Identification of a novel enterovirus E isolates HY12 from cattle with severe respiratory and enteric diseases.

In this study, a virus strain designated as HY12 was isolated from cattle with a disease of high morbidity and mortality in Jilin province. Biological and physiochemical properties showed that HY12 isolates is cytopathic with an extremely high infectivity. HY12 is resistant to treatment of organic solvent and acid, and unstable at 60°C for 1 h. Electron microscopy observation revealed the virus is an approximately 22-28 nm in diameter. The complete genome sequence of HY12 consists of 7416 nucleotides, with a typical picornavirus genome organization including a 5'-untranslated region (UTR), a large single ORF encoding a polyprotein of 2176 amino acids, and a 3'-UTR. Phylogenetic analysis clustered HY12 isolates to a new serotype/genotype within the clade of enterovirus E (formerly BEV-A). Alignment analysis revealed a unique insertion of 2 amino acid residues (NF) at the C-terminal of VP1 protein between aa 825 and 826, and several rare mutations in VP1 and VP4 of HY12 isolates in relation to known bovine enterovirus (BEV) strains. This is the first report of an enterovirus E in China, which is potentially associated with an outbreak in cattle with severe respiratory and enteric diseases

A neonatal murine model for evaluation of enterovirus E HY12 virus infection and pathogenicity.

HY12 viruses are enteroviruses recently isolated from cattle characterized by severe respiratory and digestive disease with high morbidity and mortality in China. While the viruses exhibit unique biological and molecular characters distinct from known enterovirus E, the pathogenicity and viral pathogenesis remains largely unknown.Neonatal mice of Balb/C, ICR, and Kunming strain are infected with HY12 to determine the susceptible mouse strain. The minimal infection dose, the virus infection routes, the pathogenicity and tissue tropism for HY12 were determined by infecting susceptible mice with HY12 viruses, and confirmed by different approaches including virus isolation and recovery, virus detection, histopathology, and immunohistochemistry.A murine model for HY12 infection was successfully established and employed to investigate the pathogenicity of HY12 viruses. ICR mouse strain is the most susceptible strain for HY12 infection with a minimal infective dose as 2×106TCID50/mouse. HY12 viruses have the capability of infecting ICR suckling mice via all infection routes including intranasal administration, oral administration, intraperitoneal injection, subcutaneous injection, and intramuscular injection, which are confirmed by the isolation and recovery of viruses from HY12-infected mice; detection of viruses by RT-PCR; observations of pathological lesions and inflammatory cell infiltrations in the intestine, lung, liver, and brain; uncovering of HY12 virus antigens in majority of tissues, especially in intestine, lung, and infected brain of mice by immunohistochemistry assay.A neonatal murine model for HY12 infection is successfully established for determining the susceptible mouse strain, the minimal infective dose, the infection route, the viral pathogenicity and the tropism of HY12, thus providing an invaluable model system for elucidating the pathogenesis of HY12 viruses and the elicited immunity

Full Convolution Neural Network Combined with Contextual Feature Representation for Cropland Extraction from High-Resolution Remote Sensing Images

The quantity and quality of cropland are the key to ensuring the sustainable development of national agriculture. Remote sensing technology can accurately and timely detect the surface information, and objectively reflect the state and changes of the ground objects. Using high-resolution remote sensing images to accurately extract cropland is the basic task of precision agriculture. The traditional model of cropland semantic segmentation based on the deep learning network is to down-sample high-resolution feature maps to low resolution, and then restore from low-resolution feature maps to high-resolution ideas; that is, obtain low-resolution feature maps through a network, and then recover to high resolution by up-sampling or deconvolution. This will bring about the loss of features, and the segmented image will be more fragmented, without very clear and smooth boundaries. A new methodology for the effective and accurate semantic segmentation cropland of high spatial resolution remote sensing images is presented in this paper. First, a multi-temporal sub-meter cropland sample dataset is automatically constructed based on the prior result data. Then, a fully convolutional neural network combined with contextual feature representation (HRNet-CFR) is improved to complete the extraction of cropland. Finally, the initial semantic segmentation results are optimized by the morphological post-processing approach, and the broken spots are ablated to obtain the internal homogeneous cropland. The proposed method has been validated on the Jilin-1 data and Gaofen Image Dataset (GID) public datasets, and the experimental results demonstrate that it outperforms the state-of-the-art method in cropland extraction accuracy. We selected the comparison of Deeplabv3+ and UPerNet methods in GID. The overall accuracy of our approach is 92.03%, which is 3.4% higher than Deeplabv3+ and 5.12% higher than UperNet

Unveiling of Evolution Pattern for HY12 Enterovirus Quasispecies and Pathogenicity Alteration

Enterovirus, like the majority of RNA viruses, evolves to survive the changeable environments by a variety of strategies. Here, we showed that HY12 virus evolved to alter its characteristics and pathogenicity by employing a non-synonymous mutation. Analyses of 5′UTR, VP1 and VP2 gene sequences revealed the existence of HY12 virus in an array of mutants defined as quasispecies. The determination of diversity and complexity showed that the mutation rate and complexity of HY12 virus quasispecies increased, while the proportion of HY12 VP1 and VP2 consensus (master) sequences decreased with increasing passages. Synonymous mutation and non-synonymous mutation analysis displayed a positive selection for HY12 quasispecies evolution. A comparison of HY12 virus in different passages demonstrated that HY12 virus altered its characteristic, phenotype, and pathogenicity via non-synonymous mutation. These findings revealed the evolution pattern for HY12 virus, and the alteration of HY12 virus characteristics and pathogenicity by mutation

Partial physiochemical properties for HY12 strain.

