RNA m6A modification regulates cell fate transition between pluripotent stem cells and 2-cell-like cells.

N6-methyladenosine (m6A) exerts essential roles in early embryos, especially in the maternal-to-zygotic transition stage. However, the landscape and roles of RNA m6A modification during the transition between pluripotent stem cells and 2-cell-like (2C-like) cells remain elusive. Here, we utilised ultralow-input RNA m6A immunoprecipitation to depict the dynamic picture of transcriptome-wide m6A modifications during 2C-like transitions. We found that RNA m6A modification was preferentially enriched in zygotic genome activation (ZGA) transcripts and MERVL with high expression levels in 2C-like cells. During the exit of the 2C-like state, m6A facilitated the silencing of ZGA genes and MERVL. Notably, inhibition of m6A methyltransferase METTL3 and m6A reader protein IGF2BP2 is capable of significantly delaying 2C-like state exit and expanding 2C-like cells population. Together, our study reveals the critical roles of RNA m6A modification in the transition between 2C-like and pluripotent states, facilitating the study of totipotency and cell fate decision in the future.

Menin orchestrates hepatic glucose and fatty acid uptake via deploying the cellular translocation of SIRT1 and PPARγ.

BACKGROUND: Menin is a scaffold protein encoded by the Men1 gene, which interacts with various transcriptional proteins to activate or repress cellular processes and is a key mediator in multiple organs. Both liver-specific and hepatocyte-specific Menin deficiency promotes high-fat diet-induced liver steatosis in mice, as well as insulin resistance and type 2 diabetic phenotype. The potential link between Menin and hepatic metabolism homeostasis may provide new insights into the mechanism of fatty liver disease. RESULTS: Disturbance of hepatic Menin expression impacts metabolic pathways associated with non-alcoholic fatty liver disease (NAFLD), including the FoxO signaling pathway, which is similar to that observed in both oleic acid-induced fatty hepatocytes model and biopsied fatty liver tissues, but with elevated hepatic Menin expression and inhibited FABP1. Higher levels of Menin facilitate glucose uptake while restraining fatty acid uptake. Menin targets the expression of FABP3/4/5 and also CD36 or GK, PCK by binding to their promoter regions, while recruiting and deploying the cellular localization of PPARγ and SIRT1 in the nucleus and cytoplasm. Accordingly, Menin binds to PPARγ and/or FoxO1 in hepatocytes, and orchestrates hepatic glucose and fatty acid uptake by recruiting SIRT1. CONCLUSION: Menin plays an orchestration role as a transcriptional activator and/or repressor to target downstream gene expression levels involved in hepatic energy uptake by interacting with the cellular energy sensor SIRT1, PPARγ, and/or FoxO1 and deploying their translocations between the cytoplasm and nucleus, thereby maintaining metabolic homeostasis. These findings provide more evidence suggesting Menin could be targeted for the treatment of hepatic steatosis, NAFLD or metabolic dysfunction-associated fatty liver disease (MAFLD), and even other hepatic diseases.

WD repeat domain 82 (Wdr82) facilitates mouse iPSCs generation by interfering mitochondrial oxidative phosphorylation and glycolysis.

BACKGROUND: Abundantly expressed factors in the oocyte cytoplasm can remarkably reprogram terminally differentiated germ cells or somatic cells into totipotent state within a short time. However, the mechanism of the different factors underlying the reprogramming process remains uncertain. METHODS: On the basis of Yamanaka factors OSKM induction method, MEF cells were induced and reprogrammed into iPSCs under conditions of the oocyte-derived factor Wdr82 overexpression and/or knockdown, so as to assess the reprogramming efficiency. Meanwhile, the cellular metabolism was monitored and evaluated during the reprogramming process. The plurpotency of the generated iPSCs was confirmed via pluripotent gene expression detection, embryoid body differentiation and chimeric mouse experiment. RESULTS: Here, we show that the oocyte-derived factor Wdr82 promotes the efficiency of MEF reprogramming into iPSCs to a greater degree than the Yamanaka factors OSKM. The Wdr82-expressing iPSC line showed pluripotency to differentiate and transmit genetic material to chimeric offsprings. In contrast, the knocking down of Wdr82 can significantly reduce the efficiency of somatic cell reprogramming. We further demonstrate that the significant suppression of oxidative phosphorylation in mitochondria underlies the molecular mechanism by which Wdr82 promotes the efficiency of somatic cell reprogramming. Our study suggests a link between mitochondrial energy metabolism remodeling and cell fate transition or stem cell function maintenance, which might shed light on the embryonic development and stem cell biology.

