Effect of vitrification on protein O-GlcNAcylation in mouse metaphase II oocytes.

In brief: Oocyte vitrification leads to DNA hypomethylation, which results in defect in early embryo development. This study reveals that oocyte vitrification impairs the DNA methylation pattern by influencing protein O-GlcNAcylation. Abstract: Oocyte vitrification leads to decreased DNA methylation levels, which impairs the quality and the developmental potential of oocytes. However, the underlying molecular mechanism still need to be further revealed. In this study, mouse metaphase II (M II) oocytes were frozen by vitrification technology, while fresh oocytes were used as the control group. The effect of oocyte vitrification on protein O-GlcNAcylation and its impact on the developmental potential of oocytes were elucidated. We found that the protein O-GlcNAcylation levels were significantly increased in vitrified oocytes. Increase of protein O-GlcNAcylation levels in control oocytes by PUGNAc (an O-GlcNAcase inhibitor) decreases blastocyst rate after parthenogenetic activation (20.82% in PUGNAc-treated group; 53.82% in control group, P < 0.05). We also discovered that DNA methylation was disrupted in two-cell embryos derived from vitrified oocytes, resulting in decreased 5mC and increased 5hmC, showing similar phenotypes to that derived from PUGNAc-treated oocytes. In vitrified and PUGNAc-treated oocytes, O-GlcNAcylated TET3 was significantly increased. Notably, by inhibiting protein O-GlcNAcylation in vitrified oocytes using OSMI1 (an O-GlcNAc transferase inhibitor) we restored the DNA methylation in two-cell embryos and ameliorated the developmental defects in early embryo. Thus, elevated protein O-GlcNAcylation in vitrified oocytes is an essential contributor to their declining embryonic developmental potential. Modulation of protein O-GlcNAcylation improves the developmental potential of vitrified oocytes.

Catalpol ameliorates LPS-induced inflammatory response by activating AMPK/mTOR signaling pathway in rat intestinal epithelial cells.

Intestinal inflammation is a common clinical intestinal disease. Catalpol, a natural iridoid compound, has been shown to have anti-inflammatory, anti-oxidant and anti-apoptotic functions, but the mechanism of its protection against intestinal inflammation is still unclear. This study investigated the protective effect and potential mechanism of catalpol on the lipopolysaccharide (LPS)-induced inflammatory response of intestinal epithelial cell-6 (IEC-6). The results showed that catalpol could inhibit LPS-induced inflammatory response by dose-dependently reducing the release of inflammatory factors, such as tumor necrosis (TNF)-α, interleukin (IL)-1ß and IL-6, and inhibiting the nuclear factor kappa-B (NF-κB) signaling pathway. Catalpol ameliorated cellular oxidative stress by reducing reactive oxygen species (ROS) and malondialdehyde (MDA) levels and increasing superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) expression. Meanwhile, catalpol also inhibited cell apoptosis, decreased the expression of B-cell lymphoma 2 (Bcl-2) - associated X (Bax), caspase 3 and caspase 9, and increased the expression of Bcl-2. This study found that catalpol activates AMP-activated protein kinase (AMPK) signaling pathway and inhibit mammalian target of rapamycin (mTOR) phosphorylationthe. In a further study, after inhibiting AMPK with dorsomorphin, the anti-inflammatory effects of catalpol were significantly reduced. Therefore, catalpol ameliorates LPS-induced inflammatory response by activating AMPK/mTOR signaling pathway in IEC-6 cells.

Mechanisms and effects of novel ammonifying microorganisms on nitrogen ammonification in cow manure waste composting.

