Rapid detection of human adenovirus subgroup B using recombinase polymerase amplification assay.

Human adenovirus subgroup B (HAdV B) is one of the major pathogens of human respiratory virus infections, which has considerable transmission and morbidity in a variety of populations. Therefore, rapid and specific detection of HAdV B in clinical samples is essential for diagnosis. This study aimed to develop a product for rapid nucleic acid detection of HAdV B using recombinase polymerase amplification assay (RPA) and validate the performance of this method by using clinical samples. Results showed that this method achieved a lower limit of detection (LOD) of 10 copies/µL and had no cross-reactivity with other adenovirus subgroups or respiratory pathogens. In addition to high sensitivity, it can be completed within 30 min at 40 °C. There is no need to perform nucleic acid extraction on clinical samples. Taking qPCR as the gold standard, the RPA assay possessed a high concordance (Cohen's kappa, 0.896; 95% CI 0.808-0.984; P < 0.001), with a sensitivity of 87.80% and a specificity of 100.00%. The RPA assay developed in this study provided a simple and highly specific method, making it an important tool for rapid adenovirus nucleic acid detection and facilitating large-scale population screening in resource-limited settings.

Enterovirus 71 enters human brain microvascular endothelial cells through an ARF6-mediated endocytic pathway.

Infection of the central nervous system caused by enterovirus 71 (EV71) remains the main cause of death in hand-foot-and-mouth disease. However, the mechanism responsible for how EV71 breaks through the blood-brain barrier to infect brain cells has yet to be elucidated. By performing a high-throughput small interfering RNA (siRNA) screening and validation, we found that the infection of human brain microvascular endothelial cells (HBMECs) by EV71 was independent of the endocytosis pathways mediated by caveolin, clathrin, and macropinocytosis but dependent on ADP-ribosylation factor 6 (ARF6), a small guanosinetriphosphate (GTP)-binding protein of the Ras superfamily. The specific siRNA targeting ARF6 markedly inhibited HBMECs susceptibility to EV71. EV71 infectivity was inhibited by NAV-2729, a specific inhibitor of ARF6, in a dose-dependent manner. The subcellular analysis demonstrated the co-localization of the endocytosed EV71 and ARF6, while knockdown of ARF6 with siRNA remarkably influenced EV71 endocytosis. By immunoprecipitation assays, we found a direct interaction of ARF6 with EV71 viral protein. Furthermore, ARF1, another small GTP-binding protein, was also found to participate in ARF6-mediated EV71 endocytosis. Murine experiments demonstrated that NAV-2729 significantly alleviated mortality caused by EV71 infection. Our study revealed a new pathway by which EV71 enters the HBMECs and provides new targets for drug development.

Metabolism of an insecticide fipronil by soil fungus Cunninghamella elegans ATCC36112.

In this study, the metabolic pathway of the phenylpyrazole insecticide fipronil in Cunninghamella elegans (C. elegans) was investigated. Approximately 92% of fipronil was removed within 5 days, and seven metabolites were accumulated simultaneously. The structures of the metabolites were completely or tentatively identified by GC-MS and 1H, 13C NMR. To determine the oxidative enzymes involved in metabolism, piperonyl butoxide (PB) and methimazole (MZ) were used, and the kinetic responses of fipronil and its metabolites were determined. PB strongly inhibited fipronil metabolism, while MZ weakly inhibited its metabolism. The results suggest that cytochrome P450 (CYP) and flavin-dependent monooxygenase (FMO) may participate in fipronil metabolism. Integrated metabolic pathways can be inferred from the control and inhibitor experiments. Several novel products from the fungal transformation of fipronil were identified, and similarities between C. elegans transformation and mammalian metabolism of fipronil were compared. Therefore, these results will help to gain insight into the fungal degradation of fipronil and potential applications in fipronil bioremediation. At present, microbial degradation of fipronil is the most promising approach and maintains environmental sustainability. In addition, the ability of C. elegans to mimic mammalian metabolism will assist in illustrating the metabolic fate of fipronil in mammalian hepatocytes and assess its toxicity and potential adverse effects.

Metabolic pathway of tebuconazole by soil fungus Cunninghamella elegans ATCC36112.

