Highest LD50 Research Articles

Alkaloids from Pandanus amaryllifolius Roxb Leaf as Promising Candidates for Antidyslipidemic Agents: An in silico study

Introduction: The plant Pandanus amaryllifolius Roxb (pandan), has been shown to have antidyslipidemic potency. This study explored the potential of several alkaloids from pandan leaf as antidyslipidemia as well as their safety profile in silico. Methods: Analyses were carried out by studying the binding affinity of the alkaloids to 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, peroxisome proliferator activator receptor (PPAR) alpha and Niemann Pick C1 Like 1 (NPC1L1). The structures of the alkaloids were downloaded from the Pubchem database and optimized using the ChemDraw Professional 16.0 to obtain 3D structures in protein data bank (PDB) format. The in silico testing was based on the interactions of the alkaloids with the HMG-CoA reductase (PDB ID 1HW9), PPAR alpha (PDB ID 6LX4) and NPC1L1 (PDB ID 7DFZ) proteins, downloaded from the Research Collaboratory for Structural Bioinformatics (RSCB) PDB website (http://www.rcsb.org/pdb). The preparation of protein structures was performed using the Discovery studio 2021 client and Gromacs applications, while optimization of the 3D structure of the alkaloids was carried out with the ChemDraw professional 16.0. Finally, validation was completed using AutoDock application. The safety profile was assessed by pkCSM online tool. Results: The respective root mean square deviation (RMSD) values of the 1HW9, 6LX4 and 7DFZ proteins were 1.677, 0.918 and 1.706, respectively. The alkaloids pandanusine B, pandamarilactonine A, pandamarilactonine B had respective values of binding energy for HMG-CoA of -5.52, -5.51 and -5.46 kcal/mol. The binding energy of pandamarilactonine B, pandamarilactonine A and pandanamine for PPAR alpha were -9.14, -9.10 and -8.48 kcal/mol, respectively, with the corresponding energy for t NPC1L1 of -9.63, -9.71 and -8.54 kcal/mol. The toxicity tests indicated that the alkaloids were safe, pandamarilactonines had the highest LD50 (2.736 mol/ kg). Conclusion: The studied pandan alkaloids have potential antidyslipidemic activity by interacting with HMG-CoA reductase, PPAR alpha, and NPC1L1, with good safety profile.

Determination of lambda-cyhalothrin and imidacloprid resistance and synergistic activity of diatomaceous earth in Leptinotarsa decemlineata Say, 1824 (Coleoptera: Chrysomelidae) populations

In this study conducted in 2020, the resistance of Leptinotarsa decemlineata Say, 1824 (Coleoptera: Chrysomelidae) populations collected from Karapınar, Çumra, Seydişehir, Güneysınır and Doğanhisar Districts of Konya Province, Turkey to lambda-cyhalothrin and imidacloprid was investigated. Insecticides were applied topically to fourth instar larvae. In addition, the synergistic efficiency of diatomaceous earth (DE) was investigated by applying imidacloprid plus DE (2% w/v) with the same methodology in the Karapınar population. As a result of the research, the resistance rate of all populations was found to be higher in imidacloprid than in lambda-cyhalothrin. The highest LD50 was found in the Çumra (0.212 µg/larvae) and Karapınar (0.456 µg/larvae) population for lambda-cyhalothrin and imidacloprid respectively. The highest synergism ratio was determined as 1.93 after 48 hours in imidacloprid plus DE application. As a result of the research, it is considered that the use of DE will make a great contribution to the control and resistance management of the imidacloprid resistant potato beetle.

UV and temperature effects on chloroacetanilide and triazine herbicides degradation and cytotoxicity

