Unusual matrine-adenine hybrids isolated from Sophora davidii and their inhibitory effects on human cytomegalovirus.

A phytochemical investigation on the flowers of Sophora davidii resulted in the isolation of three unusual matrine-adenine hybrids, sophovicines A-C, together with biogenetically related analogue sophocarpine. Their structures and absolute configurations were determined by NMR analysis, single-crystal X-ray diffraction, and electronic circular dichroism (ECD) data. Since sophovicines represent the first example of matrine-adenine hybrids, a putative biosynthetic pathway toward sophovicines A-C was proposed. In addition, computational molecular modeling suggested that compounds sophovicines B and C may have potent activities against human cytomegalovirus (HCMV). So, the inhibit effects of isolates on HCMV were evaluated. The results show that sophovicines B and C can inhibit HCMV replication effectively with IC50 values of 7.12 and 7.32 µM, respectively.

The Susceptibility of Primary Dermis Fibroblasts from the Chinese Tree Shrew to Human Cytomegalovirus Infection.

As a universal pathogen leading to neonatal defects and transplant failure, human cytomegalovirus (HCMV) has strict species specificity and this has prevented the development of a suitable animal model for the pathogenesis study. The mechanism of cross-species barrier remains elusive and there are so far no non-human cell culture models that support HCMV replication. The Chinese tree shrew (Tupaia belangeri chinensis) is a small laboratory animal and evolutionary closely related with primates. We investigated the susceptibility of primary tree shrew dermis fibroblasts (TSDF) to HCMV infection. Infection with a GFP-expressing HCMV virus resulted in green fluorescence in infected cells with the expression of IE1, UL44 and pp28. The titers of cell-free viruses reached 103 PFU/mL at 96 hpi, compared to titers of 104 PFU/mL observed in primary human foreskin fibroblasts. Our results suggested that TSDF was semi-permissive for HCMV infection. The TSDF model could be further used to investigate key factors influencing cross-species multiplication of HCMV.

The Conserved Proline18 in the Polerovirus P3a Is Important for Brassica Yellows Virus Systemic Infection.

ORF3a, a newly identified non-AUG-initiated ORF encoded by members of genera Polerovirus and Luteovirus, is required for long-distance movement in plants. However, the mechanism of action of P3a in viral systemic movement is still not clear. In this study, sequencing of a brassica yellows virus (BrYV) mutant defective in systemic infection revealed two-nucleotide variation at positions 3406 and 3467 in the genome. Subsequent nucleotide substitution analysis proved that only the non-synonymous substitution (C→U) at position 3406, resulting in P3aP18L, abolished the systemic infection of BrYV. Preliminary investigation showed that wild type BrYV was able to load into the petiole of the agroinfiltrated Nicotiana benthamiana leaves, whereas the mutant displayed very low efficiency. Further experiments revealed that the P3a and its mutant P3aP18L localized to the Golgi apparatus and near plasmodesmata, as well as the endoplasmic reticulum. Both P3a and P3aP18L were able to self-interact in vivo, however, the mutant P3aP18L seemed to form more stable dimer than wild type. More interestingly, we confirmed firstly that the ectopic expression of P3a of other poleroviruses and luteoviruses, as well as co-infection with Pea enation mosaic virus 2 (PEMV 2), restored the ability of systemic movement of BrYV P3a defective mutant, indicating that the P3a is functionally conserved in poleroviruses and luteoviruses and is redundant when BrYV co-infects with PEMV 2. These observations provide a novel insight into the conserved function of P3a and its underlying mechanism in the systemic infection.

Differential proteomic profiling of endometrium and plasma indicate the importance of hydrolysis in bovine endometritis.

Endometritis is an important disease of dairy cows that leads to significant economic losses in the dairy cattle industry. To investigate the alteration of proteins associated with endometritis in the dairy cow, the isobaric tags for relative and absolute quantification (iTRAQ) technique was applied to quantitatively identify differentially expressed proteins (DEP) in the endometrium and peripheral plasma of Chinese Holstein cows with endometritis. Compared with the normal (control) group, 159 DEP in the endometrium and 137 DEP in the plasma were identified in cows with endometritis. Gene ontology analysis demonstrated that the predominant endometrial DEP were primarily involved in responses to stimulus and stress processes and mainly played a role in hydrolysis in the extracellular region. The predominant plasma DEP were mainly components of the cytosol and non-membrane-bound organelles, and they were involved in the response to stress and regulation of enzyme activity. Protein-protein interaction of tissue DEP revealed that some core seed proteins, such as RAC2, ITGB2, and CDH1 in the same network as CD14, MMP3, and MMP9, had important functions in the cross-talk of pathways related to extracellular proteolysis. In summary, significant enzymatic hydrolase activity in the extracellular region is proposed as a molecular mechanism by which altered proteins may promote inflammation and hence endometritis.

Comparative proteomics analysis provide novel insight into laminitis in Chinese Holstein cows.