Tebuconazole is the most widely used fungicide in agriculture. Due to its long half-life, tebuconazole residues can be found in the environment media such as in soil and water bodies. Here, the metabolic pathway of tebuconazole was studied in Cunninghamella elegans (C. elegans). Approximately 98% of tebuconazole was degraded within 7 days, accompanied by the accumulation of five metabolites. The structures of the metabolites were completely or tentatively identified by gas chromatography-mass spectrometry (GC-MS) and ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). To identify representative oxidative enzymes that may be involved in the metabolic process, treatment with piperonyl butoxide (PB) and methimazole (MZ) was performed. PB had a strong inhibitory effect on the metabolic reactions, while MZ had a weak inhibitory effect. The results suggest that cytochrome P450 (CYP) and flavin-dependent monooxygenase are involved in the metabolism of tebuconazole. Based on the results, we propose a metabolic pathway for the fungal metabolism of tebuconazole. Data are of interest to gain insight into the toxicological effects of tebuconazole and for tebuconazole bioremediation.

Biodegradation and metabolic pathway of quinalphos by Cunninghamella elegans ATCC36112.

Quinalphos is a long-term, wide-spectrum organophosphate insecticide with residual problems in the natural environment. Cunninghamella elegans (C. elegans) is a member of Mucoromycotina. Since the degradation products of its exogenous compounds are similar to those of mammals, it is often used to simulate the metabolism pathways of mammals. In this study, the detailed metabolic pathways of quinalphos were investigated with C. elegans. Quinalphos was degraded by 92% in 7 days, while ten metabolites were produced. The metabolites were analyzed and identified by GC-MS. To determine the responsible enzymes in quinalphos metabolism, piperonyl butoxide (PB) and methimazole included in the culture flasks, and the kinetic responses of quinalphos and its metabolites by C. elegans were measured. Results indirectly demonstrated that cytochrome P450 monooxygenases were involved in the metabolism of quinalphos, but that methimazole inhibited the metabolism less efficiently. Comprehensive metabolic pathways can be deduced from the detailed analysis of metabolite profiles in control and inhibitor assays.

H-NS represses transcription of the flagellin gene lafA of lateral flagella in Vibrio parahaemolyticus.

Swarming motility is ultimately mediated by the proton-powered lateral flagellar (laf) system in Vibrio parahaemolyticus. Expression of laf genes is tightly regulated by a number of environmental conditions and regulatory factors. The nucleoid-associated DNA-binding protein H-NS is a small and abundant protein that is widely distributed in bacteria, and H-NS-like protein-dependent expression of laf genes has been identified in Vibrio cholerae and V. parahaemolyticus. The data presented here show that H-NS acts as a repressor of the swarming motility in V. parahaemolyticus. A single σ28-dependent promoter was detected for lafA encoding the flagellin of the lateral flagella, and its activity was directly repressed by H-NS. Thus, H-NS represses swarming motility by directly acting on lafA. Briefly, this work revealed a novel function for H-NS as a repressor of the expression of lafA and swarming motility in V. parahaemolyticus.

Anti-BACE1 and Antimicrobial Activities of Steroidal Compounds Isolated from Marine Urechis unicinctus.

The human ß-site amyloid cleaving enzyme (BACE1) has been considered as an effective drug target for treatment of Alzheimer's disease (AD). In this study, Urechis unicinctus (U. unicinctus), which is a Far East specialty food known as innkeeper worm, ethanol extract was studied by bioassay-directed fractionation and isolation to examine its potential ß-site amyloid cleaving enzyme inhibitory and antimicrobial activity. The following compounds were characterized: hecogenin, cholest-4-en-3-one, cholesta-4,6-dien-3-ol, and hurgadacin. These compounds were identified by their mass spectrometry, ¹H, and 13C NMR spectral data, comparing those data with NIST/EPA/NIH Mass spectral database (NIST11) and published values. Hecogenin and cholest-4-en-3-one showed significant inhibitory activity against BACE1 with EC50 values of 116.3 and 390.6 µM, respectively. Cholesta-4,6-dien-3-ol and hurgadacin showed broad spectrum antimicrobial activity, particularly strongly against Escherichia coli (E. coli), Salmonella enterica (S. enterica), Pasteurella multocida (P. multocida), and Physalospora piricola (P. piricola), with minimal inhibitory concentration (MIC) ranging from 0.46 to 0.94 mg/mL. This is the first report regarding those four known compounds that were isolated from U. unicinctus and their anti-BACE1 and antimicrobial activity, highlighting the fact that known natural compounds may be a critical source of new medicine leads. These findings provide scientific evidence for potential application of those bioactive compounds for the development of AD drugs and antimicrobial agents.

