Peniapyrones A-I, Cytotoxic Tricyclic-Fused α-Pyrone Derivatives from an Endophytic Penicillium brefeldianum F4a.

Five cyclopenta[d]pyrano[4,3-b]pyran-1,7(6H)-dione 6/6/5-fused tricyclic ring system containing metabolites peniapyrones A-E (1-5), and four previously undescribed cyclopenta[4,5]furo[3,2-c]pyran-1-one 6/5/5-fused tricyclic ring system containing compounds peniapyrones F-I (6-9), were isolated from the endophytic Penicillium brefeldianum F4a. Their structures, including absolute configurations, were determined through spectroscopic analysis and quantum chemical calculations. Peniapyrones D (4) and E (5) were a pair of diastereoisomers. Compounds 1, 3, and 5-9 showed cytotoxic activity against AsPC-1, CRL-2234, and MCF-7 cancer cell lines. Compounds 1, 3, 6, 8, and 9 inhibited the Kirsten rat sarcoma viral oncogene homologue (KRAS) mutant AsPC-1 cell line.

5-(4-Hydroxyphenyl)-3H-1,2-dithiole-3-thione derivatives of brefeldin A: Design, synthesis and cytotoxicity in MDA-MB-231 human breast cancer cells.

27 novel 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione derivatives of brefeldin A were designed and synthesized to make them more conducive to the cancer treatment. The antiproliferative activity of all the target compounds was tested against six human cancer cell lines and one human normal cell line. Compound 10d exhibited nearly the most potent cytotoxicity with IC50 values of 0.58, 0.69, 1.82, 0.85, 0.75, 0.33 and 1.75 µM against A549, DU-145, A375, HeLa, HepG2, MDA-MB-231 and L-02 cell lines. Moreover, 10d inhibited metastasis and induced apoptosis of MDA-MB-231 cells in a dose-dependent manner. The potent anticancer effects of 10d were prompted based on the aforementioned results, the therapeutic potential of 10d for breast cancer was worth further exploration.

Genome Mining and Molecular Networking Guided Isolation of Antimycin Analogs with Antifeedant Activities from the Deep-Sea-Derived Streptomyces sp. NA13.

Polyphagous insects could affect agricultural production, which leads to serious economic losses. Due to the negative effects of synthesized insecticides, finding eco-friendly and new biopesticides is emergent. To develop natural origin insecticides, an integrative approach combining antifeedant activity screening, genome mining, and molecular networking has been applied to discover antifeedant secondary metabolites from Streptomyces sp. NA13, which leads to the isolation of a novel antimycin Q (1) and six known antimycin analogs (antimycins A1a, A2a, A3a, A4a, A7a, and N-formylantimycic acid methyl ester, 2-7). Their structures were identified by high-resolution mass spectrometry (HR-MS) and nuclear magnetic resonance (NMR) spectroscopic. The absolute configuration of 1 was elucidated by the comparison of coupling constant, electronic circular dichroism (ECD) analysis, and NMR calculations. 1-6 exhibited different levels of antifeedant activities against Helicoverpa armigera, especially 1-4. At the same time, the antifeedant activity of antimycin was reported firstly.

Design and Synthesis of Brefeldin A-Isothiocyanate Derivatives with Selectivity and Their Potential for Cervical Cancer Therapy.

Brefeldin A has a wide range of anticancer activity against a variety of tumor cells. Its poor pharmacokinetic properties and significant toxicity seriously hinder its further development. In this manuscript, 25 brefeldin A-isothiocyanate derivatives were designed and synthesized. Most derivatives showed good selectivity between HeLa cells and L-02 cells. In particular, 6 exhibited potent antiproliferative activity against HeLa cells (IC50 = 1.84 µM) with no obvious cytotoxic activity to L-02 (IC50 > 80 µM). Further cellular mechanism tests indicated that 6 induced HeLa cell cycle arrest at G1 phase. Cell nucleus fragmentation and decreased mitochondrial membrane potential suggested 6 could induce apoptosis in HeLa cells through the mitochondrial-dependent pathway.

Genome mining and metabolic profiling illuminate the chemistry driving diverse biological activities of Bacillus siamensis SCSIO 05746.