Multi-omics analyses reveal that the gut microbiome and its metabolites promote milk fat synthesis in Zhongdian yak cows.

Background: Yak cows produce higher quality milk with higher concentrations of milk fat than dairy cows. Recently, studies have found the yak milk yield and milk fat percentage have decreased significantly over the past decade, highlighting the urgency for yak milk improvement. Therefore, we aimed to analyze how the gut microbiome impacts milk fat synthesis in Zhongdian yak cows. Methods: We collected milk samples from Zhongdian yak cows and analyzed the milk fat percentage, selecting five Zhongdian yak cows with a very high milk fat percentage (>7%, 8.70 ± 1.89%, H group) and five Zhongdian yak cows with a very low milk fat percentage (<5%, 4.12 ± 0.43%, L group), and then obtained gut samples of these ten Zhongdian yak cows through rectal palpation. Gut metagenomics, metabolomics, and conjoint metagenomics and metabolomics analyses were performed on these samples, identifying taxonomic changes, functional changes, and changes in gut microbes-metabolite interactions within the milk fat synthesis-associated Zhongdian yak cows gut microbiome, to identify potential regulatory mechanisms of milk fat at the gut microbiome level in Zhongdian yak cows. Results: The metagenomics analysis revealed Firmicutes and Proteobacteria were significantly more abundant in the gut of the high-milk fat Zhongdian yak cows. These bacteria are involved in the biosynthesis of unsaturated fatty acids and amino acids, leading to greater efficiency in converting energy to milk fat. The metabolomics analysis showed that the elevated gut metabolites in high milk fat percentage Zhongdian yak cows were mainly enriched in lipid and amino acid metabolism. Using a combined metagenomic and metabolomics analysis, positive correlations between Firmicutes (Desulfocucumis, Anaerotignum, Dolosiccus) and myristic acid, and Proteobacteria (Catenovulum, Comamonas, Rubrivivax, Marivita, Succinimouas) and choline were found in the gut of Zhongdian yak cows. These interactions may be the main contributors to methanogen inhibition, producing less methane leading to higher-efficient milk fat production. Conclusions: A study of the gut microbe, gut metabolites, and milk fat percentage of Zhongdian yak cows revealed that the variations in milk fat percentage between yak cows may be caused by the gut microbes and their metabolites, especially Firmicutes-myristic acid and Proteobacteria-choline interactions, which are important to milk fat synthesis. Our study provides new insights into the functional roles of the gut microbiome in producing small molecule metabolites and contributing to milk performance traits in yak cows.

Multi-Channel Metabolomics Analysis Identifies Novel Metabolite Biomarkers for the Early Detection of Fatty Liver Disease in Dairy Cows.

Fatty liver disease, a type of metabolic disorder, frequently occurs in dairy cows during the parturition period, causing a high culling rate and, therefore, considerable economic losses in the dairy industry owing to the lack of effective diagnostic methods. Here, metabolite biomarkers were identified and validated for the diagnosis of metabolic disorders. A total of 58 participant cows, including severe fatty liver disease and normal control groups, in the discovery set (liver biopsy tested, n = 18), test set (suspected, n = 20) and verification set (liver biopsy tested, n = 20), were strictly recruited and a sample collected for their feces, urine, and serum. Non-targeted GC-MS-based metabolomics methods were used to characterize the metabolite profiles and to screen in the discovery set. Eventually, ten novel biomarkers involved in bile acid, amino acid, and fatty acid were identified and validated in the test set. Each of them had a higher diagnostic ability than the traditional serum biochemical indicators, with an average area under the receiver operating characteristic curve of 0.830 ± 0.0439 (n = 10) versus 0.377 ± 0.182 (n = 9). Especially, combined biomarker panels via different metabolic pipelines had much better diagnostic sensitivity and specificity than every single biomarker, suggesting their powerful utilization potentiality for the early detection of fatty liver disease. Intriguingly, the serum biomarkers were confirmed perfectly in the verification set. Moreover, common biological pathways were found to be underlying the pathogenesis of fatty liver syndrome in cattle via different metabolic pipelines. These newly-discovered and non-invasive metabolic biomarkers are meaningful in reducing the high culling rate of cows and, therefore, benefit the sustainable development of the dairy industry.

