High-density linkage maps for <Emphasis Type="Italic">Citrus sunki</Emphasis> and <Emphasis Type="Italic">Poncirus trifoliata</Emphasis> using DArTseq markers

The construction of a high-resolution genetic map of citrus would be of great value to breeders and to associate genomic regions with characteristics of agronomic interest. Here, we describe a novel high-resolution map of citrus using a population derived from a controlled cross between Citrus sunki (female parent) and Poncirus trifoliata (male parent). The genetic linkage maps were constructed using DArTseq markers and a pseudo-testcross strategy; only markers showing the expected segregation ratio were considered. To investigate synteny, all markers from both linkage maps were aligned with the genome of Citrus sinensis. The C. sunki map has a total of 2778 molecular markers and a size of 2446.6 cM, distributed across ten linkage groups. The map of P. trifoliata was built with 3084 markers distributed in a total of nine linkage groups, with a total size of 2411.6 cM. These maps are the most saturated linkage maps available for C. sunki and P. trifoliata and have high genomic coverage. We also demonstrated that the maps reported here are closely related to the reference genome of C. sinensis.     

<Emphasis Type="Italic">Coptidis Rhizoma</Emphasis> extract inhibits replication of respiratory syncytial virus <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> by inducing antiviral state

Coptidis Rhizoma is derived from the dried rhizome of Ranunculaceous plants and is a commonly used traditional Chinese medicine. Although Coptidis Rhizoma is commonly used for its many therapeutic effects, antiviral activity against respiratory syncytial virus (RSV) has not been reported in detail. In this study, we evaluated the antiviral activities of Coptidis Rhizoma extract (CRE) against RSV in human respiratory tract cell line (HEp2) and BALB/c mice. An effective dose of CRE significantly reduces the replication of RSV in HEp2 cells and reduces the RSV-induced cell death. This antiviral activity against RSV was through the induction of type I interferon-related signaling and the antiviral state in HEp2 cells. More importantly, oral administration of CRE exhibited prophylactic effects in BALB/c mice against RSV. In HPLC analysis, we found the presence of several compounds in the aqueous fraction and among them; we confirmed that palmatine was related to the antiviral properties and immunemodulation effect. Taken together, an extract of Coptidis Rhizoma and its components play roles as immunomodulators and could be a potential source as promising natural antivirals that can confer protection to RSV. These outcomes should encourage further allied studies in other natural products.     

Interferon-mediated antiviral activities of Angelica tenuissima Nakai and its active components

Angelica tenuissima Nakai is a widely used commodity in traditional medicine. Nevertheless, no study has been conducted on the antiviral and immune-modulatory properties of an aqueous extract of Angelica tenuissima Nakai. In the present study, we evaluated the antiviral activities and the mechanism of action of an aqueous extract of Angelica tenuissima Nakai both in vitro and in vivo. In vitro, an effective dose of Angelica tenuissima Nakai markedly inhibited the replication of Influenza A virus (PR8), Vesicular stomatitis virus (VSV), Herpes simplex virus (HSV), Coxsackie virus, and Enterovirus (EV-71) on epithelial (HEK293T/HeLa) and immune (RAW264.7) cells. Such inhibition can be described by the induction of the antiviral state in cells by antiviral, IFNrelated gene induction and secretion of IFNs and pro-inflammatory cytokines. In vivo, Angelica tenuissima Nakai treated BALB/c mice displayed higher survivability and lower lung viral titers when challenged with lethal doses of highly pathogenic influenza A subtypes (H1N1, H5N2, H7N3, and H9N2). We also found that Angelica tenuissima Nakai can induce the secretion of IL-6, IFN-λ, and local IgA in bronchoalveolar lavage fluid (BALF) of Angelica tenuissima Nakai treated mice, which correlating with the observed prophylactic effects. In HPLC analysis, we found the presence of several compounds in the aqueous fraction and among them; we evaluated antiviral properties of ferulic acid. Therefore, an extract of Angelica tenuissima Nakai and its components, including ferulic acid, play roles as immunomodulators and may be potential candidates for novel anti-viral/anti-influenza agents.     