Bacillus spp. are important producers of bioactive natural products with potential applications in medicine and agriculture. Bacillus sp. SCSIO 05476 from a deep-sea sediment exhibits broad-spectrum antimicrobial activities and strong cytotoxic activity. Here, an integrative approach combining genome mining and metabolic profiling has been applied to decipher the chemical origins of this strain's varied and significant biological activities. First, genome mining revealed 19 candidate gene clusters encoding the biosynthesis of diverse secondary metabolites. Then, a series of bacillibactins, fengycins, bacillomycins, surfactins, bacillaenes, macrolactins, and related species were found by LC-DAD-MS. Finally, three new linear bacillibactins, linbacillibactins A-C (1-3), along with 11 known secondary metabolites, bacillibactin (4), normal-C13 Val7 surfactin (5), anteiso-C13 Leu7 surfactin (6), iso-C14 Leu7 surfactin (7), normal-C14 Leu7 surfactin (8), anteiso-C14 Leu7 surfactin (9), macrolactin D (10), normal-C14 bacillomycin D (11), iso-C16 bacillomycin D (12), normal-C17 bacillomycin D (13), and iso-C17 bacillomycin D (14), were obtained and elucidated by bioactivity and structure-guided isolation from the fermentation of strain SCSIO 05746. Among them, new compounds 1-3 show significant siderophore activities comparable to that of bacillibactin (4), compounds 13 and 14 exhibit strong cytotoxic activity. At the same time, the strain classification status was confirmed by genomic analyses, and the complete genome sequence of Bacillus siamensis was presented firstly. This study provides a foundation for understanding the mechanisms driving SCSIO 05746's multiple bioactivities and demonstrates a successful way of discovering bioactive metabolites using a combination of genome mining and metabolic profiling methods.

Characterization and abolishment of the cyclopiazonic acids produced by Aspergillus oryzae HMP-F28.

Extracellular alkalinization and H2O2 production are important early events during induced resistance establishment in plants. In a screen for metabolites as plant resistance activators from 98 fungal isolates associated with marine sponge Hymeniacidon perleve, the cyclopiazonic acids (CPAs) produced by Aspergillus oryzae HMP-F28 induced significant extracellular alkalinization coupled with augmented H2O2 production in tobacco cell suspensions. Bioassay-guided fractionation led to the isolation and structural elucidation of a new CPA congener (4, 3-hydroxysperadine A) and three known ones (1-3). To construct a mutasynthetic strain to generate unnatural CPA analogues, a hybrid pks-nrps gene (cpaS) was disrupted to abolish the production of the critical precursor of cyclo-acetoacetyl-L-tryptophan (cAATrp) and all the downstream CPA products. Elimination of cAATrp will allow cAATrp mimics being processed by the CPA biosynthetic machinery to produce CPA derivatives with designed structural features.

Nocardiopsis oceani sp. nov. and Nocardiopsis nanhaiensis sp. nov., actinomycetes isolated from marine sediment of the South China Sea.

Two actinomycete strains, designated 10A08AT and 10A08BT, were isolated from marine sediment samples of the South China Sea and their taxonomic positions were determined by a polyphasic approach. The two Gram-stain-positive, aerobic strains produced branched substrate mycelium and aerial hyphae, and no diffusible pigment was produced in the media tested. At maturity, spore chains were formed on aerial hyphae and all mycelium fragmented with age. Whole-cell hydrolysates of both strains contained meso-diaminopimelic acid and no diagnostic sugars. Their predominant menaquinones (>10 %) were MK-9(H4), MK-9(H6) and MK-10(H6) for strain 10A08AT and MK-9(H4), MK-9(H6), MK-10(H4) and MK-10(H6) for strain 10A08BT. The polar lipids detected from the two strains were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylcholine and unknown phosphoglycolipids and phospholipids. The major fatty acids (>10 %) of both strains were iso-C16 : 0 and summed feature 4 (iso-C17 : 1 I and/or anteiso-C17 : 1 B). The genomic DNA G+C contents of strains 10A08AT and 10A08BT were 70.9 and 71.6 mol%, respectively. On the basis of 16S rRNA gene sequence similarities, the two strains were shown to be most closely related to species of the genus Nocardiopsis. DNA­DNA hybridization relatedness values of < 70 % between these two isolates and their closest neighbour, Nocardiopsis terrae YIM 90022T, and between the two strains supported the conclusion that they represent two novel species. Based on phylogenetic analysis and phenotypic and genotypic data, it is concluded that the two isolates belong to the genus Nocardiopsis, and the names Nocardiopsis oceani sp. nov. (type strain 10A08AT = DSM 45931T = BCRC 16951T) and Nocardiopsis nanhaiensis sp. nov. (type strain 10A08BT = CGMCC 47227T = BCRC 16952T) are proposed.

