Isolation and identification of active components from Grifola frondosa and its anti-EV71 virus effect.

BACKGROUND: It is reported that anti-enterovirus 71 (EV71) drugs have some side effects on human health. Notably, fungi plays a crucial role in promoting human health and anti-virus. Grifola frondosa is a type of large medicinal and edible fungi, rich in active substances. The present study aimed to investigate the anti-EV71 effect of G. frondosa and the potential active substances. RESULTS: In the present study, the water extract of G. frondosa was subjected to ethanol precipitation to obtain the water-extracted supernatant of G. frondosa (GFWS) and water-extracted precipitation of G. frondosa. Their inhibitory effects on EV71 virus were studied based on a cell model. The results showed that GFWS had stronger security and anti-EV71 effects. In addition, the chemical constituents of GFWS were identified by ultra-high performance liquid chromatography-tandem mass spectrometry, which were selected for further separation and purification. Three compounds, N-butylaniline, succinic acid and l-tryptophan, were isolated from GFWS by NMR spectroscopy. It is noteworthy that N-butylaniline and l-tryptophan were isolated and identified from the G. frondosa fruiting bodies for the first time. Our study found that l-tryptophan has anti-EV71 virus activity, which reduced EV71-induced apoptosis and significantly inhibited the replication process after virus adsorption. Furthermore, it could also bind to capsid protein VP1 to prevent the virus from attaching to the cells. CONCLUSION: l-tryptophan was an inhibitor of the EV71 virus, which could be used in infant nutrition and possibly provide a new drug to treat hand, foot and mouth disease. © 2024 Society of Chemical Industry.

Anti-α-glucosidase, Anti-proliferative and Anti-enterovirus 71 Activity of Secondary Metabolites Identified from Grifola Frondosa.

Grifola frondosa, an edible and medicinal resource, is widely used as functional foods worldwide. To explore bioactive compounds against α-glucosidase, human tumor cells and enterovirus 71 (EV71), eight compounds were isolated from G. frondosa by chromatographic column. Among the isolated compounds, heptadecanoic acid, uridine and adenosine exhibited potent inhibition activity against α-glucosidase, ergosterols and ergosterol-5,8-peroxide showed anti-proliferative activity on tumor cells, while ergosterol and methyl linoleate displayed inhibition against the replication of EV71. Also, to our knowledge, this is the first study to report that fatty acids isolated from G. frondosa show potent inhibition against α-glucosidase and EV71. Further molecular docking results revealed that the active compounds in G. frondosa form hydrogen bonding, hydrophobic interactive and π-stacking with the active sites on the surface of α-glucosidase, CASP3 and VP1 proteins, thus promoting the active compounds to combine with the target protein to form a stable complex, thus playing an antagonistic role. Our results could provide a new active compound and mode of action for G. frondosa to treat diabetes, cancer and EV71-infected patients.

Probiotic Fermentation of Kelp Enzymatic Hydrolysate Promoted its Anti-Aging Activity in D-Galactose-Induced Aging Mice by Modulating Gut Microbiota.

SCOPE: To investigate anti-aging effects of probiotic-fermented kelp enzymatic hydrolysate culture (KMF), probiotic-fermented kelp enzymatic hydrolysate supernatant (KMFS), and probiotic-fermented kelp enzymatic hydrolysate bacteria suspension (KMFP) in D-galactose-induced aging mice. METHODS AND RESULTS: The study uses a probiotic-mixture of Lactobacillus reuteri, Pediococcus pentosaceus, and Lactobacillus acidophilus strains for kelp fermentation. KMF, KMFS, and KMFP prevent D-galactose-induced elevation of malondialdehyde levels in serum and brain tissue of aging mice, and they increase superoxide dismutase and catalase levels and total antioxidant capacity. Furthermore, they improve the cell structure of mouse brain, liver, and intestinal tissue. Compared with the model control group, the KMF, KMFS, and KMFP treatments regulate mRNA and protein levels of genes associated with aging, the concentrations of acetic acid, propionic acid, and butyric acid in the three treatment groups are more than 1.4-, 1.3-, and 1.2-fold increased, respectively. Furthermore, the treatments affect the gut microbiota community structures. CONCLUSIONS: These results suggest that KMF, KMFS, and KMFP can modulate gut microbiota imbalances and positively affect aging-related genes to achieve anti-aging effects.

Anti-Diabetic Potential of Chlorella Pyrenoidosa-Based Mixture and its Regulation of Gut Microbiota.