It is essential to reduce nitrogen losses and to improve nitrogen conversion during organic waste composting because of environmental protection and sustainable development. To reveal newly domesticated ammonifying microorganisms (AM) cultures on the ammonification and nitrogen conversion during the composting, the screened microbial agents were inoculated at 5 % concentration (in weight basis) into cow manure compost under five different treatments: sterilized distilled water (Control), Amm-1 (mesophilic fungus-F1), Amm-2 (mesophilic bacterium-Z1), Amm-3 (thermotolerant bacterium-Z2), and Amm-4 (consortium: F1, Z1, and Z2), and composted for 42 days. Compared to control, AM inoculation prolonged the thermophilic phases to 9-19 days, increased the content of NH4+-N to 1.60-1.96 g/kg in the thermophilic phase, reduced N2O and NH3 emissions by 22.85-61.13 % and 8.45-23.29 %, increased total Kjeldahl nitrogen, and improved cell count and viability by 12.09-71.33 % and 66.71-72.91 %. AM was significantly associated with different nitrogen and microbial compositions. The structural equation model (SEM) reveals NH4+-N is the preferable nitrogen for the majority of bacterial and fungal growth and that AM is closely associated with the conversion between NH3 and NH4+-N. Among the treatments, inoculation with Amm-4 was more effective, as it significantly enhanced the driving effect of the critical microbial composition on nitrogen conversion and accelerated nitrogen ammonification and sequestration. This study provided new concepts for the dynamics of microbial in the ammonification process of new AM bacterial agents in cow manure compost, and an understanding of the ecological mechanism underlying the ammonification process and its contribution to nitrogen (N) cycling from the perspective of microbial communities.

Cartridge voltage-sensitive micropump immunosensor based on a self-assembled polydopamine coating mediated signal amplification strategy.

Current biosensing detection assays were developed to focus on rapid, low-cost, stable detection for clinical diagnosis and food safety monitoring. In this work, a novel portable cartridge voltage-sensitive micropump immunosensor (CVMS) biosensing device based on the integration of the microchannel circuit biosensing principle and polydopamine (PDA) was presented for rapid and sensitive detection of pathogenic factors in real samples at trace levels. The CVMS can sensitively evaluate voltage signal changes caused by clogging effects in the closed-loop circuit when the insulated microspheres pass through the microchannel. The targets could trigger the immune reaction between antibody-antigens that leads to the change in the concentration of horseradish peroxidase (HRP). And the HRP was further employed to catalyze the polymerization of dopamine into PDA, resulting in the rapid formation of the magnetic @PDA nanoparticles (MNP@PDA) with core-shell structures. The abundant functional groups on the MNP@PDA surface can efficiently adsorb polystyrene microspheres, thus causing changes in the number of polystyrene microspheres (PS). The CVMS can accurately monitor the change of PS with a portable device, which weighs less than 0.8 kg and costs only $50. The completion of CVMS takes 90 min to complete. The limit of detection of this immunosensor for procalcitonin and ochratoxin A were 42 pg/mL and 77 pg/mL, respectively, which improved about 15 folds and 38 folds, respectively, than those of enzyme-linked immunosorbent assay.

Diluent pH affects sperm motility via GSK3 α/ß-hexokinase pathway for the efficient enrichment of X-sperm to increase the female kids rate of dairy goats.

Dairy goats are the goats bred with the ability to produce large quantities of milk, and the increase of the female kid rate of breeding dairy goats is beneficial for milk production and economic benefits of dairy goat farms. Our previous study revealed that regulating the pH of dairy goat semen diluent to 6.2 or 7.4 respectively, the proportion of X chromosome bearing sperm (X-sperm) in the up and down layers of the tube after incubation was significantly higher than that of Y chromosome bearing sperm (Y-sperm) i.e. enriched X-sperm. In this study, fresh dairy goat semen collected in different seasons was diluted in different pH solutions to calculate the number and rate of X-sperm and to measure the functional parameters of enriched sperm. The artificial insemination experiments were performed with enriched X-sperm. The mechanisms of regulating the pH of diluent affecting sperm enrichment were further studied. The results showed that the proportion of enriched X-sperm in pH 6.2 and 7.4 diluents of sperm collected in different seasons showed no significantly different, but were significantly higher than that of the control group (pH 6.8). The in vitro functional parameters of X-sperm enriched in pH 6.2 and 7.4 diluent solution were not significantly different from those of the control group (P > 0.05). After artificial insemination with X-sperm enriched in pH7.4 diluent, the proportion of female offspring was significantly higher than that of the control group. It was found that the regulating pH of the diluent affected sperm mitochondrial activity and glucose uptake capacity via phosphorylating NF-κB and GSK3α/ß proteins. The motility activity of X-sperm was enhanced under acidic conditions and weakened under alkaline conditions, which was conducive to the effective enrichment of X-sperm. This study demonstrated that the number and proportion of X-sperm enriched using pH 7.4 diluent were elevated, and the proportion of female kids was increased. This technology can be used for the reproduction and production of dairy goats in farms at large scales.