The purpose of this study was to explore the stability and toxicity of the herbicides and their degradation byproduct after exposure to different environmental factors. Triazines (atrazine, propazine, simazine) and chloroacetanilides (acetochlor, alachlor, metolachlor) which are commonly used herbicides were evaluated for cytotoxicity in different UV (254 nm and 365 nm) and temperature (4 °C, 23 °C, and 40 °C) conditions as well as degradation rates. Atrazine with the highest LD50 (4.23 μg mL−1) was less toxic than the other tested triazine herbicides Chloroacetanilides tested were more toxic than tested triazines, with LD50 0.08–1.42 μg mL−1 vs 1.44–4.23 μg mL−1, respectively. Alachlor with LD50 0.08 μg mL−1 showed the strongest toxic response as compared with other tested herbicides. Temperatures only did not alter cytotoxicity of the tested herbicides, except for acetochlor and alachlor showing about 45 % more cell death after exposure to 40 °C for 2 h. At all 3 tested temperatures, 2 h of UV treatments did not affect cytotoxic effects of the tested herbicides, except for acetochlor and alachlor. At 4 °C, acetochlor toxicity was attenuated about 63 % after UV 365 nm exposure; but alachlor toxicity was enhanced after either UV 254 or 365 nm exposure for about 40 % and 24 %, respectively. At 23 °C, acetochlor toxicity was enhanced about 35 % after UV 254 nm exposure, but attenuated about 48 % after UV 365 nm exposure. Alachlor toxicity was enhanced about 34 % after UV 254 nm and 23 °C exposure. In combination of UV 254 nm and 40 °C, acetochlor toxicity was lowered by 63 % and alachlor toxicity was no change as compared with 4 °C, no UV group. After co-treatment with UV 365 nm and 40 °C both acetochlor and alachlor toxicity was enhanced 55 % and 80 %, respectively. Through degradation analysis by LC-MS/MS, alachlor showed the most dramatic degradation (only 0.58 %–10.58 % remaining) after heat and UV treatments.

Effect of Two Essential Oils From the Asteraceaes Family Against Ectomyelois Ceratoniae Zell. (Lepidoptera, Pyralidae): Case Of Artemisia Herba-Alba Asso. And Artemisia Compestris L.

The current work was done Artemisia herba-alba and Artemisia compestris essential oils harvested from the Eastern Algerian Sahara, their insecticidal characteristics against the eggs and adults of the date moth Ectomyelois ceratoniae. Indeed, two treatment modes were used; by contact application on eggs and by inhalation against adults. It appears from the results that the hatch rates were less than the hatching rate recorded in the control (96% ±00.00). The hatching rate reported on eggs treated by the highest dose (160 μl/ml) of Artemisia herba-alba and Artemisia compestris essential oils are 16.66 ± 08.81 and 37.77 ± 13.47 respectively. Statistical treatment results by the Chi-square test (χ2), attest that the treatment by A. herba-alba and Artemisia compestris essential oils at the same dose (160 μl/ml) affect significantly (χ2 = 35.62, p = 0.00 and χ2 = 21.17, p = 0.00 respectively) the hatching rate compared to the control. The sensitivity of adults to essential oils is expressed by 100% mortality rates obtained after 10 min of treatment by the highest doses (80 μl/ml and 160 μl/ml) of A. herba-alba essential oils, the same mortality rates (100%) were notified with the same doses (80 μl/ml and 160 μl/ml) after 20 min and 15 min of treatment by Artemisia compestris essential oils respectively. The dose-dependent mortality data revealed that there was a significant difference between the five doses of A. herba-alba essential oil tested except at the last treatment time (20 min) for which it was appeared p = 0.571, while for A. compestris essential oil, a significant difference was recorded with p varying between 0.00 and 0.003. The lowest LD50 value (0.09 μl/ml and 16.71 μl/ml) were noted during the longest treatment time (20 min), while the highest LD50 value (75.85 μl/ml and 263.7 μl/ml) were found during the shortest time (5 min) of A. herba-alba and A. compestris respectively. J. Bio-Sci. 29(2): 09-17, 2021 (December)

Chemo-taxonomic and biological potential of highly therapeutic plant Pedicularis groenlandica Retz. using multiple microscopic techniques.

Pedicularis groenlandica is one of the most important medicinal plant of Deosai Plateau (Gilgit-Baltistan) Pakistan. The present study was aim to evaluate the micromorphological features, phytochemical screening and pharmacological potential of P. groenlandica by using multiple microscopic techniques. Six different solvents were used to prepare P. groenlandica extracts. Phytochemical and antioxidant activities were determined calorimetrically. To investigate antidiabetic, α-amylase inhibition assay was performed. Cytotoxicity was tested using brine shrimp assay. Anti-leishmanial via MTT assay. Disc-diffusion assay was used for protein kinase inhibitory, antibacterial and antifungal activities. Palyno-anatomical study showed significant variation for the authentication and correct identification of this highly therapeutic plant by using light and scanning electron microscopic techniques. All extracts were found rich in phytochemicals, significant amount of phenolic and flavonoid contents were found in methanol extract (PGM) 95.78 mg GAE/g and 66.90 mg QE/g. Highest DPPH scavenging potential with IC50 88.65 μg/mL, total antioxidant capacity (60.33 mg AAE/g sample) and total reducing power (83.97 mg AAE/g) were found for PGM. Disc-diffusion method showed significant antibacterial and antifungal activities. Noticeable growth inhibition in L. tropica was displayed by n-hexane extract (IC50 112 μg/mL). Brine shrimp with highest LD50 (67.65 μg/mL) in ethyl-acetate extract. Ethanol extract gives persuasive protein kinase inhibition (26 mm) against Streptomyces 85-E hyphae. Highest alpha-amylase inhibition (74.10%) was found in n-hexane extract. In conclusion, our findings scientifically support the ethno-medicinal and biological potential of P. groenlandica. In future, the plant needs to be explored for further identification and isolation of bioactive compounds to develop new drugs to treat several aliments.