<p>Partial physiochemical properties for HY12 strain.</p

Effect of exon 2b sequence on the amelogenin protein structures.

<p>(A) Hydrophilicity-plot analysis using the Kyte and Doolittle algorithm. Hydrophilicity-plots of <i>P</i>.<i>cinereus</i>-182, <i>P</i>.<i>cinereus</i>-195 and mouse (D31768.1) were generated and compared. In relation to <i>P</i>.<i>cinereus</i>-182 and mouse amelogenin, the region underlined with a black line (middle panel) is the exon 2b sequence, which was hydrophilic. (B) Exon 2b has no significant effect on the secondary structure of P.cinereus-195 predicted by Psipred. Similar to that of mouse amelogenin (AA 4~12), one potential helical region for <i>P</i>.<i>cinereus</i>-182 (AA 4~11) and <i>P</i>.<i>cinereus</i>-195 (AA 4~11) was revealed by Psipred prediction. (C) Exon 2b sequence effects on the tertiary structures of <i>P</i>.<i>cinereus</i>-195. <i>P</i>.<i>cinereus</i>-182 and <i>P</i>.<i>cinereus</i>-195 were used as query sequence for homology detection and structure prediction by HMM-HMM comparison using HHpred. A bar graph summarizes the positions and color-coded significances of the database matches with the probability. A tabular hit lists probabilities, E-values, scores, and match regions in queries and templates.</p

Outsplicing of exon 6 in <i>P</i>.<i>cinereus</i>-50 has dramatic effects on secondary and tertiary structure.

<p>(A–B) <b>Outsplicing</b> of exon 6 dramatically affects amelogenin secondary structure of <i>P</i>.<i>cinereus</i>-50 predicted by Psipred in relation to <i>P</i>.<i>cinereus</i>-182. One potential helical region was observed in <i>P</i>.<i>cinereus</i>-182 (AA 4-11), while two potential helical regions were revealed for <i>P</i>.<i>cinereus</i>-50 (AA 3~15, 38~48) in addition to a beta-strand region (AA 28~35) as revealed by Psipred prediction. (C–D) Exon 6 outsplicing affects the tertiary structure of <i>P</i>.<i>cinereus</i>-50. <i>P</i>.<i>cinereus</i>-182 and P.cinereus-50 were used as query sequence for homology detection and structure prediction by HMM-HMM comparison using HHpred. A bar graph summarizes the positions and color-coded significances of the database matches with the probability.</p

PCR amplification of potential amelogenin transcripts in <i>Plethodon</i><i>cinereus</i>.

<p>The cDNA was synthesized from total RNA isolated from tooth organs of <i>Plethodon</i><i>cinereus</i> and used to perform gradient PCR. PCR products amplified with different annealing temperatures were shown in lane 1(50°C), lane 2~3 (52°C, 54°C), lane 4 (56°C), and lane 5 (58°C), respectively. The arrow on the left showed the PCR products corresponding to AMEL-L, M and S (from top to bottom). Lane 6 is 100bp DNA plus ladder (Invitrogen).</p

Novel amelogenin gene splicing forms in <i>Plethodon</i><i>cinereus</i>.

<p>(A) Alignment analysis of the full-length salamander amelogenin gene cDNA sequence of <i>P</i><i>. cinereus</i>-M with the novel salamander amelogenin cDNA sequence <i>P</i><i>. cinereus</i>-L, and <i>P</i><i>. cinereus</i>-S. The full-length of <i>P</i><i>. cinereus</i>-M transcript is 812 bp, while the <i>P</i><i>. cinereus</i>-L and <i>P</i><i>. cinereus</i>-S transcripts are 851 bp and 416 bp, respectively. In relation to <i>P</i><i>. cinereus</i>-M, the <i>P</i><i>. cinereus</i>-L contains an additional 39 bp located between nucleotide 136 and 137, and <i>P</i><i>. cinereus</i>-S is short of 396 nucleotides between nucleotides 230 and 629. The 5’-untranslated region contains 82 nucleotides upstream of the translation start codon ATG. The 3’-untranslated region contains 181 nucleotides downstream stop codon TAA. (B) Sequence analysis of the deduced amino acid sequence of <i>P</i><i>. cinereus</i>-M, <i>P</i><i>. cinereus</i>-L, and <i>P</i><i>. cinereus</i>-S. In relation to <i>P</i>.<i>cinereus</i>-182 encoded by <i>P</i><i>. cinereus</i>-M, the <i>P</i>.<i>cinereus</i>-195 encoded by <i>P</i><i>. cinereus</i>-L contains an additional 13 amino acid residues located between amino acid residue 18 and 19. <i>P</i>.<i>cinereus</i>-50 was short of 123 amino acid residues between AA 49 and 181.</p