Magnetic Relaxation Switching Immunosensors via a Click Chemistry-Mediated Controllable Aggregation Strategy for Direct Detection of Chlorpyrifos.

Chlorpyrifos (CPF) is the most frequently found organophosphate pesticide residue in solid food samples and can cause increasing public concerns about potential risks to human health. Traditional detection signals of such small molecules are mostly generated by target-mediated indirect conversion, which tends to be detrimental to sensitivity and accuracy. Herein, a novel magnetic relaxation switching detection platform was developed for target-mediated direct and sensitive detection of CPF with a controllable aggregation strategy based on a bioorthogonal ligation reaction between tetrazine (Tz) and trans-cyclooctene (TCO) ligands. Under optimal conditions, this sensor can achieve a detection limit of 37 pg/mL with a broad linear range of 0.1-500 ng/mL in 45 min, which is approximately 51-fold lower than that of the gas chromatography analysis and 13-fold lower than that of the enzyme-linked immunosorbent assay. The proposed click chemistry-mediated controllable aggregation strategy is direct, rapid, and sensitive, indicating great potential for residue screening in food matrices.

Alkaline Dilution Alters Sperm Motility in Dairy Goat by Affecting sAC/cAMP/PKA Pathway Activity.

In dairy goat farming, increasing the female kid rate is beneficial to milk production and is, therefore, economically beneficial to farms. Our previous study demonstrated that alkaline incubation enriched the concentration of X-chromosome-bearing sperm; however, the mechanism by which pH affects the motility of X-chromosome-bearing sperm remains unclear. In this study, we explored this mechanism by incubating dairy goat sperm in alkaline dilutions, examining the pattern of changes in sperm internal pH and Ca2+ concentrations and investigating the role of the sAC/cAMP/PKA pathway in influencing sperm motility. The results showed that adding a calcium channel inhibitor during incubation resulted in a concentration-dependent decrease in the proportion of spermatozoa with forward motility, and the sperm sAC protein activity was positively correlated with the calcium ion concentration (r = 0.9972). The total motility activity, proportion of forward motility, and proportion of X-chromosome-bearing sperm decreased (p < 0.05) when cAMP/PKA protease activity was inhibited. Meanwhile, the enrichment of X-chromosome-bearing sperm by pH did not affect the sperm capacitation state. These results indicate that alkaline dilution incubation reduces Ca2+ entry into X-sperm and the motility was slowed down through the sAC/cAMP/PKA signaling pathway, providing a theoretical foundation for further optimization of the sex control method.

Contamination-free V-shaped ultrafast reaction cascade transferase signal amplification driven CRISPR/Cas12a magnetic relaxation switching biosensor for bacteria detection.