BACKGROUND: Laminitis is considered as the most important cause of hoof lameness in dairy cows, which causes abundant economic losses in husbandry. Through intense efforts in past decades, the etiology of laminitis is preliminarily considered to be subacute ruminal acidosis; however, the pathogenesis of laminitis needs further research. The differentially expressed proteins (DEP) were detected in plasma of healthy cows and clinical laminitis cows by two-dimensional gel electrophoresis (2-DE) and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. RESULTS: Nineteen protein spots were differentially expressed, and 16 kinds of proteins were identified after peptide mass fingerprint search and bioinformatics analysis. Of these, 12 proteins were differentially up-regulated and 4 down-regulated. Overall, these differential proteins were involved in carbohydrate metabolism, lipids metabolism, molecular transport, immune regulation, inflammatory response, oxidative stress and so on. CONCLUSIONS: The DEPs were closely related to the occurrence and development of laminitis and the lipid metabolic disturbance may be a new pathway to cause laminitis in dairy cows. The results provide the theory foundation for further revealing the mechanism of laminitis and screening the early diagnostic proteins and therapeutic target.

Development of three full-length infectious cDNA clones of distinct brassica yellows virus genotypes for agrobacterium-mediated inoculation.

Brassica yellows virus is a newly identified species in the genus of Polerovirus within the family Luteoviridae. Brassica yellows virus (BrYV) is prevalently distributed throughout Mainland China and South Korea, is an important virus infecting cruciferous crops. Based on six BrYV genomic sequences of isolates from oilseed rape, rutabaga, radish, and cabbage, three genotypes, BrYV-A, BrYV-B, and BrYV-C, exist, which mainly differ in the 5' terminal half of the genome. BrYV is an aphid-transmitted and phloem-limited virus. The use of infectious cDNA clones is an alternative means of infecting plants that allows reverse genetic studies to be performed. In this study, full-length cDNA clones of BrYV-A, recombinant BrYV5B3A, and BrYV-C were constructed under control of the cauliflower mosaic virus 35S promoter. An agrobacterium-mediated inoculation system of Nicotiana benthamiana was developed using these cDNA clones. Three days after infiltration with full-length BrYV cDNA clones, necrotic symptoms were observed in the inoculated leaves of N. benthamiana; however, no obvious symptoms appeared in the upper leaves. Reverse transcription-PCR (RT-PCR) and western blot detection of samples from the upper leaves showed that the maximum infection efficiency of BrYVs could reach 100%. The infectivity of the BrYV-A, BrYV-5B3A, and BrYV-C cDNA clones was further confirmed by northern hybridization. The system developed here will be useful for further studies of BrYV, such as host range, pathogenicity, viral gene functions, and plant-virus-vector interactions, and especially for discerning the differences among the three genotypes.

Complete genomic sequence analysis reveals a novel fabavirus infecting cucurbits in China.

The complete genome sequence of a cucurbit-infecting fabavirus was determined. Sequence analysis revealed that it had a genomic organization typical of fabaviruses, with genome segment sizes of 5870 nt (RNA-1) and 3294 nt (RNA-2). It shared CP and Pro-Pol amino acid sequence identities of 52.0-58.9% with those of reported fabaviruses. ELISA and western blots gave no cross-reactions between this cucurbit virus and broad bean wilt viruses 1 and 2. Based on molecular and serological criteria for species demarcation in the genus Fabavirus, the virus represents a distinct species, for which the species name Cucurbit mild mosaic virus (CuMMV) is proposed.

Molecular characterization of two genotypes of a new polerovirus infecting brassicas in China.

The genomic RNA sequences of two genotypes of a brassica-infecting polerovirus from China were determined. Sequence analysis revealed that the virus was closely related to but significantly different from turnip yellows virus (TuYV). This virus and other poleroviruses, including TuYV, had less than 90% amino acid sequence identity in all gene products except the coat protein. Based on the molecular criterion (>10% amino acid sequence difference) for species demarcation in the genus Polerovirus, the virus represents a distinct species for which the name Brassica yellows virus (BrYV) is proposed. Interestingly, there were two genotypes of BrYV, which mainly differed in the 5'-terminal half of the genome.

Molecular characterization of two Chinese isolates of Beet western yellows virus infecting sugar beet.

Beet western yellows virus (BWYV) has previously been reported as an agent of sugar beet yellowing disease in China. In this article, the complete genomic RNA sequences of two Chinese BWYV isolates infecting beet from Inner Mongolia (BWYV-IM) and Gansu (BWYV-GS) were determined and compared with three beet poleroviruses (BMYV, BChV and BWYV-US) and other non-beet-infecting poleroviruses. The genomes of the two isolates were 5,668 nt in length, and had almost the same genomic organization and characteristics as BWYV-US. The full length of BWYV-IM shared nucleotide sequence identities of 97.4, 86.6, 64.4 and 70.8% with BWYV-GS, BWYV-US, BChV and BMYV, respectively. Further sequence analysis indicated that the Chinese BWYV isolates were more closely related to BWYV-US; however, the identity of any gene product between the Chinese isolates and BWYV-US was <90%. Therefore, on the basis of genome sequence, we propose that these Chinese isolates are a distinct strain of BWYV that infect sugar beet. In addition, recombinant detection analysis revealed that BWYV-IM might be a recombinant virus.