Trachelogenin, a novel inhibitor of hepatitis C virus entry through CD81.

Although much progress has been made in antiviral agents against hepatitis C virus (HCV) in recent years, novel HCV inhibitors with improved efficacy, optimized treatment duration and more affordable prices are still urgently needed. Here, we report the identification of a natural plant-derived lignan, trachelogenin (TGN), as a potent entry inhibitor of HCV without genotype specificity, and with low cytotoxicity. TGN was extracted and purified from Caulis trachelospermi, a traditional Chinese herb with anti-inflammatory and analgesic effects. A crucial function of TGN was the inhibition of HCV entry during a post-binding step without affecting virus replication, translation, assembly and release. TGN blocked virus infection by interfering with the normal interactions between HCV glycoprotein E2 and the host entry factor CD81, which are key processes for valid virus entry. In addition, TGN diminished HCV cell-to-cell spread and exhibited additional synergistic effects when combined with IFN or telaprevir. In conclusion, this study highlights the effect of a novel HCV entry inhibitor, TGN, which has a target that differs from those of the current antiviral agents. Therefore, TGN is a potential candidate for future cocktail therapies to treat HCV-infected patients.

The role of lipid rafts in the early stage of Enterovirus 71 infection.

BACKGROUND/AIMS: Although it has been widely accepted that Enterovirus 71 (EV71) enters permissive cells via receptor-mediated endocytosis, the details of entry mechanism for EV71 still need more exploration. This study aimed to investigate the role of lipid rafts in the early stage of EV71 Infection. METHODS: The effect of cholesterol depletion or addition of exogenous cholesterol was detected by immunofluorescence assays and quantitative real-time PCR. Effects of cholesterol depletion on the association of EV71 with lipid rafts were determined by flow cytometry and co-immunoprecipitation assays. Localization and internalization of EV71 and its receptor were assayed by confocal microscpoy and sucrose gradient analysis. The impact of cholesterol on the activation of phosphoinositide 3'-kinase/Akt signaling pathway during initial virus infection was analyzed by Western-blotting. RESULTS: Disruption of membrane cholesterol by a pharmacological agent resulted in a significant reduction in the infectivity of EV71. The inhibitory effect could be reversed by the addition of exogenous cholesterol. Cholesterol depletion post-infection did not affect EV71 infection. While virus bound equally to cholesterol-depleted cells, EV71 particles failed to be internalized by cholesterol-depleted cells. EV71 capsid protein co-localized with cholera toxin B, a lipid-raft-dependent internalization marker. CONCLUSION: Lipid rafts play a critical role in virus endocytosis and in the activation of PI3K/Akt signaling pathway in the early stage of EV71 infection.

Potential Dermal Exposure to Flonicamid and Risk Assessment of Applicators During Treatment in Apple Orchards.

Exposure and risk assessments of flonicamid for applicators were performed in apple orchards in Korea. Fifteen experiments were done with two experienced applicators under typical field conditions using a speed sprayer. In this study, cotton gloves, socks, masks, and dermal patches were used to monitor potential dermal exposure to flonicamid, and personal air samplers with XAD-2 resin and glass fiber filter were used to monitor potential inhalation exposure. The analytical methods were validated for the limit of detection, limit of quantitation, reproducibility, linearity of the calibration curve, and recovery of flonicamid from various exposure matrices. The results were encouraging and acceptable for an exposure study. The applicability of XAD-2 resin was evaluated via a trapping efficiency and breakthrough test. During the mixing/loading, the average total dermal exposure was 22.6 µg of flonicamid, corresponding to 4.5×10(-5)% of the prepared amount. For the spraying, the potential dermal exposure was 9.32 mg, and the ratio to applied amount was 1.9 × 10(-2%). The primary exposed body parts were the thigh (2.90 mg), upper arm (1.75 mg), and lower leg (1.66 mg). By comparison, absorbable quantity of exposure was small, only 1.62 µg (3.2×10(-6)%). The margin of safety (MOS) were calculated for risk assessment, in all sets of trials, MOS > 1, indicating the exposure level of flonicamid was considered to be safe in apple orchards. Although this was a limited study, it provided a good estimate of flonicamid exposure for orchard applicators.