Menin regulates lipid deposition in mouse hepatocytes via interacting with transcription factor FoxO1.

Non-alcoholic fatty liver disease (NAFLD) is rapidly being recognized as the leading cause of chronic liver disease worldwide. Men1, encoding protein of menin, is a key causative gene of multiple endocrine neoplasia type 1 syndrome including pancreatic tumor. It is known that insulin that secretes by endocrine tissue pancreatic islets plays a critical role in hepatic metabolism. Mouse model of hemizygous deletion of Men1 was shown to have severe hepatic metabolism disorders. However, the molecular function of menin on lipid deposition in hepatocytes needs to be further studied. Transcriptome sequencing does show that expression suppression of Men1 in mouse hepatocytes widely affect signaling pathways involved in hepatic metabolism, such as fatty acid metabolism, insulin response, glucose metabolism and inflammation. Further molecular studies indicates that menin overexpression inhibits expressions of the fat synthesis genes Srebp-1c, Fas, and Acc1, the fat differentiation genes Pparγ1 and Pparγ2, and the fat transport gene Cd36, thereby inhibiting the fat accumulation in hepatocytes. The biological process of menin regulating hepatic lipid metabolism was accomplished by interacting with the transcription factor FoxO1, which is also found to be critical for lipid metabolism. Moreover, menin responds to insulin in hepatocytes and mediates its regulatory effect on hepatic metabolism. Our findings suggest that menin is a crucial mediation factor in regulating the hepatic fat deposition, suggesting it could be a potential important therapeutic target for NAFLD.

Inducing somatic cells into pluripotent stem cells is an important platform to study the mechanism of early embryonic development.

The early embryonic development starts with the totipotent zygote upon fertilization of differentiated sperm and egg, which undergoes a range of reprogramming and transformation to acquire pluripotency. Induced pluripotent stem cells (iPSCs), a nonclonal technique to produce stem cells, are originated from differentiated somatic cells via accomplishment of cell reprogramming, which shares common reprogramming process with early embryonic development. iPSCs are attractive in recent years due to the potentially significant applications in disease modeling, potential value in genetic improvement of husbandry animal, regenerative medicine, and drug screening. This review focuses on introducing the research advance of both somatic cell reprogramming and early embryonic development, indicating that the mechanisms of iPSCs also shares common features with that of early embryonic development in several aspects, such as germ cell factors, DNA methylation, histone modification, and/or X chromosome inactivation. As iPSCs can successfully avoid ethical concerns that are naturally present in the embryos and/or embryonic stem cells, the practicality of somatic cell reprogramming (iPSCs) could provide an insightful platform to elucidate the mechanisms underlying the early embryonic development.

Proteome analysis identified proteins associated with mitochondrial function and inflammation activation crucially regulating the pathogenesis of fatty liver disease.

