard environmental samples collected in 2015 in Canada, along with 22 previously released complete genomes in the GenBank. With this combined data set, the evolution of the SVA over a 12-month period in 2015/2016 was evaluated. These SVA isolates were characterized by a rapid accumulation of genetic variations driven mainly by a high nucleotide substitution rate and purifying selection. The SVA sequences clustered in clearly defined geographical areas with reported cases of SVA infection. No transmission links were identified between assembly yards, suggesting that point source introductions may have occurred. In addition, 25 fixed non-synonymous mutations were identified across all analyzed strains when compared to the prototype SVA strain (SVV-001). This study highlights the importance of monitoring SVA mutations for their role in increased virulence and impact on SVA diagnostics.