Foodborne pathogenic bacteria seriously endanger human health and must be rapidly identified for control. Magnetic relaxation switching biosensors (MRS) are ideal for rapid bacteria detection due to their high signal-to-noise ratio and immunity to sample matrix signal interference. However, conventional MRS still has some challenges in terms of sensitivity, specificity, and stability due to insufficient cross-linking or non-specific binding of magnetic nanoparticles (MNPs) to the target. To address these challenges, we firstly proposed a novel contamination-free uracil-DNA glycosylase (UDG) assisted V-shaped PCR driven CRISPR/Cas12a-MRS (UPC-MRS) biosensor, which combines contamination-free ultrafast nucleic acid amplification and powerful CRISPR/Cas12a system. It has an extremely specific quadruple signal guarantee realized by the merits of UDG anti-contamination, PCR primer specificity matching, the CRISPR/Cas12a system's precise recognition abilities, and magnetic probe signal unaffected by the sample matrix. As a cascade combined with original terminal deoxynucleotidyl transferase (Tdt)-mediated signal amplification technology, this platform can achieve Salmonella detection at concentrations as low as 53 CFU/mL, which is more sensitive than most existing MRS sensors, and it displays accuracy and applicability in real sample detection. This novel UPC-MRS biosensors avoid the common aerosol pollution problem of previous CRISPR/Cas12a systems which after combining with nucleic acid amplification, hence not only offers an alternative toolbox for Salmonella and other pathogen detection with satisfactory specificity and sensitivity, but also has potential for future applications across diverse fields.

Effects of MICU1-Mediated Mitochondrial Calcium Uptake on Energy Metabolism and Quality of Vitrified-Thawed Mouse Metaphase II Oocytes.

BACKGROUND: Oocyte vitrification has been widely used in the treatment of infertility and fertility preservation. However, vitrification-induced mitochondrial damage adversely affects oocyte development. Several studies have reported that mitochondrial calcium uptake protein 1 (MICU1) regulates the uptake of mitochondrial calcium by the mitochondrial calcium uniporter (MCU) and subsequently controls aerobic metabolism and oxidative stress in mitochondria, but research considering oocytes remains unreported. We evaluated whether the addition of MICU1 modulators enhances mitochondrial activity, pyruvate metabolism, and developmental competence after warming of MII oocytes. METHODS: Retrieved MII oocytes of mice were classified as vitrified or control groups. After thawing, oocytes of vitrified group were cultured with or without DS16570511 (MICU1 inhibitor) and MCU-i4 (MICU1 activator) for 2 h. RESULTS: Mitochondrial Ca2+ concentration, pyruvate dephosphorylation level, and MICU1 expression of MII oocytes were significantly increased after vitrification. These phenomena were further exacerbated by the addition of MCU-i4 and reversed by the addition of DS16570511 after warming. However, the mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) in vitrified-warmed MII oocytes drop significantly after vitrification, which was improved after MCU-i4 treatment and decreased significantly after DS16570511 treatment. The vitrification process was able to elicit a development competence reduction. After parthenogenetic activation, incubation of the thawed oocytes with MCU-i4 did not alter the cleavage and blastocyst rates. Moreover, incubation of the thawed oocytes with DS16570511 reduced the cleavage and blastocyst rates. CONCLUSIONS: MICU1-mediated increasing mitochondrial calcium uptake after vitrification of the MII oocytes promoted the pyruvate oxidation, and this process may maintain oocyte development competence by compensating for the consumption of ATP under stress state.

Advancements in mammalian X and Y sperm differences and sex control technology.

Mammal sex determination depends on whether the X sperm or Y sperm binds to the oocyte during fertilization. If the X sperm joins in oocyte, the offspring will be female, if the Y sperm fertilizes, the offspring will be male. Livestock sex control technology has tremendous value for livestock breeding as it can increase the proportion of female offspring and improve the efficiency of livestock production. This review discusses the detailed differences between mammalian X and Y sperm with respect to their morphology, size, and motility in the reproductive tract and in in vitro conditions, as well as 'omics analysis results. Moreover, research progress in mammalian sex control technology has been summarized.

Cattle manure compost humification process by inoculation ammonia-oxidizing bacteria.