The aim of the present study was to investigate the anti-diabetic effect of CGSGCG and its beneficial effects on gut microbiota in type 2 diabetes (T2D) mice induced by streptozotocin and high sucrose and high fat diet. The results showed that treatment with CGSGCG reduced fasting blood glucose, improved oral glucose tolerance test, protected the liver from injury, and reduced inflammation in T2D mice. The contents of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid and isovaleric acid in CGSGCG group were 2.49-, 1.74-, 3.31-, 1.93-, 1.36- and 1.30-fold than that of the model group. Moreover, administration of CGSGCG up-regulated the expression of INSR/IRS-1/PI3K/AKT/GLUT4 and mTOR but down-regulated the P38MAPK expression. Furthermore, the abundance of beneficial bacteria such as Verrucomicrobia, Proteobacteria, Osillibacter, Dubosiella and Lactococcus in intestinal tract increased, indicating that CGSCGG regulated and improved the bacterial community structure of T2D mice, which were closely related to glycometabolism.

Analytical validation of a reverse transcriptase droplet digital PCR (RT-ddPCR) for quantitative detection of infectious hematopoietic necrosis virus.

Infectious hematopoietic necrosis virus (IHNV) is an important pathogen of salmonid fishes. A validated universal reverse transcriptase quantitative PCR (RT-qPCR) assay that can quantify levels of IHNV in fish tissues has been previously reported. In the present study, we adapted the published set of IHNV primers and probe for use in a reverse-transcriptase droplet digital PCR (RT-ddPCR) assay for quantification of the virus in fish tissue samples. The RT-ddPCR and RT-qPCR assays detected 13 phylogenetically diverse IHNV strains, but neither assay produced detectable amplification when RNA from other fish viruses was used. The RT-ddPCR assay had a limit of detection (LOD) equating to 2.2 plaque forming units (PFU)/µl while the LOD for the RT-qPCR was 0.2 PFU/µl. Good agreement (69.4-100%) between assays was observed when used to detect IHNV RNA in cell culture supernatant and tissues from IHNV infected rainbow trout (Oncorhynchus mykiss) and arctic char (Salvelinus alpinus). Estimates of RNA copy number produced by the two assays were significantly correlated but the RT-qPCR consistently produced higher estimates than the RT-ddPCR. The analytical properties of the N gene RT-ddPCR test indicated that this method may be useful to assess IHNV RNA copy number for research and diagnostic purposes. Future work is needed to establish the within and between laboratory diagnostic performance of the RT-ddPCR assay.

Identification of Infectious Salmon Anaemia Virus from Imported Iced Atlantic Salmons.

Infectious Salmon anaemia virus (ISAV) has become a threat to the salmon industry worldwide and has caused considerable economic loss. In the present study, 9 suspect cases of ISAV infection were identified from iced Atlantic salmons imported from Norway in 2014 through Shenzhen port (Shenzhen, China) using methods recommended by the World Organization for Animal Health. However, the results of virus isolation were negative., Based on the sequence analysis of ISAV segment 6, the 9 ISAV isolates belonged to the HPRO type, had high homology (98.3%~100.0%) and closest relationship with Norway strains. We identified the 9 positive HPRO ISAVs from 491 iced Atlantic salmons (1. 8%). Therefore, we should strengthen the quarantine of iced Atlantic salmons from Norway in case of HPRO ISAV into China.

Determination of the complete genome sequence of infectious hematopoietic necrosis virus (IHNV) Ch20101008 and viral molecular evolution in China.

This study determined the complete genomic sequence of the infectious hematopoietic necrosis virus (IHNV) strain Ch20101008 isolated from farmed brook trout (Salvelinus fontinalis) that died from a disease caused by the virus in northeast China. The sequence was determined from 10 overlapping clones obtained through RT-PCR amplification. The whole genome length of Ch20101008 comprised 11,129 nucleotides (nt), and the overall organization was typical of that observed for all other IHNV strains. The phylogenetic analysis results of the 65 IHNV glycoprotein genes and 47 IHNV partial nucleoprotein genes presented five major genogroups (J, U, L, E and M). The J genogroup included the J Nagano and J Shizuoka subgroups. The IHNV Ch20101008 strain belonged to the J Nagano subgroup of the J genogroup and was significantly related to other Chinese IHNV strains. All Chinese IHNV isolates are monophyletic, with a recent common ancestor, except for the BjLL strain. The N, P, M, G, NV and L genes of Ch20101008 were compared with the available IHNV sequences in GenBank. The results indicated that 198 nt were substituted, 53 of which exhibited amino acid change in the Ch20101008 genome. An adenine nucleotide deletion was found at position 4959 of the 5' UTR of the L gene. In the G gene, specific nucleotides and amino acid variations of the Chinese IHNV strains were observed when compared with 23 isolates from other countries. Of the 15 nucleotide sites that changed, seven resulted in amino acid substitution. The data further demonstrated that the J genogroup IHNV was introduced to and evolved in salmon farm environments in China.