BACKGROUND: Fatty liver disease prevalently occurs in commercial postpartum dairies, resulting in a worldwide high culling rate because of their subsequent limitations of production and reproduction performance. RESULTS: Fatty liver-specific proteome and acetylome analysis revealed that energy metabolism suppression closely associated with mitochondrial dysfunction and inflammation activation were shown to be remarkable biological processes underlying the development of fatty liver disease, furthermore, acetylation modification of proteins could be one of the main means to modulate these processes. Twenty pivotal genetic factors/genes that differentially expressing and being acetylation modified in liver were identified and proposed to regulate the pathogenesis of fatty liver dairies. These proteins were confirmed to be differentially expressing in individual liver tissue, eight of which being validated via immunohistochemistry assay. CONCLUSIONS: This study provided a comprehensive proteome and acetylome profile of fatty liver of dairy cows, and revealed potential important biological processes and essential regulators in the pathogenesis of fatty liver disease. Expectantly, understanding the molecular mechanisms of the pathogenesis of fatty liver disease in dairies, as an animal model of non-alcoholic fatty liver disease (NAFLD) in human beings, which is a clinico-pathologically defined process associated with metabolic syndrome, could inspire and facilitate the development of efficacious therapeutic drugs on NAFLD.

AGPAT3 Gene polymorphisms are associated with milk production traits in Chinese Holstein cows.

The current study reports the identification of previously undiscovered single-nucleotide polymorphisms (SNPs) in the bovine AGPAT3 gene and further investigates their associations with milk production traits. Our results demonstrate that the major allele C of the SNP g.12264 C > T is positively correlated with test-day milk yield, protein percentage and 305-day milk yield. Importantly, in silico analysis showed that the C/T transition at this locus gives rise to two new transcription factor binding sites (TFBS), E2F1 and Nkx3-2. Polymorphism g.18658 G > A was the only SNP associated with milk urea nitrogen (MUN) with the G allele related to an increase in milk urea nitrogen as well as fat percentage. The GG genotype of SNP g.28731 A > G was associated with the highest fat and protein percentage and lowest 305-day milk yield and somatic cell score (SCS). The association between AGPAT3 locus and milk production traits could be utilized in marker-assisted selection for the genetic improvement of milk production traits and, probably in conjunction with other traits, for selection to improve fitness of dairy cattle.

Dcaf11 activates Zscan4-mediated alternative telomere lengthening in early embryos and embryonic stem cells.

Telomeres play vital roles in ensuring chromosome stability and are thus closely linked with the onset of aging and human disease. Telomeres undergo extensive lengthening during early embryogenesis. However, the detailed molecular mechanism of telomere resetting in early embryos remains unknown. Here, we show that Dcaf11 (Ddb1- and Cul4-associated factor 11) participates in telomere elongation in early embryos and 2-cell-like embryonic stem cells (ESCs). The deletion of Dcaf11 in embryos and ESCs leads to reduced telomere sister-chromatid exchange (T-SCE) and impairs telomere lengthening. Importantly, Dcaf11-deficient mice exhibit gradual telomere erosion with successive generations, and hematopoietic stem cell (HSC) activity is also greatly compromised. Mechanistically, Dcaf11 targets Kap1 (KRAB-associated protein 1) for ubiquitination-mediated degradation, leading to the activation of Zscan4 downstream enhancer and the removal of heterochromatic H3K9me3 at telomere/subtelomere regions. Our study therefore demonstrates that Dcaf11 plays important roles in telomere elongation in early embryos and ESCs through activating Zscan4.

TGF-ß1 controls porcine granulosa cell states: A miRNA-mRNA network view.

TGF-ß1, an important multi-functional cytokine of the TGF-ß signaling pathway, has been reported to be crucial for ovarian granulosa cell (GC) states and female fertility. However, the molecular mechanism underlying TGF-ß1 regulation of GC states remains largely unknown. Here, we provide a comprehensive transcriptomic view on TGF-ß1 regulation of cell states in porcine GCs. We first confirmed that TGF-ß1 can control GC states (apoptosis and proliferation) in pig ovary. RNA-seq showed that 909 differentially expressed genes (DEGs), including 890 DEmRNAs and 19 DEmiRNAs, were identified in TGF-ß1-treated porcine GCs. Functional annotation showed that these DEGs were mainly involved in regulating cell states. In addition, multiple hub genes were identified by constructing the protein-protein interaction network, DEmiRNA-DEmRNAs regulatory network, and gene-pathway-function co-expression networks, which were further found to be enriched in FoxO, TGF-ß, Wnt, PIK3-Akt, p53 and Ras signaling pathways that play important roles in regulating cell states, cell cycle, proliferation, stress-responses and inflammation. The current research deeply reveals the effects of TGF-ß1 on porcine GCs, and also identifies potential therapeutic RNA molecules for inhibiting and rescuing female infertility.