Structure determination of two new indole-diterpenoids from Penicillium sp. CM-7 by NMR spectroscopy.

Two new indole-diterpenoids 4b-deoxy-1'-O-acetylpaxilline (1) and 4b-deoxypenijanthine A (2) were isolated from the fermentation broth and the mycelia of the soil fungus Penicillium sp. CM-7, along with three known structurally related compounds, 1'-O-acetylpaxilline (3), paspaline (4) and 3-deoxo-4b-deoxypaxilline (5). The structures of compounds 1 and 2 were elucidated by extensive spectroscopic methods, especially 2D NMR, and their absolute configurations were suggested on the basis of the circular dichroism spectral analysis and the NOESY data.

Impact of Temperature Elevation on Microbial Communities and Antibiotic Degradation in Cold Region Soils of Northeast China.

This study systematically investigated the effects of temperature changes on the degradation of antibiotics in soil, as well as the alterations in microbial community structure and aggregation, through a field warming experiment in a greenhouse. Compared to non-warming soil, the warming treatment significantly accelerated the degradation rate of tetracyclines during soil freezing and mitigated the impact of environmental fluctuations on soil microbial communities. The greenhouse environment promoted the growth and reproduction of a wide range of microbial taxa, but the abundance of Myxococcota was positively correlated with antibiotic concentrations in both treatments, suggesting a potential specific association with antibiotic degradation processes. Long-term warming in the greenhouse led to a shift in the assembly process of soil microbial communities, with a decrease in dispersal limitation and an increase in the drift process. Furthermore, co-occurrence network analysis revealed a more loosely structured microbial community in the greenhouse soil, along with the emergence of new characteristic taxa. Notably, more than 60% of the key taxa that connected the co-occurrence networks in both groups belonged to rare taxa, indicating that rare taxa play a crucial role in maintaining community structure and function.

Description of Streptomonospora sediminis sp. nov. and Streptomonospora nanhaiensis sp. nov., and reclassification of Nocardiopsis arabia Hozzein & Goodfellow 2008 as Streptomonospora arabica comb. nov. and emended description of the genus Streptomonospora.

Two actinomycete strains isolated from marine sediment samples, designated YIM M11335(T) (from the Indian Ocean) and 12A09(T) (from the South China Sea), were obtained and examined by a polyphasic approach. The two Gram-staining-positive, aerobic strains produced branched substrate mycelia and aerial hyphae that were not fragmented, and no diffusible pigment was produced on the media tested. At maturity, spore chains and single spores were formed on aerial hyphae and substrate mycelium, respectively. Whole-organism hydrolysates of both strains contained meso-diaminopimelic acid and the diagnostic sugars glucose and galactose. Their predominant menaquinones were MK-10(H4), MK-10(H6), MK-11(H4), MK-11(H6) and MK-11(H8) for strain YIM 11335(T) and MK-10(H4), MK-10(H6), MK-11(H4), MK-11(H6) and MK-11(H8) for strain 12A09(T). The polar lipids detected in the two strains were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol mannoside, phosphatidylinositol, phosphatidylcholine, an unknown phosphoglycolipid and several unknown glycolipids, phospholipids and polar lipids. The major fatty acids (>10%) were iso-C16 : 0 and C16:0 for strain YIM 11335(T) and iso-C16:0 for strain 12A09(T). The G+C contents of the genomic DNA of strains YIM 11335(T) and 12A09(T) were 70.7% and 74.4%, respectively. DNA-DNA hybridization relatedness values of these two isolates with the type strains Nocardiopsis arabia DSM 45083(T) and Streptomonospora halophila YIM 91355(T) supported the hypothesis they are representatives of two different species. Based on phylogenetic analysis, phenotypic and genotypic data, it is concluded that the two isolates belong to the genus Streptomonospora of the family Nocardiopsaceae and that the type strain of N. arabia should be reclassified as a representative of Streptomonospora arabica comb. nov. The names proposed for the two novel species are Streptomonospora sediminis sp. nov. (type strain YIM M11335(T) = DSM 45723(T) = CCTCC AB 2012051(T)) and Streptomonospora nanhaiensis sp. nov. (type strain 12A09(T) = KCTC 29145(T) = CCTCC AB 2013140(T)), respectively. An emended description of the genus Streptomonospora is also proposed in the light of the new data.