Chalcones as novel influenza A (H1N1) neuraminidase inhibitors from Glycyrrhiza inflata

The emergence of highly pathogenic influenza A virus strains, such as the new H1N1 swine influenza (novel influenza), represents a serious threat to global human health. During our course of an anti-influenza screening program on natural products, one new licochalcone G (1) and seven known (2-8) chalcones were isolated as active principles from the acetone extract of Glycyrrhiza inflata. Compounds 3 and 6 without prenyl group showed strong inhibitory effects on various neuraminidases from influenza viral strains, H1N1, H9N2, novel H1N1 (WT), and oseltamivir-resistant novel H1N1 (H274Y) expressed in 293T cells. In addition, the efficacy of oseltamivir with the presence of compound 3 (5 μM) was increased against H274Y neuraminidase. This evidence of synergistic effect makes this inhibitor to have a potential possibility for control of pandemic infection by oseltamivir-resistant influenza virus.     

Dense Granule Protein-7 (GRA-7) of <Emphasis Type="Italic">Toxoplasma gondii</Emphasis> inhibits viral replication <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis>

Dense granule protein-7 (GRA-7) is an excretory protein of Toxoplasma gondii. It is a potential serodiagnostic marker and vaccine candidate for toxoplasmosis. Previous reports demonstrated that GRA-7 induces innate immune responses in macrophages by interacting with TRAF6 via the MyD88-dependent pathway. In the present study, we evaluated the antiviral activity and induction of an antiviral state by GRA-7 both in vitro and in vivo. It was observed that GRA-7 markedly reduced the replication of vesicular stomatitis virus (VSV-GFP), influenza A virus (PR8-GFP), coxsackievirus (H3-GFP), herpes simplex virus (HSV-GFP), and adenovirus-GFP in epithelial (HEK293T/HeLa) and immune (RAW264.7) cells. These antiviral activities of GRA-7 were attributed to the induction of type I interferon (IFN) signaling, resulting in the secretion of IFNs and pro-inflammatory cytokines. Additionally, in BALB/c mice, intranasal administration of GRA-7 prevented lethal infection by influenza A virus (H1N1) and exhibited prophylactic effects against respiratory syncytial virus (RSV-GFP). Collectively, these results suggested that GRA-7 exhibits immunostimulatory and broad spectrum antiviral activities via type I IFN signaling. Thus, GRA-7 can be potentially used as a vaccine adjuvant or as a candidate drug with prophylactic potential against viruses.     

Effects of heat-killed <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> against influenza viruses in mice

The potential use of dietary measures to treat influenza can be an important alternative for those who lack access to influenza vaccines or antiviral drugs. Lactobacillus plantarum (Lp) is one of many lactic acid bacteria that grow in ‘kimchi’, an essential part of Korean meal, and several strains of Lp reportedly show protective effects against influenza. Using heat-killed Lp (nF1) isolated from kimchi, which is known for its immunomodulatory effects, we investigated whether regular oral intake of nF1 could influence the outcome of influenza virus infection in a mouse model. In a lethal challenge with influenza A (H1N1 and H3N2 subtypes) and influenza B (Yamagata lineage) viruses, daily oral administration of nF1 delayed the mean number of days to death of the infected mice and resulted in increased survival rates compared with those of the non-treated mice. Consistent with these observations, nF1 treatment also significantly reduced viral replication in the lungs of the infected mice. Taken together, our results might suggest the remedial potential of heatkilled Lactobacillus probiotics against influenza.     

Synthesis and antiviral activity of novel glycyrrhizic acid conjugates with D-amino acid esters

Glycyrrhizic acid (GA) conjugates with methyl and ethyl esters of D-amino acids (D-Trp, D-Phe, D-Tyr, D-Val, D-Leu) have been synthesized by the activated esters method using mixtures of N-hydroxybenzotriazole or N-hydroxysuccinimide with N,N′-dicyclohexylcarbodiimide. GA conjugate with D-Trp ethyl ester exhibited antiviral activity against influenza viruses A/H3N2, A/H1N1/pdm09, A/H5N1, B (SI > 10–29), and HRSV (SI > 25). GA conjugate with D-Trp methyl ester inhibited influenza virus A/H1N1/pdm09 (SI > 30).     