The effectiveness of newly isolated ammonia-oxidizing bacteria (AOBs; T-AOB-2, M-AOB-4 and MT-AOB-2-4) in promoting organic matter degradation and humification of cattle manure compost was explored. The results show that, compared with the control, the inoculation of AOBs (5%, v/w) promoted the humification process, particularly in the MT-AOB-2-4, which showed the lowest total organic carbon (19.13%) and dissolved organic carbon (2.61%), whereby humic substances (CEX) and humic acid (CHA) increased to 89.84 g/kg and 85.20 g/kg, and fulvic acid (CFA) decreased to 4.63 g/kg. The high-throughput sequencing and quantitative PCR showed that the abundance of Bacillaceae, amoA and nirS had a significant correlation with humification factors. Among the treatments, the inoculation of MT-AOB-2-4 provided the driving force for the composting process by enhancing the bacterial activity and had the most significant effect on the formation of humic substances and the efficiency of organic matter decomposition.

Effects of Diluent pH on Enrichment and Performance of Dairy Goat X/Y Sperm.

In this paper, on the basis of the differences in the hydrogen ion concentration (pH) of the diluent dairy goat semen on X/Y sperm motility, an X/Y sperm enrichment study was conducted to establish a simple and effective method for gender control in dairy goats. Dairy goat semen was diluted using different pH dilutions and was incubated. Then, the X/Y sperm ratio in the isolated upper sperm was determined using the double TaqMan qPCR method. The internal pH change pattern of sperm cells at different pH dilutions was measured using BCECF-AM probe, and the functional parameters of the isolated sperm were tested with the corresponding kit. Next, an in vitro fertilization test was conducted using isolated spermatozoa and oocytes to determine their fertilization rates, the percentages of female embryos, and the expression of genes related to developing potentially fertilized embryos. Results showed that the percentages of the X sperm cells in the upper sperm layer were 67.24% ± 2.61% at sperm dilution pH of 6.2 and 30.45% ± 1.03% at sperm dilution pH of 7.4, which was significantly different from 52.35% ± 1.72% of the control group (pH 6.8) (P < 0.01). Results also showed that there is a relationship between the external pHo and internal pHi of sperm cells. Furthermore, the percentages of female embryos after the in vitro fertilization of the isolated upper sperm with mature oocytes at pH 6.2 and 7.4 were 66.67% ± 0.05 and 29.73% ± 0.04%, respectively, compared with 48.57% ± 0.02% in the control group (pH 6.8). Highly significant differences occurred between groups (P < 0.01). Additionally, no significant difference was observed during the expression of genes related to embryonic development between the blastocysts formed from sperm isolated by changing the pH of the diluent and the control sperm (P > 0.05). Therefore, this study successfully established a simple and effective method for enriched X/Y sperms from dairy goats, which is important for regulating the desired sex progeny during dairy goat breeding and for guiding dairy goat production.

Effect of oocyte vitrification on glucose transport in mouse metaphase II oocytes.

Oocyte vitrification has significantly improved the survival rate and become the mainstream method for cryopreserving oocytes. Previous studies have demonstrated that the ultrastructure, mitochondrial function, DNA methylation, and histone modification exhibit an irreversible effect after oocyte vitrification. However, little is known about the effects of oocyte vitrification on glucose transport and metabolism. This study aims to determine whether mouse oocyte vitrification causes abnormal glucose metabolism and identify a strategy to correct abnormal glucose metabolism. Furthermore, this study further investigates the effects of oocyte vitrification on glucose uptake, and glucose metabolism, and energy levels. The results indicated that vitrification significantly reduced the glucose transport activity, NADPH, glutathione, and ATP levels, and increased reactive oxygen species levels in oocytes (P < 0.01). Vitrification also reduced the expression of glucose transporter isoform 1 (GLUT1) (P < 0.01). Adding a GLUT1 inhibitor reduced the glucose uptake capacity of oocytes. Furthermore, the inclusion of vitamin C into thawing and culture solutions restored abnormal glucose transportation and metabolism and improved the survival, two-cell embryo, and blastocyst rates of the vitrified groups via parthenogenesis (P < 0.05). Overall, this method may improve the quality and efficiency of oocyte vitrification.

First detection of neboviruses in yak (Bos grunniens) and identification of a novel neboviruses based on complete genome.