New spirotetronate antibiotics, lobophorins H and I, from a South China Sea-derived Streptomyces sp. 12A35.

Strain 12A35 was isolated from a deep-sea sediment collected from the South China Sea and showed promising antibacterial activities. It was identified as Streptomyces sp. by the 16S rDNA sequence analysis. Bioassay-guided fractionation using HP20 adsorption, flash chromatography over silica gel and octadecylsilyl (ODS) and semi-preparative HPLC, led to the isolation and purification of five metabolites from the fermentation culture of 12A35. Two new spirotetronate antibiotics, lobophorins H (1) and I (2), along with three known analogues, O-ß-kijanosyl-(1→17)-kijanolide (3), lobophorins B (4) and F (5) were characterized by 1D, 2D-NMR and MS data. These compounds exhibited significant inhibitory activities against Bacillus subtilis. Compounds 1 and 5 exhibited moderate activities against Staphylococcus aureus. In particular, the new compound lobophorin H (1) showed similar antibacterial activities against B. subtilis CMCC63501 to ampicillin.

Polyketides from the halotolerant fungus Myrothecium sp. GS-17.

Two new polyketides, myrothecol (1) and 5-hydroxy-3-methyl-4-(1-hydroxylethyl)-furan-2(5H)-one (2), were isolated from the fermentation broth of the halotolerant fungus Myrothecium sp. GS-17 along with three known compounds, 5-hydroxyl-3-[(1S)-1-hydroxyethyl]-4-methylfuran-2(5H)-one (3), 3,5-dimethyl-4- hydroxylmethyl-5-methoxyfuran-2(5H)-one (4), and 3,5-dimethyl-4-hydroxymethyl-5- hydroxyfuran-2(5H)-one (5). Compound 1 is the first natural occurring polyketide with a unique furylisobenzofuran skeleton. The structures of these compounds were established via extensive spectroscopic analyses including 1D-, 2D-NMR, HRESI-MS, and crystal X-ray diffraction analysis.

Structure determination of two new trichothecenes from a halotolerant fungus Myrothecium sp. GS-17 by NMR spectroscopy.

Two new trichothecenes, named 8α-hydroxyroridin H and myrothecin A, along with six known compounds, 8α-acetoxy roridin H, isororidin K, verrucarin A, verrucarin J, verrucarin L and 8α-acetoxy verrucarin L, were isolated from the fermentation broth of a halotolerant fungus Myrothecium sp. GS-17, which was separated from the soil sample of a salina. Structure elucidation and NMR signal assignments were achieved on the basis of spectroscopy. In addition, compounds 1 and 2 were active against plant pathogenic fungi Rhizoctonia solani and Fusarium oxysporum.

Penipentenone A and brefeldin A derivatives potently inhibit KRAS mutant cancer cells from an endophytic fungus Penicillium brefeldianum F4a.

Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation is one of the most important carcinogenic factors in many solid tumors, which leads to the poor prognosis and therapy resistance of cancer. In order to develop direct or indirect KRAS inhibitors, one unique asymmetric dicyclopentenone penipentenone A, three undescribed brefeldin A (BFA) derivatives, and five known BFA derivatives were discovered from the endophytic fungus Penicillium brefeldianum guided by LC-MS/MS and cytotoxic activities. Their structures were elucidated by optical rotation, mass spectrometry, and NMR spectroscopic data. The absolute configurations of four undescribed compounds were elucidated by comparison of the experimental and calculated ECD spectra. The antiproliferative activities of obtained compounds against three KRAS mutant tumor cell lines and two BFA derivative-sensitive cell lines were evaluated. Besides 4-epi-15-epi-brefeldin A, the other compounds showed significant inhibitory activities against those tumor cell lines with IC50 values ranging from 0.82 to 18.87 µM. Intriguingly, penipentenone A selectively inhibited KRAS mutant cancer cells SW620 (KRASG12V) and ASPC-1 (KRASG12D). BFA and four derivatives showed potent cytotoxic activities against all selected tumor cell lines H358 (KRASG12C), SW620 (KRASG12V), ASPC-1 (KRASG12D), PC-3, and HepG-2. These findings will provide undescribed lead compounds for developing drugs that target KRAS mutations.