Genome-wide identification and expression analysis of the molecular chaperone binding protein <Emphasis Type="Italic">BiP</Emphasis> genes in <Emphasis Type="Italic">Citrus</Emphasis>

Here, we report for the first time the genome-wide identification and expression analysis of the molecular chaperone BiP genes in Citrus. Six genes encoding the conserved protein domain family GPR78/BiP/KAR2 were identified in the genome of Citrus sinensis and C. clementina. Two of them, named here as CsBiP1 and CsBiP2, were classified as true BiPs based on their deduced amino acid sequences. Alignment of the deduced amino acid sequences of CsBiP1 and CsBiP2 with BiP homologs from soybean and Arabidopsis showed that they contain all the conserved functional motifs of BiPs. Analysis of the promoter region of CsBiPs revealed the existence of cis-acting regulatory sequences involved in abiotic, heat-shock, and endoplasmic reticulum (ER) stress responses. Publicly available RNA-seq data indicated that CsBiP1 is abundantly expressed in leaf, flower, fruit, and callus, whereas CsBiP2 expression is rarely detected in any tissues under normal conditions. Comparative quantitative real-time PCR (qPCR) analysis of expression of these genes between C. sinensis grafted on the drought-tolerant “Rangpur” lime (C. limonia) and -sensitive “Flying Dragon” trifoliate orange (Poncirus trifoliata) rootstocks showed that CsBiP1 was upregulated by drought stress on the former but downregulated on the latter, whereas the CsBiP2 mRNA levels were downregulated on drought-stressed “Flying Dragon,” but remained constant on “Rangpur.” CsBiP2 upregulation was only observed in C. sinensis seedlings subjected to osmotic and cold treatments. Taken together, these results indicate the existence of two highly conserved BiP genes in Citrus that are differentially regulated in the different tissues and in response to abiotic stresses.     

Antiviral activity of hemolymph of <Emphasis Type="Italic">Podalia</Emphasis> against rubella virus

Many active principles produced by animals, plants and microorganisms have been employed in the development of new drugs for the treatment of human diseases. Among animals known to produce pharmacologically active molecules that interfere in human cell physiology. Rubella virus (genus Rubivirus, family Togaviridae) is a single stranded RNA virus of positive genome polarity. Rubella virus infection of susceptible women during the first trimester of pregnancy often results in long-term virus persistence in the fetus causing multiple organ abnormalities. Potent antiviral activity against rubella virus (RV) has been observed in the hemolymph of Podalia sp. (Lepidoptera: Megalopygidae). This study evaluated the effect of hemolymph on RV infected Statens Serum Institute Rabbit Cornea (SIRC) cells. Results of cell viability and cell proliferation assays indicated that hemolymph was not toxic to cultured SIRC cells. Viral binding assay, antiviral assay, PCR, real-time PCR, and transmission electron microscopy were used to demonstrate that hemolymph in post-treatment could inhibit the production of infectious RV particles. Specifically, hemolymph was found to inhibit RV adsorption to the SIRC cells.     

Effect of influenza A virus and bacterial lipopolysaccharide on proliferation and expression of cytokines and other cellular factors in the endothelial cell line ECV-304

Viral infection and bacterial lipopolysaccharide (LPS) cause endothelial-cell dysfunction. The aim of the current study was to investigate the effect of influenza A virus and LPS from Escherichia coli on the proliferative activity and gene expression of cytokines and cellular factors (TNFα, TGFβ, IFN-γ, MMP-9, NF-κB, Rho A, eNOS, and iNOS) in human endothelial cells ECV-304. It was found that ECV-304 cells infected with very low infectious doses of influenza virus acquired the capacity for the long-term active proliferation (over eight passages). Addition of LPS from E. coli reduced the virus-stimulated cell proliferation. It was shown that influenza virus and LPS affected the gene expression of cytokine and other cellular factors. When endothelial cells were infected with influenza A virus in the presence of LPS, there was a significant increase in the expression of several genes and the expression pattern of certain genes was modified. Expression of MMP-9 gene inhibited by the virus and LPS separate exposure significantly increased during the first day after addition of the virus and LPS simultaneously. The same was true for the IFN-γ gene expression. TNFα gene was active only for 1–3 days whereas the expression of TGFβ, eNOS, iNOS, NF-κB and Rho A genes increased significantly on the fifth day, as it was observed with the cells treated with LPS only. Thus, the influenza A virus and LPS change the physiological state of endothelial cells. This occurred during various time periods (as well as at various degrees of viral infection) produced by different cellular factors and, possibly, involved different signaling pathways.     