Impact of chemical mutagenesis using ethyl methane sulphonate on tepary bean seedling vigour and adult plant performance

Tepary bean is an important food legume. The genetic improvement of the crop is limited by a narrow genetic base but this genetic base can be broadened through induced mutagenesis. This study was designed to determine the (i) effects of chemical mutagenesis using ethyl methane sulfonate (EMS) on M1 seedlings (ii) the adult plant performance of M2 - M4 generations and (iii) the relationships between growth attributes at the seedling and adult plant stage. Mutagenized seed with varying doses of EMS was germinated at room temperature in order to raise M1 seedlings. There were highly significant (P < 0.01) differences due to dose effects among the seedlings. The highest LD50 (3.37 % EMS v/v) was observed for ‘Genotype 3’. Under field conditions, all the three factors influenced the plant performance. The results demonstrated the potential of EMS to induce genotypic variation and desirable agronomic traits in tepary bean.

Effects of acetamiprid on Lipaphis pseudobrassicae (Hemiptera: Aphididae) resistant and susceptible to the parasitoid Diaeretiella rapae (Hymenoptera: Braconidae, Aphidiinae)

ABSTRACT This study evaluated the relationship between the resistance of Lipaphis pseudobrassicae to the parasitoid Diaeretialla rapae and the effects of the insecticide acetamiprid on the aphid. Four groups of L. pseudobrassicae collected in Uberlândia, MG, were used. The first two were formed by individuals of the same clone (C1), which were resistant (C1R) or susceptible (C1S) to the parasitoid. The third group was formed by resistant individuals to the parasitoid descended from a clone collected in canola (C2R) and the fourth group, from a population collected in a commercial plantation of collard greens (P1). Determination of LD50 was done with three replications of 10 aphids, subjected to dose-response assays, with the application of five concentrations of the insecticide acetamiprid (0.01, 0.1, 1, 10 or 100 ng a.i./aphid) and a control treatment consisting of acetone alone. There was no significant difference in the LD50 of individuals of the same clone, C1R and C1S (0.06 ng a.i./aphid for both clones). C2R had the highest LD50 (0.14 ng a.i./aphid), while P1 had the lowest (0.01 ng a.i./aphid). These results suggest that resistance against the parasitoid is not, therefore, associated with the effects of insecticide on L. pseudobrassicae.

Toxicology study of nanoclays adsorbed with the antimicrobial peptide nisin on Caenorhabditis elegans

Halloysite and montmorillonite nanoclays have a wide range of industrial and medical applications, including food packaging. To ensure safe incorporation of these clays into nanocomposites, toxicological screening must be performed. In this study, the acute toxicity of three nanoclays, namely halloysite, bentonite and montmorillonite modified with octadecylamine (Mt-ODA), was evaluated using the free-living nematode Caenorhabditis elegans as in vivo model. Nanoclays were tested pure and adsorbed with nisin, an important antimicrobial peptide used as food preservative. Lethal concentration 50% (LC50), nematode development, reactive oxygen species (ROS) production, quantification of catalase (CAT) and superoxide dismutase (SOD) enzymes, and lipid peroxidation were the toxicological endpoints evaluated in C elegans. Halloysite and Mt-ODA had the lowest and highest LD50, respectively. The adsoption of nisin on halloysite and bentonite provoked an increase in surviving nematodes. All treatments altered the normal growth, in different degrees, being nematodes exposed to bentonite the most affected. ROS production increased in all treatments, especially Mt-ODA, where lipid peroxidation was enhanced, suggesting high oxidative stress.