Selection and characterization of single-chain recombinant antibodies against infectious haematopoietic necrosis virus from mouse phage display library.

Six single-chain fragment variable (scFv) antibodies against infectious haematopoietic necrosis virus (IHNV) were selected from an antibody phage display library by phage display technology. The soluble scFv antibodies showed a molecular weight 32kDa by Western blot. Dot blot analysis revealed that the six scFv antibodies could recognize IHNV. For enzyme linked immunosorbent assay (ELISA), four scFv antibodies (P1A4, P1A12, P1D5 and P3E2) showed cross-reactivity with spring viraemia of carp virus (SVCV). However, none of the six scFv antibodies had cross-reaction with Pike fry rhabdovirus (PFRV), Soft-shelled turtle iridovirus (STIV), viral haemorrhagic septicemia virus (VHSV), or viral nervous necrosis virus (VNNV). Indirect immunofluorescence results showed that all of these scFv antibodies reacted positively with virus in the IHNV-infected cells. These scFv antibodies will be useful in diagnostic test development and pathogenesis studies for IHNV.

Development and evaluation of a loop-mediated isothermal amplification assay for diagnosis of Cyprinid herpesvirus 2.

Goldfish Haematopoietic Necrosis caused by Cyprinid herpesvirus 2 (CyHV-2) is a severe fish disease with high level of mortality. This is the first study on detection of this disease by loop-mediated isothermal amplification (LAMP). A set of six primers targeting terminase gene (accession no. EU349285.1) was determined after a serial of tests. Detection limit was 1.09 × 10(-4)µg/µL, which was superior to conventional PCR and real-time PCR. No cross reaction with 28 other viruses or bacteria commonly found in fish was observed. The application of commercial kit and instrument for the LAMP assay could reduce the risk of cross contamination, which is suitable for detection of infection under field conditions.

Complete sequence of a viral nervous necrosis virus (NNV) isolated from red-spotted grouper (Epinephelus akaara) in China.

A nodavirus isolated from red-spotted grouper (Epinephelus akaara) larvae in China has been subjected to genome analysis. The full-length genome sequences of RNA1 and RNA2 were determined, and the 5'-non-coding region (NCR) and 3'NCR sequences were determined by 5' rapid amplification of cDNA ends (RACE) and 3'RACE. RNA1 is 3,103 nt in length and contains a 982-amino-acid open reading frame (ORF) encoding protein A with a calculated molecular mass of 110.74 kDa. RNA2 is 1,433 nt long and contains a 338-amino-acid major ORF encoding coat protein with a calculated molecular mass of 37.059 kDa. Multiple alignment and phylogenetic analysis clearly supported including this virus in the species Redspotted grouper nervous necrosis virus, genus Betanodavirus, family Nodaviridae.

Evaluation of a loop-mediated isothermal amplification assay for rapid diagnosis of soft-shelled turtle iridovirus.

Softshelled turtle iridovirus (STIV) is the first Asian iridovirus isolated from reptiles, which infects soft-shelled turtles severely and leads to "Red neck disease" associated with high mortality. A set of four specific primers was designed by targeting the STIV Thymidine kinase (TK) gene and amplified STIV DNA specifically under optimized amplification conditions at 63°C for 60 min. The sensitivity of the loop-mediated isothermal amplification (LAMP) assay was found to be 20 copies/µl of STIV DNA. To evaluate the application of the LAMP assay for detection of STIV in clinical samples, 223 samples suspected of STIV infection from turtle tissues were tested by the LAMP assay and by cell-based virus isolation. A 78.5% concordance was observed between the results of the two methods. In this study, a robust and simple LAMP assay for rapid detection of STIV was developed and evaluated, which is the first suitable for potential diagnosis and helping to monitor STIV infections in the aquaculture industry.

Characterization of the complete genome sequence of pike fry rhabdovirus.