Bioprospecting of <Emphasis Type="Italic">Diaporthe terebinthifolii</Emphasis> LGMF907 for antimicrobial compounds

Antibiotic-resistant bacteria have been observed with increasing frequency over the past decades, driving the search for new drugs and stimulating the interest in natural products sources. Endophytic fungi from medicinal plants represent a great source of novel bioactive compounds useful to pharmaceutical and agronomical purposes. Diaporthe terebinthifolii is an endophytic species isolated from Schinus terebinthifolius, a plant used in popular medicine for several health problems. The strain D. terebinthifolii LGMF907 was previously reported by our group to produce secondary metabolites with biological activity against phytopathogens. Based on these data, strain LGMF907 was chosen for bioprospecting against microorganisms of clinical importance and for characterization of major secondary metabolites. In this study, different culture conditions were evaluated and the biological activity of this strain was expanded. The crude extracts demonstrated high antibacterial activity against Escherichia coli, Micrococcus luteus, Saccharomyces cerevisiae, methicillin-sensitive Staphylococcus aureus, and methicillin-resistant S. aureus. The compounds diaporthin and orthosporin were characterized and also showed activity against the clinical microorganisms evaluated. This study discloses the first isolation of diaporthin and orthosporin from D. terebinthifolii, and revealed the potential of this endophytic fungus to produce secondary metabolites with antimicrobial activity.     

Molecular cloning,expression, and subcellular localization of a PAL gene from Citrus reticulata under iron deficiency

Phenylalanine ammonia lyase (PAL) is a specific branch point enzyme of primary and secondary metabolism. The Citrus reticulata Blanco PAL gene was cloned and designated as CrPAL1. The cDNA sequence of CrPAL1 was 2 166 bp, encoding 721 amino acid residues. Sequence alignment indicates that CrPAL1 shared a high identity with PAL genes found in other plants. Both the dominant and catalytic active sites of CrPAL1 were similar to PAL proteins observed in Petroselinum crispum. Phylogenetic tree analysis indicates that CrPAL1 was more closely related to PALs in Citrus clementina × C. reticulata and Poncirus trifoliata than to those from other plants. Subcellular localization reveals that CrPAL1-green fluorescent protein fusion protein was specifically localized in the plasma membrane. Activity of PAL as well as CrPAL1 expression increased under Fe deficiency. A similar result was noted for total phenolic content. The root exudates of C. reticulata strongly promoted reutilization of apoplastic Fe in roots. Furthermore, Fe was more desorbed from the cell wall under Fe deficiency than in sufficient Fe supply.     

Acaricidal active fractions from acetone extract of <Emphasis Type="Italic">Aloe vera</Emphasis> L. against <Emphasis Type="Italic">Tetranychus cinnabarinus</Emphasis> and <Emphasis Type="Italic">Panonychus citri</Emphasis>

This study aimed to isolate acaricidal active fractions from acetone extract of Aloe vera L. and investigate the toxicity of these fractions against Tetranychus cinnabarinus (T. cinnabarinus) and Panonychus citri (P. citri). Acetone extract of A. vera L. was isolated by immersing in acetone for 72 h, and diverse fractions were fractionated by column chromatography. The acaricidal activity of each fractions was evaluated by corrected mortality of T. cinnabarinus through slide-dip bioassay. The 8th and 13th fractions of acetone extract with good acaricidal activity were indentified by LC/MS, and the toxicity of these two fractions to T. cinnabarinus and P. citri was identified by regression analysis. Acetone extract of A. vera L. exhibited obvious acaricidal activity, from which a total of 18 fractions were isolated. The 8th and 13th fractions with strong acaricidal activity against T. cinnabarinus were identified to be 3-O-alpha-d-mannopyranosyl-d-mannopyranose (OAMM) and aloe emodin. When compared with spirodiclofen, both OAMM and aloe emodin exhibited higher toxicity to T. cinnabarinus, while only OAMM exhibited a higher toxicity to P. citri (P < 0.05). OAMM and aloe emodin isolated from acetone extract of A. vera L. exhibited obvious acaricidal activities against T. cinnabarinus and P. citri.     

An antiviral protein having deoxyribonuclease and ribonuclease activity from leaves of the post-flowering stage of <Emphasis Type="Italic">Celosia cristata</Emphasis>

An antiviral protein named CCP-27 was purified from the leaves of Celosia cristata at the post-flowering stage by anion-exchange, cation-exchange, and gel-filtration chromatography. It exhibited resistance against sunnhemp rosette virus in its test host Cyamopsis tetragonoloba. It also exhibited deoxyribonuclease activity against supercoiled p BlueScript SK⁺ plasmid DNA. It was found to nick supercoiled DNA into nicked circular form at lower prote in concentration followed by nicked to linear form conversion at higher protein concentration. CCP-27 also possesses strong ribonuclease activity against Torula yeast rRNA.     