Nigirpexin E, a new azaphilone derivative with anti-tobacco mosaic virus activity from soil-derived fungus Trichoderma afroharzianum LTR-2.

A new compound classified as one new azaphilone derivative, nigirpexin E (1), was obtained from the soil-derived fungus Trichoderma afroharzianum LTR-2, together with seven known compounds (2-8). The structures of 1-8 were determined by their HRESIMS, optical rotation, and NMR spectroscopic data. The absolute configuration of nigirpexin E (1) was determined on the basis of comparisons of experimental and theoretically calculated ECD spectra. Compound 3 was firstly isolated from Trichoderma. Bioactivities of the isolated compounds were assayed their anti-tobacco mosaic virus (anti-TMV) activities. The results showed that compound 1 exhibited significant inactivation effect against TMV with an inhibition rate of 67.25% (0.5 mg ml-1), which was higher than that of positive control ribavirin (56.74%). This is the first report of the anti-TMV activity of azaphilone derivatives.

Cytotoxic and antimicrobial activities of secondary metabolites isolated from the deep-sea-derived Actinoalloteichus cyanogriseus 12A22.

A new deep-sea-derived actinomycete 12A22 was isolated from the sediment of the South China Sea which showed potential cytotoxic and antimicrobial activities. The actinomycete was identified as Actinoalloteichus cyanogriseus by investigating morphological characteristics and phylogenetic analyses based on its 16S rRNA gene sequence. Two compounds, cyclo-(L-Pro-D-Pro-L-Tyr-L-Tyr) (1) and 2-hydroxyethyl-3-methyl-1,4-naphthoquinone (2), were isolated and characterized from the fermentation broth of the strain 12A22. Compound 2 exhibited significant inhibitory activities against a variety of phytopathogenic fungi (Fusarium oxysporum f. sp. cucumerinum, Setosphaeria turcica, and Botrytis cinerea) and Gram-positive bacterium (Bacillus subtilis). In particular, this compound showed better antifungal activity against Botrytis cinerea than positive control amphotericin B. Besides, compound 2 showed moderate cytotoxic activity against human breast cancer MDA-MB-435 cells with IC50 10.59 µM, weaker than the positive control diaminedichloroplatinum with 5.91 µM. Our results suggested that this naphthoquinone could be used as a potential antimicrobial and antitumor agent. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02846-0.

Novel Antioxidants and α-Glycosidase and Protein Tyrosine Phosphatase 1B Inhibitors from an Endophytic Fungus Penicillium brefeldianum F4a.

Oxidative stress plays a very important role in the progression of diabetes and its complications. A therapeutic agent that is both antidiabetic and antioxidant would be the preferred choice for the treatment of diabetes. The crude extract of the endophytic fungus Penicillium brefeldianum F4a has significant antioxidant and α-glycosidase and protein tyrosine phosphatase 1B (PTP1B) inhibition activities. Chemical investigation of P. brefeldianum F4a using an activity-guided isolation led to the discovery of three new compounds called peniorcinols A-C (1-3) along with six known compounds: penialidins A (4), penialidin F (5), myxotrichin C (6), riboflavin (7), indole-3-acetic acid (8), and 2-(4-hydroxy-2-methoxy-6-methylphenyl) acetic acid (9). Their chemical structures were established by their NMR and HRESIMS. The absolute configurations of 1 and 3 were determined by experimental and calculated electronic circular dichroism (ECD). Their antioxidant activities were evaluated by DPPH• and ABTS•+ scavenging assays. Compounds 1-6 and 8-9 showed moderate to strong free radical scavenging activities. Significantly, 4-6 exhibited more potent ABTS•+ scavenging activity than that of the positive control. Their α-glycosidase and PTP1B inhibition activities were tested. Among them, compound 3 showed α-glucosidase inhibition activity, and compounds 7 and 8 showed PTP1B inhibitory activity for the first time. It is worth noting that 3 and 8 displayed both antioxidant and α-glycosidase or PTP1B inhibition activities. These finding suggest that compounds 3 and 8 could be used as lead compounds to generate new potent drugs for the treatment of oxidative stress-related diabetes.