Multiresidue method for the determination of 227 pesticides in hot pepper (Capsicum annuum L.) by liquid chromatography with tandem mass spectrometry.

A high-throughput, rapid, and efficient modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) method with a simple cleanup procedure has been developed for simultaneously determining 227 pesticides in pepper samples by liquid chromatography with tandem mass spectrometry (running time: 10 min). Pesticide residues were extracted/partitioned with an acetonitrile/DisQuE QuEChERS pouch, and the resulting samples were cleaned up with different methods: dispersive solid-phase extraction with primary secondary amines or multiwalled carbon nanotubes and graphitized carbon solid mini cartridge column. The results indicated that multiwalled carbon nanotubes dispersive sorbents achieved the best recoveries and had less matrix interference. The numbers of pesticides with a recovery in the range of 70-120% were 199 at a spiked level of 40 µg/kg. The correlation coefficients (r(2)) for 227 pesticides were above 0.99, while the limits of quantitation of pesticides in pepper samples ranged from 0.13 to 13.51 µg/kg (S/N = 10), and the limits of detection ranged from 0.04 to 4.05 µg/kg (S/N = 3). The relative standard deviations of approximately 197 pesticides were below 20% at spiked levels of 40 µg/kg. Based on these results, the proposed method was chosen as the most suitable cleanup procedure for the determination of multiresidue pesticides in pepper samples.

Three different functional microdomains in the hepatitis C virus hypervariable region 1 (HVR1) mediate entry and immune evasion.

High genetic heterogeneity is an important characteristic of hepatitis C virus (HCV) that contributes to its ability to establish persistent infection. The hypervariable region 1 (HVR1) that includes the first 27 amino acid residues of the E2 envelope glycoprotein is the most variable region within the HCV polyprotein. HVR1 plays a major role in both HCV cell entry and immune evasion, but the respective contribution of specific amino acid residues is still unclear. Our mutagenesis analyses of HCV pseudoparticles and cell culture-derived HCV using the H77 isolate indicate that five residues at positions 14, 15, and 25-27 mediate binding of the E2 protein to the scavenger receptor class B, type I receptor, and any residue herein is indispensable for HCV cell entry. The region spanning positions 16-24 contains the sole neutralizing epitope and is dispensable for HCV entry, but it is involved in heparan binding. More importantly, this region is necessary for the enhancement of HCV entry by high density lipoprotein and interferes with virus neutralization by E2-neutralizing antibodies. Residues at positions 1-13 are also dispensable for HCV entry, but they can affect HCV infectivity by modulating binding of the envelope protein to scavenger receptor class B, type I. Mutations occurring at this site may confer resistance to HVR1 antibodies. These findings further our understanding about the mechanisms of HCV cell entry and the significance of HVR1 variation in HCV immune evasion. They have major implications for the development of HCV entry inhibitors and prophylactic vaccines.

Japanese encephalitis virus enters rat neuroblastoma cells via a pH-dependent, dynamin and caveola-mediated endocytosis pathway.

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus and one of the most common agents of viral encephalitis. The infectious entry process of JEV into host cells remains largely unknown. Here, we present a systemic study concerning the cellular entry mechanism of JEV to B104 rat neuroblastoma cells. It was observed that JEV internalization was inhibited by chloroquine and ammonium chloride, both of which can elevate the pH of acidic organelles. However, JEV entry was not affected by chlorpromazine, overexpression of a dominant-negative form of EPS 15 protein, or silencing of the clathrin heavy chain by small interfering RNA (siRNA). These results suggested that JEV entry depended on the acidic intracellular pH but was independent of clathrin. We found that endocytosis of JEV was dependent on membrane cholesterol and was inhibited by inactivation of caveolin-1 with siRNA or dominant-negative mutants. It was also shown, by using the inhibitor dynasore, the K44A mutant, and specific siRNA, that dynamin was required for JEV entry. Phagocytosis or macropinocytosis did not play a role in JEV internalization. In addition, we showed that JEV entry into the neuroblastoma cells is not virus strain specific by assessing the effect of the pharmacological inhibitors on the internalization of JEV belonging to different genotypes. Taken together, our results demonstrate that JEV enters B104 cells through a dynamin-dependent caveola-mediated uptake with a pH-dependent step, which is distinct from the clathrin-mediated endocytosis used by most flaviviruses.

Virus-host Interactions in Early Japanese Encephalitis Virus Infection.

Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic virus that can cause severe viral encephalitis. Initial interactions between JEV and host cells are required for productive viral infection and initiation of the viral life cycle. The elucidation of these interactions is critical, not only to understand the pathogenesis of JEV infection, but also to design efficient antiviral strategies. In this review, we outline the known viral and cellular components involved in JEV entry into host cells, with a particular focus on the initial virus-host cell interaction on the cell surface and the downstream early events such as endocytosis, membrane fusion, and viral genome release.

Recent Advances in Antivirals for Japanese Encephalitis Virus.

Culex mosquitoes are the primary vectors of the Japanese encephalitis virus (JEV). Since its discovery in 1935, Japanese encephalitis (JE), caused by JEV, has posed a significant threat to human health. Despite the widespread implementation of several JEV vaccines, the transmission chain of JEV in the natural ecosystem has not changed, and the vector of transmission cannot be eradicated. Therefore, JEV is still the focus of attention for flaviviruses. At present, there is no clinically specific drug for JE treatment. JEV infection is a complex interaction between the virus and the host cell, which is the focus of drug design and development. An overview of antivirals that target JEV elements and host factors is presented in this review. In addition, drugs that balance antiviral effects and host protection by regulating innate immunity, inflammation, apoptosis, or necrosis are reviewed to treat JE effectively.

Metagenomic surveillance and comparative genomic analysis of Chlamydia psittaci in patients with pneumonia.

Chlamydia psittaci, a strictly intracellular bacterium, is an underestimated etiologic agent leading to infections in a broad range of animals and mild illness or pneumonia in humans. In this study, the metagenomes of bronchoalveolar lavage fluids from the patients with pneumonia were sequenced and highly abundant C. psittaci was found. The target-enriched metagenomic reads were recruited to reconstruct draft genomes with more than 99% completeness. Two C. psittaci strains from novel sequence types were detected and these were closely related to the animal-borne isolates derived from the lineages of ST43 and ST28, indicating the zoonotic transmissions of C. psittaci would benefit its prevalence worldwide. Comparative genomic analysis combined with public isolate genomes revealed that the pan-genome of C. psittaci possessed a more stable gene repertoire than those of other extracellular bacteria, with ~90% of the genes per genome being conserved core genes. Furthermore, the evidence for significantly positive selection was identified in 20 virulence-associated gene products, particularly bacterial membrane-embedded proteins and type three secretion machines, which may play important roles in the pathogen-host interactions. This survey uncovered novel strains of C. psittaci causing pneumonia and the evolutionary analysis characterized prominent gene candidates involved in bacterial adaptation to immune pressures. The metagenomic approach is of significance to the surveillance of difficult-to-culture intracellular pathogens and the research into molecular epidemiology and evolutionary biology of C. psittaci.

Association of heat-shock protein 70 with lipid rafts is required for Japanese encephalitis virus infection in Huh7 cells.

Japanese encephalitis virus (JEV) is an enveloped flavivirus and the most common agent of viral encephalitis. It enters cells through receptor-mediated endocytosis and low pH-triggered membrane fusion. Although lipid rafts, cholesterol-enriched lipid-ordered membrane domains, have been shown to participate in JEV entry, the mechanisms of the early events of JEV infection, including the cellular receptors of JEV, remain largely unknown. In the current study, it was demonstrated that heat-shock protein 70 (HSP70), rather than other members of the HSP70 family, was required for JEV entry into a human cell line. Cell-surface expression of HSP70 and a direct interaction between JEV envelope (E) protein and HSP70 were observed. Biochemical fractionation showed that HSP70 clearly migrated into the raft fraction after virus infection and co-fractioned with E protein. Depletion of cholesterol shifted the E protein and HSP70 to a non-raft membrane and decreased JEV entry without affecting virus binding to host cells. Notably, recruitment of HSP70 into lipid rafts was required for activation of the phosphoinositide 3-kinase/Akt signalling pathway in the early stage of JEV infection. These results indicate that lipid rafts facilitate JEV entry, possibly by providing a convenient platform to concentrate JEV and its receptors on the host-cell membrane.

Tupaia CD81, SR-BI, claudin-1, and occludin support hepatitis C virus infection.