Host range extension of <Emphasis Type="Italic">Cydia pomonella</Emphasis> granulovirus: adaptation to Oriental Fruit Moth, <Emphasis Type="Italic">Grapholita molesta</Emphasis>

Among various Cydia pomonella granulovirus (CpGV) isolates, the Mexican isolate (CpGV-M) has demonstrated a significant ability to reduce damage induced by the oriental fruit moth, Grapholita molesta (Busck) (=Cydia molesta) in peach crops. To obtain a more efficient virus for G. molesta control, an experimental virus population was constructed by mixing various CpGV isolates. This mixture was then selected for replication in a G. molesta laboratory colony. After 12 successive passages on this alternative host, the insecticidal efficacy of the virus population had improved. The concentration of virus occlusion bodies required to kill 90 % of neonate larvae was 450-fold lower than that of the original isolate mixture, and 120-fold lower than that of the CpGV-M isolate alone. Following adaptation to this alternative host, the efficacy against its natural host, the codling moth, C. pomonella, was conserved. This mixed isolate population can be produced on C. pomonella without loss of efficacy, which is useful from a commercial production perspective. This adapted virus isolate mixture is likely to prove more effective than individual component isolates at controlling G. molesta.     

Antifungal and Biocontrol Evaluation of Four <Emphasis Type="Italic">Lysobacter</Emphasis> Strains Against Clubroot Disease

The effect of crude extract (Ce), seed coating agent (SCA) and whole bacterial broth culture (WBC) of Lysobacter strains was evaluated against the causal agent of clubroot formation in Cruciferous vegetables. The ability of four Lysobacter strains (L. antibioticus 6-B-1, L. antibioticus 6-T-4, L. antibioticus 13-B-1 and L. capsici ZST1-2) inhibited Plasmodiophora brassicae of resting spores and disease. Application of WBC of four Lysobacter strains inhibited clubroot disease, indicating that the disease suppression was due to antifungal compounds produced by the biocontrol bacterium in the culture. Development of clubroot on Chinese cabbage was inhibited when the WBC and SCA were applied before P. brassicae inoculation. Crude extract (Ce) of culture filtrate was effective in arresting the germination of resting spores of P. brassicae on slides. However, Lysobacter strains differed in their biocontrol effects, the strain L. capsci ZST1-2 recorded a high level of disease limiting effect.     

Novel Pandemic Influenza A(H1N1) Viruses Are Potently Inhibited by DAS181, a Sialidase Fusion Protein

Background

The recent emergence of a novel pandemic influenza A(H1N1) strain in humans exemplifies the rapid and unpredictable nature of influenza virus evolution and the need for effective therapeutics and vaccines to control such outbreaks. However, resistance to antivirals can be a formidable problem as evidenced by the currently widespread oseltamivir- and adamantane-resistant seasonal influenza A viruses (IFV). Additional antiviral approaches with novel mechanisms of action are needed to combat novel and resistant influenza strains. DAS181 (Fludase™) is a sialidase fusion protein in early clinical development with in vitro and in vivo preclinical activity against a variety of seasonal influenza strains and highly pathogenic avian influenza strains (A/H5N1). Here, we use in vitro, ex vivo, and in vivo models to evaluate the activity of DAS181 against several pandemic influenza A(H1N1) viruses.

Methods and Findings

The activity of DAS181 against several pandemic influenza A(H1N1) virus isolates was examined in MDCK cells, differentiated primary human respiratory tract culture, ex-vivo human bronchi tissue and mice. DAS181 efficiently inhibited viral replication in each of these models and against all tested pandemic influenza A(H1N1) strains. DAS181 treatment also protected mice from pandemic influenza A(H1N1)-induced pathogenesis. Furthermore, DAS181 antiviral activity against pandemic influenza A(H1N1) strains was comparable to that observed against seasonal influenza virus including the H274Y oseltamivir-resistant influenza virus.

Conclusions

The sialidase fusion protein DAS181 exhibits potent inhibitory activity against pandemic influenza A(H1N1) viruses. As inhibition was also observed with oseltamivir-resistant IFV (H274Y), DAS181 may be active against the antigenically novel pandemic influenza A(H1N1) virus should it acquire the H274Y mutation. Based on these and previous results demonstrating DAS181 broad-spectrum anti-IFV activity, DAS181 represents a potential therapeutic agent for prevention and treatment of infections by both emerging and seasonal strains of IFV.