Neboviruses (NeVs) is an important causative agent of calf diarrhea. Here, 354 diarrhoeic samples were collected from yak on 55 farms in the Qinghai-Tibet Plateau, China. 22.0% of the diarrhoeic samples were detected as NeVs-positive by RT-PCR assay. Phylogenetic analysis of 78 NeVs RdRp fragments showed that 69 strains were closely related to NB-like strains, and the remaining 9 strains were clustered into an independent branch, which may represent a novel RdRp genotype. Two complete NeVs genomes (YAK/NRG-17/17/CH and YAK/HY1-2/18/CH) were successfully sequenced with 7459 nt and 7460 nt in length, respectively. The genomes of the two strains only shared 68.1%-69.3% nt identity with all six known NeVs genomes, and phylogenetic trees based on its genome, VP1, RdRp, VP2, P34, NTPase, P30, VPg and 3CLpro proteins suggested that the two strains may represent a novel NeVs strain with novel VP1 genotype and novel RdRp genotype. Notably, 11.5% NeVs strains were screened as the novel NeVs strains based VP1 and RdRp sequences. These novel NeVs strains were detected from 6 farms in two counties, indicating that the novel NeVs has spread in local region. To best of our knowledge, this is the first detection of NeVs in yak. Moreover, a novel NeVs strain was identified based on complete genome. These results contribute to further understand the prevalence and genetic evolution of NeVs.

First detection of bovine coronavirus in Yak (Bos grunniens) and a bovine coronavirus genome with a recombinant HE gene.

The yak (Bosgrunniens) is a unique domestic bovine species that plays an indispensable role for herdsmen in the Qinghai-Tibet Plateau. Here, 336 diarrhoeic samples were collected from yaks on 29 farms in the Qinghai-Tibet Plateau from 2015 to 2017. Approximately 69.05 % (232/336) of the diarrhoeic samples were assessed as bovine coronavirus (BCoV)-positive by RT-PCR assay, and most of the detected strains showed a unique evolution based on 40 spike (S), nucleocapsid (N) and haemagglutinin-esterase (HE) gene fragments. Notably, the 12 complete S genes detected shared 1 identical amino acid mutation (E121V) in the S1 subunit compared with the other 150 complete S genes in the GenBank database. Furthermore, a BCoV strain (designated YAK/HY24/CH/2017) was isolated from one diarrhoeic sample (virus titre : 108.17TCID50 ml-1), and a phylogenetic analysis based on complete genome sequences revealed that strain YAK/HY24/CH/2017 has the closest genetic relationship with the BCoV prototype strain Mebus. Interestingly, 2 significant characteristics were observed in the genome of strain YAK/HY24/CH/2017 :  (1) the strain had 26 unique amino acid variations in the S gene compared with the other 150 BCoV S genes in the GenBank database and (2) a recombination event was identified between the esterase and lectin domains of the HE gene. In conclusion, this study revealed the high prevalence of BCoV in yaks in the Qinghai-Tibet Plateau. To the best of our knowledge, this is the first description of the molecular prevalence of BCoV in yaks and of a BCoV genome with an HE gene recombination.

Detection and molecular characteristics of neboviruses in dairy cows in China.

In this study, 98 diarrhoeic and 70 non-diarrhoeic samples were collected from 13 dairy farms located across 5 provinces in China from April 2017 to May 2018. Approximately 41.8 % (41/98) of diarrhoeic samples and 5.7 % (4/70) of non-diarrhoeic samples were nebovirus-positive based on RT-PCR results, and some diarrhoeic samples were co-infected with bovine rotavirus (73.2 %), bovine coronavirus (36.6 %) and/or bovine viral diarrhoea virus (31.7 %). A phylogenetic analysis of 23 nebovirus RdRp fragments showed that these strains were closely related to Nebraska-like (NB-like) strains but were all located in a unique large branch. Moreover, a phylogenetic analysis of the 18 complete VP1 sequences from this study revealed that 14 strains belonged to lineage 1 and 4 strains belonged to lineage 3. Notably, all four lineage 3 strains shared the same recombination event, with a breakpoint located within the P1A domain. The complete genome of one nebovirus strain, Bo/YLA-2/17/CH, which had a recombination event within the P1A domain of its VP1, was successfully sequenced and was found to be 7453 nt in length, and this may represent a novel nebovirus strain based on the phylogenetic analysis of its complete genome sequence. In conclusion, this study reveals that neboviruses circulate widely in dairy cows in China and exhibit a unique evolution of RdRp. To the best of our knowledge, this is the first reported recombination event located within the P1A domain of nebovirus VP1.