Identification of Crucial Genetic Factors, Such as PPARγ, that Regulate the Pathogenesis of Fatty Liver Disease in Dairy Cows Is Imperative for the Sustainable Development of Dairy Industry.

Frequently occurring fatty liver disease in dairy cows during the perinatal period, a typical type of non-alcoholic fatty liver disease (NAFLD), results in worldwide high culling rates of dairy cows (averagely about 25%) after calving. This has been developing into a critical industrial problem throughout the world, because the metabolic disease severely affects the welfare and economic value of dairy cows. Findings about the molecular mechanisms how the fatty liver disease develops would help scientists to discover novel therapeutic targets for NAFLD. Studies have shown that PPARγ participates or regulates the fat deposition in liver by affecting the biological processes of hepatic lipid metabolism, insulin resistance, gluconeogenesis, oxidative stress, endoplasmic reticulum stress and inflammation, which all contribute to fatty liver. This review mainly focuses on crucial regulatory mechanisms of PPARγ regulating lipid deposition in the liver via direct and/or indirect pathways, suggesting that PPARγ might be a potential critical therapeutic target for fatty liver disease, however, it would be of our significant interest to reveal the pathology and pathogenesis of NAFLD by using dairy cows with fatty liver as an animal model. This review will provide a molecular mechanism basis for understanding the pathogenesis of NAFLD.

Identification of Whole-Genome Significant Single Nucleotide Polymorphisms in Candidate Genes Associated With Serum Biochemical Traits in Chinese Holstein Cattle.

A genome-wide association study (GWAS) was conducted on 23 serum biochemical traits in Chinese Holstein cattle. The experimental population consisted of 399 cattle, each genotyped by a commercial bovine 50K SNP chip, which had 49,663 SNPs. After data cleaning, 41,092 SNPs from 361 Holstein cattle were retained for GWAS. The phenotypes were measured values of serum measurements of these animals that were taken at 11 days after parturition. Two statistical models, a fixed-effect linear regression model (FLM) and a mixed-effect linear model (MLM), were used to estimate the association effects of SNPs. Genome-wide significant and suggestive thresholds were set up to be 1.22E-06 and 2.43E-06, respectively. In the Chinese Holstein population, FLM identified 81 genome-wide significant (0.05/41,092 = 1.22E-06) SNPs associated with 11 serum traits. Among these SNPs, five SNPs (BovineHD0100005950, ARS-BFGL-NGS-115158, BovineHD1500021175, BovineHD0800028900, and BTB-00442438) were also identified by the MLM to have genome-wide suggestive effects on CHE, DBIL, and LDL. Both statistical models pinpointed two SNPs that had significant effects on the Holstein population. The SNP BovineHD0800028900 (located near the gene LOC101903458 on chromosome 8) was identified to be significantly associated with serum high- and low-density lipoprotein (HDL and LDL), whereas BovineHD1500021175 (located in 73.4Mb on chromosome 15) was an SNP significantly associated with total bilirubin and direct bilirubin (TBIL and DBIL). Further analyses are needed to identify the causal mutations affecting serum traits and to investigate the correlation of effects for loci associated with fatty liver disease in dairy cattle.

MiR-24-3p regulates cell proliferation and milk protein synthesis of mammary epithelial cells through menin in dairy cows.

