Development and Evaluation of Stable Sugarcane Mosaic Virus Mild Mutants for Cross-Protection Against Infection by Severe Strain.

Sugarcane mosaic virus (SCMV; genus Potyvirus) induces maize dwarf mosaic disease that has caused serious yield losses of maize in China. Cross-protection is one of the efficient strategies to fight against severe virus strains. Although many mild strains have been identified, the spontaneous mutation is one of the challenging problems affecting their application in cross-protection. In this study, we found that the substitution of cysteine (C) at positions 57 or 60 in the zinc finger-like motif of HC-Pro with alanine (A; C57A or C60A) significantly reduced its RNA silencing suppression activity and SCMV virulence. To reduce the risk of mild strains mutating to virulent ones by reverse or complementary mutations, we obtained attenuated SCMV mutants with double-mutations in the zinc finger-like and FRNK motifs of HC-Pro and evaluated their potential application in cross-protection. The results showed that the maize plants infected with FKNK/C60A double-mutant showed symptomless until 95 days post-inoculation and FKNK/C60A cross-protected plants displayed high resistance to severe SCMV strain. This study provides theoretical and material bases for the control of SCMV through cross-protection.

Development and application of a full-length infectious clone of potato virus Y isolate belonging to SYR-I strain.

Potato virus Y (PVY) causes huge damage to potato and tobacco production worldwide. The complete genome sequence of GZ, a PVY isolate (strain SYR-I) from Guizhou province, China, was cloned into the binary vector pCambia0390. Three introns were individually inserted into the P3 and CI ORFs to produce plasmid pCamPVY-GZ. The plasmid could infect plants of Nicotiana benthamiana, N. tabacum via agroinfiltration and plants of pepper and potato by mechanical inoculation. The green fluorescence protein gene of Aequoria victoriae was cloned into the encoding regions between nuclear inclusion body 'b' and coat protein genes in pCamPVY-GZ to produce pCamPVY-GZ-GFP, which could infect plants of N. benthamiana, N. tabacum, potato and tomato, and produce green fluorescence in the systemic leaves of inoculated plants. Mutations were introduced to pCamPVY-GZ to make the lysine (K) 391 and glutamic acid (E)410 of helper component-proteinase to arginine (R) and asparagic acid (E), respectively. Unlike wild type PVY-GZ, the mutant PVY-K391R/E410D could not induce veinal necrosis in N. tabacum plants. With an interval of 14 days, mutant PVY-K391R/E410D could protect N. tabacum plants from the infection of severe PVY strain. The results presented here provide a promising alternate for the prevention of diseases caused by PVY.

'Hands-off/poised' or 'Hands-on' method among Chinese midwives: A cross-sectional survey.

AIMS AND OBJECTIVES: To determine midwives' preference for "Hands-on" and "Hands-off/poised" methods and to explore the impact factors. BACKGROUND: With the increasing rate of obstetric anal sphincter injuries, great controversy surrounds the "Hands-on" or "Hands-off" method during childbirth to decrease obstetric anal sphincter injuries incidence. Without regular records and related research, determining the use of this technique in China is difficult. DESIGN: This study used a quantitative study design, following the EQUATOR guidelines (STROBE). METHODS: A nationwide cross-sectional online survey was conducted using respondent-driven sampling across 31 provinces in China from 1st October 2017-31st December 2017. A total of 6,425 midwives were involved. Descriptive analyses, chi-square test and binary logistic regression were undertaken. RESULTS: A total of 5,225 questionnaires were returned; 55.8% of the participants preferred the "Hands-off/poised" method. The impact factors included hospital categories, total work experience in a birth unit in years, theoretical education and skill training. In the study, for situations with increased OASI risk, 100% of the midwives in the "Hands-off" group expressed willingness to change to the "Hands-on" method whether there was concern about impending obstetric anal sphincter injuries. CONCLUSION: The "Hands-off/poised" method is heavily practised by Chinese midwives. However, majority of the midwives adopt "Hands-on" method in the face of high risk for obstetric anal sphincter injuries. Further studies are needed to determine the association between obstetric anal sphincter injuries rate and perineal management for low-risk birth. RELEVANCE TO CLINICAL PRACTICE: The result indirectly illustrates the application of "Hands-off/poised" technique in China and provides evidence for the international midwifery organisation to understand the status of Chinese midwifery to some extent. It also provides the latest data for further study of these two methods and the study of the relationship between hands-off/poised method and obstetric anal sphincter injuries.

Caffeine Treatment Promotes Differentiation and Maturation of Hypoxic Oligodendrocytes via Counterbalancing Adenosine 1 Adenosine Receptor-Induced Calcium Overload.