Comparative studies on the toxicological, antiinflammatory and analgesic properties of three sources of Xuedan in mice and their rapid identification by electronic tongue

Purpose: To compare the toxicological, anti-inflammatory and analgesic properties of three sources of Xuedan, viz, Hemsleya omeiensis (HO), Hemsleya giganth (HG) and Hemsleya dolichocarpa (HD) in mice, and to study their rapid identification based on electronic tongue (E-tongue). Methods: After 7 days of administration, the median lethal doses (LD 50 ) of the three xuedan decoctions in mice were determined. In addition, the anti-inflammatory and analgesic effects of the three xuedans were evaluated in mice using xylene-induced ear edema and acetic acid-induced pain. Furthermore, Etongue technology was used to identify HO, HG and HD. Principal component analysis (PCA) and discriminant factor analysis (DFA) were used to analyze the data acquired by E-tongue. Results: The median lethal dose (LD 50 ) values of H. omeiensis , H. gigantha and H. dolichocarpa were 32.3, 17.4 and 13.7g/kg, respectively. Compared with normal control group, the anti-inflammatory effects of Xuedan were obvious in xylene-induced ear edema ( p < 0.05), and pain sensation was significantly inhibited in acetic acid-induced writhing test ( p < 0.05). Furthermore, E-tongue technology effectively identified HO, HG and HD. Conclusion: H. omeiensis exhibits the highest LD50 value and best analgesic effect among the three sources of xuedan. E-tongue technology is effective and rapid in identifying HO, HG and HD. Keywords: Xuedan, Hemsleya omeiensis , Hemsleya gigantha , Hemsleya dolichocarpa , Antiinflammation, Analgesia, Electronic tongue

Molecular characteristics and virulence analysis of eight Aeromonas hydrophila isolates obtained from diseased Amur sturgeon Acipenser schrenckii Brandt, 1869.

Aeromonas hydrophila is an opportunistic pathogen of a variety of aquatic animals that displays extreme diversity in drug resistance, phenotypes, virulence genes, and virulence. In this study, eight pathogenic A. hydrophila strains were isolated from diseased Amur sturgeons and investigated for their sensitivity to select antibiotics, their phenotype, virulence genes, and virulence. According to the phylogenetic analysis of the DNA gyrase subunit B protein, the eight isolates formed a single branch in the A. hydrophila group. The antibiotics ceftazidime, cefuroxime, cefoperazone, cefotaxime, ceftriaxone, aztreonam, and cefepime appeared effective against them. All of the isolates possessed the virulence genes for aerolysin, flagellin, heat-stable cytotonic enterotoxin, heat-labile cytotonic enterotoxin, hemolysin, and elastase, while only one isolate, HZ8, possessed the gene for lateral flagella. The cytolytic enterotoxin and lipase genes were present in all isolates, except in ZJ10 and ZJ12. Enterobacterial repetitive intergenic consensus sequence PCR indicated that the eight A. hydrophila isolates could be divided into four types. Isolates YW2, TR3, HZ8 and ZJ10, each representing a different type, were selected for challenge experiments. The challenge tests revealed that isolate HZ8 had the lowest lethal dose, causing 50% mortality at 2.30 × 104 colony forming units (cfu)/ml. The isolate ZJ10 had the highest LD50, 1.25 × 106 cfu/ml. Knowledge of the characteristics of the A. hydrophila isolates obtained from Amur sturgeon will be beneficial in developing potential disease control strategies.

Cytotoxic and Phytotoxic Activities of &amp;lt;i&amp;gt;Reinwardtia trigyna&amp;lt;/i&amp;gt; (ROXB.)

The present study was carried out to check the cytotoxic and phytotoxic activities of the ethanolic extract of leaf of Reinwardtia trigyna. The cytotoxic potential checked through Artemia salina mortality test which showed low cytotoxic effect (30% and 43.33%) at the doses of 10µg/ml, and 100µg/ml, while moderate cytotoxic effect (53.33%) was observed at 1000µg/ml with highest LD50, value 4496.29µg/ml as compared to the control having normal saline that shows no mortality. The same extracts showed significant dose dependent phytotoxicity 32.25%, 61.29% and 70.96% inhibition of Lemna minor at the doses of 10, 100 and 1000µg/ml as compared to the positive control “Atrazine sulphate” that shows 100% frond inhibition. The FI50 value of plant extract was very low as 58.448 µg/ml. it is concluded from the present work that Reinwardtia trigyna could be used as a natural herbicide and weedicide as it shows a good phytotoxic activity.

Comparative study of the fungicide Benomyl toxicity on some plant growth promoting bacteria and some fungi in pure cultures.