MiR-24-3p, a broadly conserved, small, noncoding RNA, is abundantly expressed in mammary tissue. However, its regulatory role in this tissue remains poorly understood. It was predicted that miR-24-3p targets the 3' untranslated region (3'-UTR) of multiple endocrine neoplasia type 1 (MEN1), an important regulatory factor in mammary tissue. The objective of this study was to investigate the function of miR-24-3p in mammary cells. Using a luciferase assay in mammary epithelial cells (MAC-T), miR-24-3p was confirmed to target the 3'-UTR of MEN1. Furthermore, miR-24-3p negatively regulated the expression of the MEN1 gene and its encoded protein, menin. miR-24-3p enhanced proliferation of MAC-T by promoting G1/S phase progression. MiR-24-3p also regulated the expression of key factors involved in phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin and Janus kinase/signal transducer and activators of transcription signaling pathways, therefore controlling milk protein synthesis in epithelial cells. Thus, miR-24-3p appears to act on MAC-T by targeting MEN1. The expression of miR-24-3p was controlled by MEN1/menin, indicating a negative feedback loop between miR-24-3p and MEN1/menin. The negatively inhibited expression pattern of miR-24-3p and MEN1 was active in mammary tissues at different lactation stages. The feedback mechanism is a new concept to further understand the lactation cycle of mammary glands and can possibly to be manipulated to improve milk yield and quality.

Effect of hay supplementation timing on rumen microbiota in suckling calves.

An animal feeding trial was conducted on 18 seven-day-old Holstein dairy bull calves weighing 42 ± 3 kg each. Calves were randomly assigned into three groups (n = 6 each). The dietary treatments were as follows: (1) milk and starter for the control group (MS), (2) supplementation of oat hay from week 2 on the basis of milk and starter (MSO2), and (3) supplementation of oat hay from week 6 on the basis of milk and starter (MSO6). All animals were fed starter and oat hay ad libitum. The major phyla in the different groups of rumen fluid included Firmicutes, Actinobacteria, Bacteroidetes, Proteobacteria, and Euryarchaeota. The major genera were identified, and major genera proportions in the three groups were as follows: Methanobrevibacter (Euryarchaeota), 2.1%, 1.7%, and 2.1%; Olsenella (Actinobacteria), 23.9%, 17.7%, and 12.8%; Prevotella (Bacteroidetes), 10.5%, 16.5%, and 19.2%; Dialister (Firmicutes), 3.3%, 4.1%, and 2.8%; Succiniclasticum (Firmicutes), 3.8%, 4.7%, and 9.2%; and Sharpea (Firmicutes), 0.4%, 2.5%, and 0.2%, respectively. There were no significant differences in the various phyla among the three groups (p > .05). The results showed that calves hay supplementation time did not affect the diversity of the rumen microbiota in the suckling calves. However, the hay supplementation altered the proportion of the various microbial populations, supplementation of oat hay from week 2 on the basis of milk and starter could improve calves rumen pH.

Menin Modulates Mammary Epithelial Cell Numbers in Bovine Mammary Glands Through Cyclin D1.

Menin, the protein encoded by the MEN1 gene, is abundantly expressed in the epithelial cells of mammary glands. Here, we found MEN1/menin expression slowly decreased with advancing lactation but increased by the end of lactation. It happened that the number of bovine mammary epithelial cells decreases since lactation, suggesting a role of menin in the control of mammary epithelial cell growth. Indeed, reduction of menin expression through MEN1-specific siRNA transfection in the bovine mammary epithelial cells caused cell growth arrest in G1/S phase. Decreased mRNA and protein expression of Cyclin D1 was observed upon MEN1 knockdown. Furthermore, menin was confirmed to physically bind to the promoter region of Cyclin D1 through a ChIP assay, indicating that menin plays a regulatory role in mammary epithelial cell cycle progression. Moreover, lower expression of MEN1/menin induced increased epithelial cell apoptosis and caused extracellular matrix remodeling by down-regulating its associated genes, such as DSG2 and KRT5, suggesting that menin's role may also be involved in the control of cell-cell adhesion in normal mammary glands. Taken together, our data revealed an unknown molecular function of menin in epithelial cell proliferation, which may be important in the regulation of lactation behavior of mammary glands.

MEN1/Menin regulates milk protein synthesis through mTOR signaling in mammary epithelial cells.