BACKGROUND We aimed to explore the involvement of adenosine 1 adenosine receptor (A1AR) in hypoxia-induced poor differentiation of oligodendrocytes (OLs), and the underlying mechanism of caffeine treatment in hypoxic injuries. MATERIAL AND METHODS Real-time polymerase chain reaction (RT-PCR) was used to assess the alterations of AR expression in cultured hypoxic OLs with or without caffeine treatment. Then, intracellular alterations of Ca²âº concentrations ([Ca²âº) were detected by confocal Fluo-3 imaging. The subsequent changes of myelin related protein expression were determined by western blot and immunofluorescence. RESULTS Three hours after hypoxia, significantly upregulated expression of A1AR was observed, accompanied with significantly decreased expression of oligodendrocyte transcription factor (Olig2). In addition, either hypoxia stimulation or 100 µM adenosine induced apparent elevation of resting [Ca²âº] in cultured OLs. However, pretreatment with DPCPX (A1AR selective antagonist) or caffeine abolished the [Ca²âº] increase, and the subsequent adenosine of high dose induced Ca²âº activity in developing OLs. Furthermore, caffeine or DPCPX improved the expression MBP and CNPase proteins after hypoxia stimulation, which resulted in the morphological maturation of OLs. CONCLUSIONS Caffeine treatment exerted protective effects on neonatal hypoxia injuries. It prevented Ca²âº overload injury, kept Ca²âº homeostasis in hypoxic developing OLs, and facilitated optimal expression of myelin related proteins by inhibiting A1AR in vitro. This study also provided experimental evidence for clinical application of caffeine in early treatment of neonatal hypoxia, and highlighted the potential significance of A1AR in anti-hypoxic drug discovery.

Recombination of strain O segments to HCpro-encoding sequence of strain N of Potato virus Y modulates necrosis induced in tobacco and in potatoes carrying resistance genes Ny or Nc.

Hypersensitive resistance (HR) to strains O and C of Potato virus Y (PVY, genus Potyvirus) is conferred by potato genes Ny(tbr) and Nc(tbr), respectively; however, PVY N strains overcome these resistance genes. The viral helper component proteinases (HCpro, 456 amino acids) from PVY(N) and PVY(O) are distinguished by an eight-amino-acid signature sequence, causing HCpro to fold into alternative conformations. Substitution of only two residues (K269R and R270K) of the eight-amino-acid signature in PVY(N) HCpro was needed to convert the three-dimensional (3D) model of PVY(N) HCpro to a PVY(O) -like conformation and render PVY(N) avirulent in the presence of Ny(tbr), whereas four amino acid substitutions were necessary to change PVY(O) HCpro to a PVY(N) -like conformation. Hence, the HCpro conformation rather than other features ascribed to the sequence were essential for recognition by Ny(tbr). The 3D model of PVY(C) HCpro closely resembled PVY(O), but differed from PVY(N) HCpro. HCpro of all strains was structurally similar to ß-catenin. Sixteen PVY(N) 605-based chimeras were inoculated to potato cv. Pentland Crown (Ny(tbr)), King Edward (Nc(tbr)) and Pentland Ivory (Ny(tbr)/Nc(tbr)). Eleven chimeras induced necrotic local lesions and caused no systemic infection, and thus differed from both parental viruses that infected King Edward systemically, and from PVY(N) 605 that infected Pentland Crown and Pentland Ivory systemically. These 11 chimeras triggered both Ny(tbr) and Nc(tbr) and, in addition, six induced veinal necrosis in tobacco. Further, specific amino acid residues were found to have an additive impact on necrosis. These results shed new light on the causes of PVY-related necrotic symptoms in potato.

Antioxidants: potential antiviral agents for Japanese encephalitis virus infection.

Japanese encephalitis (JE) is prevalent throughout eastern and southern Asia and the Pacific Rim. It is caused by the JE virus (JEV), which belongs to the family Flaviviridae. Despite the importance of JE, little is known about its pathogenesis. The role of oxidative stress in the pathogenesis of viral infections has led to increased interest in its role in JEV infections. This review focuses mainly on the role of oxidative stress in the pathogenesis of JEV infection and the antiviral effect of antioxidant agents in inhibiting JEV production. First, this review summarizes the pathogenesis of JE. The pathological changes include neuronal death, astrocyte activation, and microglial proliferation. Second, the relationship between oxidative stress and JEV infection is explored. JEV infection induces the generation of oxidants and exhausts the supply of antioxidants, which activates specific signaling pathways. Finally, the therapeutic efficacy of a variety of antioxidants as antiviral agents, including minocycline, arctigenin, fenofibrate, and curcumin, was studied. In conclusion, antioxidants are likely to be developed into antiviral agents for the treatment of JE.

Genetic determinants of Potato virus Y required to overcome or trigger hypersensitive resistance to PVY strain group O controlled by the gene Ny in potato.

Potato virus Y (PVY) (genus Potyvirus) is the most economically damaging and widely distributed virus in potato. Spread of PVY in the field is controlled by growing resistant cultivars. The dominant potato gene Ny(tbr) for hypersensitive resistance (HR) controls ordinary PVY strains (PVY(O)) but is overcome by PVY(N) strains. Studies with infectious PVY chimeras and mutants indicated that the viral determinants necessary and sufficient to overcome Ny(tbr) reside within the helper component proteinase (HC-Pro) (residues 227 to 327). Specifically, eight residues and the modeled three-dimensional conformation of this HC-Pro region distinguish PVY(N) from PVY(O) strains. According to the model, the conserved IGN and CCCT motifs implicated in potyvirus replication and movement, respectively, are situated in a coiled structure and an α-helix, respectively, within this region in PVY(O); however, their locations are reversed in PVY(N). Two residues (R269 and K270) are crucial for the predicted PVY(O)-specific HC-Pro conformation. Two viral chimeras triggered Ny(tbr) and induced veinal necrosis in tobacco, which is novel for PVY. One chimera belonged to strain group PVY(E). Our results suggest a structure-function relationship in recognition of PVY(O) HC-Pro by Ny(tbr), reveal HC-Pro amino acid signatures specific to PVY(O) and PVY(N), and facilitate identification of PVY strains overcoming Ny(tbr).