The complete genome sequence of pike fry rhabdovirus (PFRV), consisting of 11,097 nucleotides, was determined. The genome contains five genes, encoding the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA-dependent RNA polymerase (L) protein in the order 3'-N-P-M-G-L-5'. 3' leader- and 5' trailer-sequences in the PFRV genome show inverse complementarity. The PFRV proteins share the highest homology to the proteins of spring viremia of carp virus (SVCV), ranging from 55.3 to 91.4%. Phylogenetic analysis of the five proteins showed that PFRV clusters with SVCV and is closely related to the mammalian vesiculoviruses, 903/87, STRV and SCRV.

[Comparison of characteristics of SVCV strains isolated in China and in Europe].

In the nationwide epidemiological investigation, SVCV-741 was for the first time isolated in Beijing region, China in 2003, and designated as SVCV Asian strain. In this paper, we compared SVCV-741 (Asian strains isolated in China) with SVCV-10/3 (Europe reference strain) on their physico-chemical, biological and morphological characteristics. The results indicated that there were no distinct differences between two SVCV strains on phycico-chemical and morphological characteristics. The main existing differences were: (1) The stability of SVCV-741 to temperature in cell culture was higher than that of SVCV-10/3, which might have some evolutionary and biological implication of SVCV; (2) No SVC outbreak ever occurred caused by SVCV-741;Furthermore we found that both SVCV-741 and SVCV-10/3 grew faster and produced higher virus titer in CO cells than other cell lines. It indicated that CO cell lines might be useful tool for SVCV research.

Development of a sensitive and quantitative assay for spring viremia of carp virus based on real-time RT-PCR.

A one-step real-time reverse transcription-polymerase chain reaction (RT-PCR) using a TaqMan probe to quantitatively detect spring viremia of carp virus (SVCV) is described. In this assay, a pair of primers amplifying an 81-bp DNA fragment and a TaqMan probe was designed targeting the conserved region at the SVCV glycoprotein (G) gene. To avoid the disadvantages arising from plasmids, an extension adding a T7 phage polymerase promoter to the 5' end of the antisense primer was carried out to obtain viral cRNA. Standardized cycle threshold (Ct) values for 10-fold serial dilutions of SVCV cRNA were achieved by real-time RT-PCR and used to create standard curves. A regression line between the mean Ct values and viral template concentrations over a 1:10(7) dilution range with an r(2) value (0.9916) and a slope (-3.36) and the coefficient of variation (intra- or inter-assay is <2% and <4%, respectively) indicated that the assay was highly reproducible. The assay was specific to SVCV and there was no cross-reactivity with other fish viruses (viral hemorrhagic septicemia virus, VHSV; infectious pancreatic necrosis virus, IPNV; grass carp reovirus, GCRV; epizootic haematopoietic necrosis virus, EHNV). The standard curve allows precise absolute quantitation and shows that the detection limit of the assay is 40 copies of the viral RNA. This one-step RT-PCR assay was evaluated using 60 clinical common carp samples collected during the year 2006, indicating such technology offers considerable advantages over conventional RT-PCR methods in current routine use for SVCV surveillance. This is the first report of the development of a one-step TaqMan RT-PCR for SVCV detection.

Simultaneous detection of three fish rhabdoviruses using multiplex real-time quantitative RT-PCR assay.

Spring viremia of carp virus (SVCV), infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are three important fish rhabdoviruses, causing serious Office International des Epizooties (OIE) classified diseases in wild and farmed fish. Here, a new multiplex real-time quantitative RT-PCR (mqRT-PCR) assay was developed for simultaneous detection, identification and quantification of these three rhabdoviruses. The sets of primers and probes were targeted to conserved regions of glycoprotein (G) gene of SVCV, nucleoprotein (N) gene of IHNV and G gene of VHSV and used to amplify. The sensitivity, specificity and interference test of mqRT-PCR assay was analyzed. It was shown that the detection levels of 100 copies of SVCV, 220 copies of IHNV and 140 copies of VHSV were achieved, and there was no non-specific amplification and cross-reactivity using RNA of pike fry rhabdovirus (PFRV), infectious pancreatic necrosis virus (IPNV) and grass carp reovirus (GCRV). A total of 80 clinical fish samples were tested using the mqRT-PCR assay and the results were confirmed by antigen-capture ELISA and cell culture assay. This assay has the potential to be used for both research applications and diagnosis.