The MEN1 gene, which encodes the protein Menin, was investigated for its regulatory role in milk protein synthesis in mammary glands. Menin responds to nutrient and hormone levels via the PI3K/Akt/mTOR pathway. Bovine mammary epithelial cells and tissues were used as experimental models in this study. The results revealed that the milk protein synthesis capacity of mammary epithelial cells could be regulated by MEN1/Menin. The overexpression of Menin caused significant suppression of factors involved in the mTOR pathway, as well as milk protein κ-casein (CSNK). In contrast, a significant increase in these factors and CSNK was observed upon MEN1/Menin knockdown. The repression of MEN1/Menin on the mTOR pathway was also observed in mammary gland tissues. Additionally, MEN1/Menin was found to elicit a negative response on prolactin (PRL) and/or insulin (INS), which caused a similar downstream impact on mTOR pathway factors and milk proteins. Collectively, our data indicate that MEN1/Menin could play a regulatory role in milk protein synthesis through mTOR signaling in the mammary gland by mediating the effects of hormones and nutrient status. The discovery of Menin's role in mammary glands suggests Menin could be potential new target for the improvement of milk performance and adjustment of lactation period of dairy cows.

Structural alteration of a BYDV-like translation element (BTE) that attenuates p35 expression in three mild Tobacco bushy top virus isolates.

To identify the molecular effects of Tobacco bushy top virus (TBTV) evolution on the degeneration of tobacco bushy top disease, three TBTV isolates with mild virulence were compared with wild-type TBTV to assess the translation of p35, which relies on a BYDV-like translation element (BTE) in a cap-independent manner. The in vitro expression of p35 in the mild isolates was only 20% to 40% of the expression observed in wt TBTV. Based on translation data from chimeric TBTV RNA, low-level p35 expression in the three mild isolates was associated with two regions: the 5' terminal 500 nt region (RI) and the 3' internal region (RV), which included the BTE. For the RV region, low level p35 expression was mainly associated with structural alterations of the BTE instead of specific sequence mutations within the BTE based on SHAPE structural probing and mutation analysis. Additionally, structural alteration of the TBTV BTE resulted from mutations outside of the BTE, implying structural complexity of the local region surrounding the BTE. This study is the first report on the structural alteration of the 3' cap-independent translation element among different isolates of a given RNA virus, which is associated with variations in viral virulence.

Identification of three new isolates of Tomato spotted wilt virus from different hosts in China: molecular diversity, phylogenetic and recombination analyses.

BACKGROUND: Destructive diseases caused by Tomato spotted wilt virus (TSWV) have been reported associated with many important plants worldwide. Recently, TSWV was reported to infect different hosts in China. It is of value to clone TSWV isolates from different hosts and examine diversity and evolution among different TSWV isolates in China as well as worldwide. METHODS: RT-PCR was used to clone the full-length genome (L, M and S segments) of three new isolates of TSWV that infected different hosts (tobacco, red pepper and green pepper) in China. Identity of nucleotide and amino acid sequences among TSWV isolates were analyzed by DNAMAN. MEGA 5.0 was used to construct phylogenetic trees. RDP4 was used to detect recombination events during evolution of these isolates. RESULTS: Whole-genome sequences of three new TSWV isolates in China were determined. Together with other available isolates, 29 RNA L, 62 RNA M and 66 RNA S of TSWV isolates were analyzed for molecular diversity, phylogenetic and recombination events. This analysis revealed that the entire TSWV genome, especially the M and S RNAs, had major variations in genomic size that mainly involve the A-U rich intergenic region (IGR). Phylogenetic analyses on TSWV isolates worldwide revealed evidence for frequent reassortments in the evolution of tripartite negative-sense RNA genome. Significant numbers of recombination events with apparent 5' regional preference were detected among TSWV isolates worldwide. Moreover, TSWV isolates with similar recombination events usually had closer relationships in phylogenetic trees. CONCLUSIONS: All five Chinese TSWV isolates including three TSWV isolates of this study and previously reported two isolates can be divided into two groups with different origins based on molecular diversity and phylogenetic analysis. During their evolution, both reassortment and recombination played roles. These results suggest that recombination could be an important mechanism in the evolution of multipartite RNA viruses, even negative-sense RNA viruses.