Identification and genomic characterization of a novel CRESS DNA virus from a calf with severe hemorrhagic enteritis in China.

In this study, a novel circular replication-associated protein (Rep)-encoding single stranded (CRESS) DNA virus was discovered in diarrheic sample of a calf with severe hemorrhagic enteritis. The virus, named Bo-Circo-like virus CH, has a circular genome with 3909 nucleotides (nt). Six putative open reading frames (ORFs) were identified, including Rep, capsid (Cap) and four proteins of unknown function. Both the genome size and the number as well as the organization of encoded ORFs, Bo-Circo-like virus CH is most closely related to Po-Circo-like virus 21 detected in pig faeces. A preliminary survey using specific primers for the Rep region showed that 5.3% (4/75) of diarrheic samples were positive for Bo-Circo-like virus, and all 42 healthy samples were negative. In conclusion, our results indicate that Bo-Circo-like virus CH may represent a new virus in bovine. Further investigation is needed to determine the relationship between the virus infection and diarrhea.

Genomic characterization of a RdRp-recombinat nebovirus strain with a novel VP1 genotype.

Nebovirus is a new genus within the family Caliciviridae and is a causative agent of calf diarrhea. The limited nebovirus genomic sequences that are currently available has hampered understanding of nebovirus genetic evolution. The aim of the present study was to determine the genomic characterization of strain Bo/LZB-1/17/CH, which was previously identified as being similar to the novel genotype strain Bo/DijonA216/06/FR based on partial capsid sequences. Our results show that the complete RNA genome of strain Bo/LZB-1/17/CH is 7453 nucleotides (nt) in length and shares 79.0%-83.5% nt identity with all available nebovirus genomes in the GenBank database. A phylogenetic analysis based on its complete genome sequence revealed that strain Bo/LZB-1/17/CH clustered into an independent branch. Two interesting characteristics were observed in the genome of strain Bo/LZB-1/17/CH. First, the major capsid protein (VP1) of strain Bo/LZB-1/17/CH shares 96.6% amino acid (aa) identity with strain Bo/DijonA216/06/FR but shares only 75.2%-76.8% aa identity with other nebovirus strains and has an even lower identity in the P2 domain (61.1%-65% aa identity). Second, the RNA-dependent RNA polymerase (RdRp) of strain Bo/LZB-1/17/CH is more closely related to NB-like strains than it is to strain Bo/DijonA216/06/FR, and a recombination event was identified within the 3' end of the RdRp in strain Bo/LZB-1/17/CH. In conclusion, the results in this study indicate that strain Bo/LZB-1/17/CH may represent a novel nebovirus strain. To the best of our knowledge, this is the first description of a recombinant event in nebovirus RdRp.

First detection of Nebovirus and Norovirus from cattle in China.

Neboviruses and genogroup III noroviruses (NoVsGIII) are causative agents of calf diarrhea. The purpose of this study was to investigate the presence of neboviruses and noroviruses in cattle in China. Twenty-eight diarrhea fecal samples collected from 5 different farms were analyzed by RT-PCR. The results showed that 3 nebovirus positive samples were detected on 2 farms, with two strains being related to Bo/DijonA216/06/FR strain and the other one clustering with NB-like strains. Meanwhile, 3 norovirus positive samples were detected on 3 farms, all of which belonged to genotype 1. Our results confirmed the presence of neboviruses and NoVsGIII in China for the first time, and supported the presence of a novel "DijonA216-like" nebovirus genotype.