Six laboratory experiments were carried out to investigate the effect of the fungicide Benomyl on pure cultures of some plant growth promoting bacteria (PGPB) and some fungi. The highest LD50 was recorded for Bacillus circulans and proved to be the most resistant to the fungicide, followed by Azospirillum braziliense, while Penicillium sp. was the most affected microorganism. LD50 values for the affected microorganisms were in 21–240 orders of magnitude lower in comparison with the LD50 value for Azospirillum braziliense. The results indicate a strong selectivity for Benomyl against Rhizobium meliloti and Penicillium sp. when compared to other microorganisms tested. The highest safety coefficient was recorded for Bacillus circulans followed by Azospirillum braziliense, while Rhizobium meliloti, showed the lowest safety coefficient value compared to other bacteria. The lowest toxicity index was recorded for Bacillus circulans and Azospirillum braziliense. The slope of the curves for Bacillus sp. and Rhizobium meliloti was steeper than that of the other curves, suggesting that even a slight increase of the dose of the fungicide can cause a very strong negative effect. In conclusion, Benomyl could be applied without restriction when using inocula based on growth promoting bacteria such as symbiotic nitrogen fixers (Rhizobium meliloti), non-symbiotic nitrogen fixers (Azospirillum braziliense) or potassium solibilizers (Bacillus circulans), given that the fungicide is applied within the range of the recommended field dose.

Effects of UV wavelength on cell damages caused by UV irradiation in PC12 cells

Ultraviolet (UV) radiations present in sunlight are a major etiologic factor for many skin diseases and induce DNA damage through formation of cyclobutane pyrimidine dimer (CPD). This study was conducted to determine the toxicological effects of different wavelengths (250, 270, 290, and 310nm) and doses of UV radiation on cell viability, DNA structure, and DNA damage repair mechanisms in a PC12 cell system. For this, we evaluated cell viability and CPD formation. Cell survival rate was markedly decreased 24h after UV irradiation in a dose-dependent manner at all wavelengths (except at 310nm). Cell viability increased with increasing wavelength in the following order: 250<270<290<310nm. UV radiation at 250nm showed the highest cell killing ability, with a median lethal dose (LD50) of 120mJ/cm2. The LD50 gradually increased with increase in wavelength. Among the 4 wavelengths tested, the highest LD50 (6000mJ/cm2) was obtained for 310nm. CPD formation decreased substantially with increasing wavelength. Among the 4 wavelengths, the proportion of CPD formation was highest at 250nm and lowest at 310nm. On the basis of LD50 values for each wavelength, PC12 cells irradiated with UV radiation of 290nm showed maximum DNA repair ability, whereas those irradiated with the 310-nm radiation did not show any repair ability. Toxicity of UV radiation varied with wavelengths and exposure doses.

Use of Residue Profile Analysis to Identify Modes of Insecticide Activity Contributing to Control of Plum Curculio in Apples

Residue profile analysis techniques were developed, along with laboratory and field-based bioassays to describe the modes of insecticidal activity responsible for the control of the plum curculio, Conotrachelus nenuphar (Herbst), in apples (Malus spp.). Adult plum curculios were treated in laboratory topical bioassays to determine acute contact activity and lethal time for five insecticides. Azinphosmethyl had the highest levels of toxicity and shortest lethal time values, followed by the neonicotinoids thiacloprid, thiamethoxam, and imidacloprid, whereas indoxacarb had the highest LD50 and LT50 values for topical exposure. Field-based residual activity bioassays assessed adult mortality, and fruit and leaf injury from plum curculio exposed to 4 h, 7 d, and 14 d field-aged residues. All compounds caused significant levels of mortality to plum curculio when adults were exposed to fruit clusters 4 h post-application. Thiacloprid, thiamethoxam, and imidacloprid showed oviposition deterrence, antifeedant, and repellency effects in the 7- and/or 14 d residual bioassays and protected fruit in the absence of significant lethal activity. Indoxacarb maintained lethal activity throughout the study intervals, with the incidence of plum curculio feeding, suggesting that ingestion is an important mode of entry. For the neonicotinoids thiacloprid, thiamethoxam and imidacloprid plum curculio mortality was highly correlated with fruit and leaf surface residues. As surface residues declined, sublethal effects such as oviposition deterrence and antifeedant effect remained. The value of the plant–insect–chemistry triad model for describing the temporal dimensions of insecticidal modes of activity and understanding a compound’s critical performance characteristics is discussed.