Structure-based molecular networking for the target discovery of novel germicidin derivatives from the sponge-associated streptomyces sp. 18A01.

Four new α-pyrone derivatives, named germicidins P-S (1-4) along with nine known analogues (5-13) were discovered from the sponge-associated Streptomyces sp. 18A01 guided by Global Natural Products Social (GNPS) molecular networking, the LC-DAD-MS profile, and hexokinase II (HK2) inhibitory activity. The structures of 1-13 were elucidated by analysis of their HRMS, optical rotation, and NMR spectroscopic data. The absolute configurations of germicidin P (1) and germicidin Q (2) were determined on the basis of comparisons of experimental and theoretically calculated ECD spectra. Bioactivities of the isolated compounds were assayed against human HK2. The bioassay results showed that compounds 1-4 and 11-13 exhibited significant inhibitory activities against HK2, with IC50 values ranging from 5.1 to 11.0 µM. A molecular docking simulation demonstrated that these germicidins were docked in the inner active site tunnel of HK2. Interestingly, the amino residue Arg91 has a better binding affinity and efficacy than the amino residue Asn89 in the process of HK2 binding to the ligands, resulting in better hexokinase inhibitory activity. This result provided a valuable perspective for better understanding their HK2 inhibition activity.

Molecular cloning and sequence analysis of the duck enteritis virus UL5 gene.

Duck enteritis virus (DEV) is a herpesvirus that causes an acute, contagious, and fatal disease. In the present article, the DEV UL5 gene was cloned and sequenced from a vaccine virus. According to the consensus sequence of herpesvirus UL5 and UL3 gene degenerate oligonucleotide primers were designed and were used in the polymerase chain reaction (PCR) to amplify DNA products with 4577 bp in size. DNA sequence analysis revealed a 2568 bp open reading frame (ORF) encoding a 855 amino acid polypeptide homologous to herpesvirus UL5 proteins. The DEV UL5 gene has a base composition of 769 adenine (29.95%), 556 cytosine (21.65%), 533 guanine (20.76%) and 710 thymine (27.65%). Sequence comparison revealed that the nucleotide sequence of the DEV UL5 gene was highly similar to other alphaherpesviruses. Phylogenetic tree analysis showed that the fifteen herpesviruses viruses analyzed fell into four large groups, and the duck enteritis virus itself branched and was most closely related to meleagrid herpesvirus 1, gallid herpesvirus 2 and gallid herpesvirus 3 subtrees.

[Prokaryotic expression of N-terminal antigenic domain of duck plague virus gB protein and the establishment of putative indirect ELISA assay].

Based on the antigenic analysis of duck plague virus (DPV) gB protein, we designed a pair of primers to amplify the gene fragment encoding high antigenic domain of DPV N-terminal gB protein from the DPV genome. The cloned gene was digested with EcoR I and Hind III and then inserted into pET32a vector to obtain the recombinant pET-gB1 plasmid. The recombinant plasmid was transformed into E. coli BL21, and expressed in very high level after induced with IPTG. The expressed product was analyzed by SDS- PAGE and Western blotting. The result indicated that the fusion protein (pET-gB1) existed as inclusion body, which was about 42.4kDa and showed specific immunoreactivity with anti-DPV sera. The recombinant gB1 protein was purified with His-Bind resin protein purification procedure. Then an indirect ELISA was established to detect antibody against DPV with the purified gB1 protein as the coating antigen. The result showed that the optimal concentration of coated antigen was 6.5 microg/mL and the optimal dilution of serum was 1 : 80. The positive criterion of this ELISA assay was OD(the tested serum) > 0.4 and OD(the tested serum)/OD(the negative serum) > 2.0. The ELISA was done on 700 sera that were preserved in Shandong, Jiangsu Provinces, and were detected by igB1-ELISA and iDPV-ELISA with duck plague virus as the coating antigen respectively. The agreement ratio between the two methods was 95.6%.