Hepatitis C virus (HCV)-related research has been hampered by the lack of appropriate small-animal models. It has been reported that tree shrews, or tupaias (Tupaia belangeri), can be infected with serum-derived HCV. However, these reports do not firmly establish the tupaia as a reliable model of HCV infection. Human CD81, scavenger receptor class B type I (SR-BI), claudin 1 (CLDN1), and occludin (OCLN) are considered essential receptors or coreceptors for HCV cell entry. In the present study, the roles of these tupaia orthologs in HCV infection were assessed. Both CD81 and SR-BI of tupaia were found to be able to bind with HCV envelope protein 2 (E2). In comparison with human CD81, tupaia CD81 exhibited stronger binding activity with E2 and increased HCV pseudoparticle (HCVpp) cell entry 2-fold. The 293T cells transfected with tupaia CLDN1 became susceptible to HCVpp infection. Moreover, simultaneous transfection of the four tupaia factors into mouse NIH 3T3 cells made the cells susceptible to HCVpp infection. HCVpp of diverse genotypes were able to infect primary tupaia hepatocytes (PTHs), and this infection could be blocked by either anti-CD81 or anti-SR-BI. PTHs could be infected by cell culture-produced HCV (HCVcc) and did produce infectious progeny virus in culture supernatant. These findings indicate that PTHs possess all of the essential factors required for HCV entry and support the complete HCV infection cycle. This highlights both the mechanisms of susceptibility of tupaia to HCV infection and the possibility of using tupaia as a promising small-animal model in HCV study.

Isolation and characterization of cyromazine degrading Acinetobacter sp. ZX01 from a Chinese ginger cultivated soil.

Cyromazine, a symmetrical triazine insecticide, is used to control dipteran larvae in chicken manure by feeding to the poultry, flies on animals, and leafminers in vegetables. Its extensive use has resulted in the widespread contamination in the environment. In the current study, a cyromazine degrading bacterium (designated strain ZX01) was isolated and characterized from a Chinese ginger cultivated soil by selective enrichment culture method. On the basis of morphological, biochemical characteristics, and 16S rRNA gene sequence, this bacterium showed strong similarity to the Pseudomonadales members and was closely related to the Acinetobacter baumannii group. Spectrophotometric and HPLC analyses revealed that strain ZX01 degraded cyromazine and utilized it as the sole carbon source for its growth. This process hydrolyzes cyromazine to melamine. Strain ZX01 degraded most of the cyromazine in 60 h. Besides, its substrate specificity against four symmetrical triazine herbicides, one triazinone herbicide, as well as 10 insecticides and its antibiotic sensitivity towards eight commercial antibiotics were also tested. At the concentration of 100 µg/mL for 60 h, it could effectively degrade a variety of different pesticides, including atrazine, prometon, simazine, prometryn, enitrothion, diazinon, cypermethrin, and acetamiprid, and the degradation was in the range of 71-87%. In particular, melamine, the main degradation product of cyromazine, was degraded by 47.3%. This microorganism was sensitive to chloramphenicol and tetracycline and intermediate to amoxicillin and trimethoprim. These results highlight that strain ZX01 can be used as a potential biological agent for the remediation of soil, water, or crop contaminated with cyromazine and other symmetrical triazine insecticides.

Structure-inhibitory activity relationships of pyrrolnitrin analogues on its biosynthesis.

Pyrrolnitrin is a bacterial metabolite, served as a natural lead of agricultural fungicides. In a previous study, fenpiclonil was proven to inhibit the oxidative transformation of aminopyrrolnitrin to pyrrolnitrin, catalyzed by aminopyrrolnitrin oxidase (PrnD). This monooxygenase has an interesting catalytic activity of selective oxidation of aromatic amines, rather than aliphatic amines. However, its structural details are not well understood. In this study, various analogues of pyrrolnitrin were prepared to elucidate the structures of active site of PrnD through structure-activity relationships. In vivo pyrrolnitrin biosynthesis inhibition was determined with Burkholderia sp. O33 and Pseudomonas fluorescens Pf-5. Quantitative analysis of pyrrolnitrin and precursors indicates that 2,3-disubstituted phenyl at 3rd carbon and small substituents at 4th carbon of pyrrole are strictly required to give strong inhibitory effects. In addition, dissociable proton of pyrrole is also critical for inhibitory activity. Molecular simulation with homology-based PrnD model suggests a highly restricted conformational space in active site. The results may help more detailed understanding of this unusual enzyme. In addition, the information will be useful for the development of novel fungicide, compatible with pyrrolnitrin-producing bacterium.