Use of Residue Profile Analysis to Identify Modes of Insecticide Activity Contributing to Control of Plum Curculio in Apples

Residue profile analysis techniques were developed, along with laboratory and field-based bioassays to describe the modes of insecticidal activity responsible for the control of the plum curculio, Conotrachelus nenuphar (Herbst), in apples (Malus spp.). Adult plum curculios were treated in laboratory topical bioassays to determine acute contact activity and lethal time for five insecticides. Azinphosmethyl had the highest levels of toxicity and shortest lethal time values, followed by the neonicotinoids thiacloprid, thiamethoxam, and imidacloprid, whereas indoxacarb had the highest LD50 and LT50 values for topical exposure. Field-based residual activity bioassays assessed adult mortality, and fruit and leaf injury from plum curculio exposed to 4 h, 7 d, and 14 d field-aged residues. All compounds caused significant levels of mortality to plum curculio when adults were exposed to fruit clusters 4 h post-application. Thiacloprid, thiamethoxam, and imidacloprid showed oviposition deterrence, antifeedant, and repellency effects in the 7- and/or 14 d residual bioassays and protected fruit in the absence of significant lethal activity. Indoxacarb maintained lethal activity throughout the study intervals, with the incidence of plum curculio feeding, suggesting that ingestion is an important mode of entry. For the neonicotinoids thiacloprid, thiamethoxam and imidacloprid plum curculio mortality was highly correlated with fruit and leaf surface residues. As surface residues declined, sublethal effects such as oviposition deterrence and antifeedant effect remained. The value of the plant-insect-chemistry triad model for describing the temporal dimensions of insecticidal modes of activity and understanding a compound's critical performance characteristics is discussed.

Responses of Populations of the Tobacco Budworm (Lepidoptera: Noctuidae) from Northwest Mexico to Pyrethroids

Dose-mortality bioassays were begun in 1984 to determine the susceptibility of the tobacco budworm, Heliothts virescens (F.), to commonly used pyrethroids. Responses of different populations from the northwestern region of Mexico were compared with responses of two susceptible strains. The LD50’s indicated that the populations have been and are being selected for resistance to varying degrees. A population from Valle de Mexicali had the highest LD50 and resistance ratio for deltamethrin. The highest LD50’s for permethrin were obtained from 1987 populations from Valle del Yaqui and Valle de Mexicali (resistance ratios of 55.4 and 48.7, respectively). Resistance ratios from Valle del Yaqui for cypermethrin indicated that the 1987 tobacco budworm population was 32 times more resistant than the susceptible colony. Although control failures have not been reported from these areas, the dose-mortality data suggest that control failures may occur. Permethrin resistance appears to be increasing in Valle del Yaqui, Costa de Hermosillo, and Valle de Mexicali. Resistance to cypermethrin and cyfluthrin seems to be increasing in Valle del Yaqui and Valle de Mexicali. On the basis of LD50’s, deltamethrin resistance remains low except for Valle de Mexicali but should be carefully monitored.

日本腦炎ウイルスの卵殼を用いての培養並びに増殖法 (II)

Employing deembryonated eggs, H. Bernkoph (1949-1950) has succeeded in cultivating influenza virus in a similar manner to the tissue culture. The authors, attempted to cultivate Japanese B encephalitis virus (Nakayama strain) by the modifications of the above method.All of the contents, except the chorioallantoic membrane, of the fertilized eggs incubated for 12 to 15 days were removed from the egg shell, and the eggs were refilled with the medium mainly composed of 2% yolk saline solution. In addition, Japanese B encephalitis virus was inoculated into the eggs in an amount to hold the virulence at 10-3, and the eggs were incubated at 37°C. The following is the results thus obtained.Better results in the multiplication of the virus were obtained with the developing eggs incubated for 13 or 14 days when compared with those obtained with the eggs incubated for 12 days or for 15 days. The LD50 determined in the former cases were 10-6.0 and 10-5.8 respectively, while in the latter, they were 10-5.5 and 10-4.6 respectively.As to the incubation period of the culture, the multiplication of the virus was found more favorable when incubated for 24 hours or 48 hours than 72 hours, showing the LD50 values at 10-6.0 and 10-5.5 respectively and 10-4.5 for the last case.When 100-1, 000units/cc of the medium of penicillin was added, an increase in the LD50 value was recorded showing the highest value in the LD50 at 10-7.0.Further, the highest LD50 value attained by the addition of 10% extract of the developing chick embryo (7-11 days old) at the rate of 1% of the medium was 10-7.8, and the average was 10-7.2.All of the above experiments were conducted with the eggs not infected by Japanese B encephalitis virus. With the eggs infected by Japanese B encephalitis virus, almost identical results, though slightly lower in the LD50 values, were obtained.