Changes in cerebral level of monoamines by Japanese encephalitis virus infection

The changes in monoamine levels of different brain regions following Japanese encephalitis virus (JEV) intraperitoneal inoculation were examined in experimentally JEV-infected mice. In addition, virus distribution was studied using infectivity assay and immuno-histochemistry of viral antigen. 1) The level of monoamines in brain tissues was not affected by 48 hours after viral inoculation, but marked effects were elicited at 96 hours after the inoculation. The cerebral concentration of 5-hydroxyindole-3-acetic acid (5 HIAA) was increased, while that of dopamine (DA) showed a decrease. Especially these alteration were observed in the cerebral cortex, but not in the cerebellum. 2) The viral growth in the brain was observed at 48 hours after the inoculation. The growth in the cerebellum, however, was found to be lower than those in other cerebral regions. 3) The viral antigen was detected in the cerebral cortex, hippocampus, mesencephalon and diencephalon in addition to the substantia nigra and striatum. From these results, it is presumed that clinical manifestation of JEV infection may involve the changes in the metabolism of neurotransmitter, especially those of DA and serotonin in the brain.     

The protective effects of nutritional antioxidant therapy on Ehrlich solid tumor-bearing mice depend on the type of antioxidant therapy chosen: histology, genotoxicity and hematology evaluations

Strong evidence indicates that reactive oxygen species (ROS) play an important role in the initiation as well as the promotion phase of carcinogenesis. Studies support the role of ROS in cancer, in part, by showing that dietary antioxidants act as cancer-preventive agents. Although results are promising, the research on this topic is still controversial. Thus, the aim of this study was to investigate whether vitamins C, E and pequi oil can, individually, provide prevention and/or be used afterward as an adjuvant in cancer therapy. Ehrlich solid tumor-bearing mice received antioxidant as follows: before tumor inoculation, before and after tumor inoculation (continuous administration), and after tumor inoculation; morphometric analyses of tumor, genotoxicity and hematology were then carried out. Antioxidant administrations before tumor inoculation effectively inhibited its growth in the three experimental protocols, but administrations after the tumor's appearance accelerated tumor growth and favored metastases. Continuous administration of pequi oil inhibited the tumor's growth, while the same protocol with vitamins E and C accelerated it, favoring metastasis and increasing oxidative stress on erythrocytes. Except for continuous administration with vitamin E, the development of ascites tumor metastases was linked with increased inflammation. Results suggest that the efficiency and applicability of antioxidants in the medical clinic can depend not only on the nature of the antioxidant, the type and stage of cancer being treated and the prevailing oxygen partial pressure in the tissues, but also on the type of antioxidant therapy chosen.     

Effect of Temperature on Susceptibility of the Primary Leaves ofPhaseolus vulgaris L. to Red Clover Mottle Virus

Red clover mottle virus isolated in Czechoslovakia was studied in relation to its reaction to varying temperature on primary French bean leaves (Phaseolus vulgaris L.) on which it forms local necrotic lesions. The plants were kept 24 or 48 h before, or 24 or 48 h after inoculation at the temperatures 23, 25, 27, 30, 33 and 36°C. After such exposures the French beans were kept at a constant temperature of 25°C. The lesions were counted at various intervals. In the experiment the optimal temperature for the maximum number of lesions seems to be 36°C 48 h before inoculation. The temperature above 25°C applied 24 h after inoculation seems to have a decreasing effect upon the number of lesions formed by RCMV on primary leaves of French beans and the lesions appeared several hours later, especially at 30, 33 and 36°C. The temperatures 27, 30 and 33°C applied 48 h after inoculation have a further decreasing effect on the number of lesions. The temperature of 36°C applied 48 h after inoculation has an inactivating effect upon RCMV inoculated on French bean leaves and no lesions appeared 5 days after inoculation.     

人三叶因子3在毕赤酵母中表达条件的研究

为提高人三叶因子 3 (HumanTrefoilfactor 3 ,hTFF3 )在毕赤酵母中的表达量 ,研究了转化子生长的培养条件 ,包括不同碳源对转化子生长的影响和接种量、甲醇浓度、pH值、摇瓶转速及不同诱导时间对人三叶因子 3表达的影响。结果表明转化子在生长阶段加入葡萄糖生长旺盛 ,培养 14h后OD₆₀₀ 就可达到 50。在 100mL生长培养基上的菌液以 1∶1接入诱导培养基时蛋白表达量最高 ;转化子在 1%的甲醇、pH60、摇瓶转速240r/min的条件下诱导4 8h ,菌体密度OD₆₀₀为 15 ,目的蛋白表达量达到 20mg L。用 5L发酵罐进行了高密度发酵 ,经2%甲醇32h诱导 ,最终菌体密度OD₆₀₀ 达到 120 ,每升发酵液中含目的蛋白100mg。     

ASSEMBLY AND AGGREGATION OF TOBACCO MOSAIC VIRUS IN TOMATO LEAFLETS

Cells of tomato leaflets (Lycopersicum esculentum Mill.) were studied by phase and electron microscopy at various intervals after inoculation with a common strain of tobacco mosaic virus (TMV). Forty-eight hours after inoculation, prior to the development of assayable virus, individual TMV particles, and also particle aggregates, were observed in the ground cytoplasm of mesophyll cells. The most rapid synthesis of virus occurred between 80 and 300 hours after inoculation. Cytological changes during this time were characterized by an increased number of individual particles in the cytoplasm, growth of some aggregates, distortion and vacuolation of chloroplasts, and formation of filaments in the cytoplasm which were approximately four times the size of TMV. These filaments were interpreted as possible developmental forms of the TMV particle. Vacuoles in chloroplasts commonly contained virus particles. Evidence indicated that TMV was assembled in the ground cytoplasm and, in some cases, subsequently was enveloped by distorted chloroplasts.     

The intensity of respiration and heat emission from seedlings of Festuca pratensis (Hud.) and Hordeum vulgare L. during pathogenesis caused by Bipolaris sorokiniana (Sacc.) Shoem

The presented work was conducted on seedlings of spring barley and meadow fescue which differ in the degree of sensitivity to leaf spot pathogen Bipolaris sorokiniana (Sacc.) Shoem. The seedling reaction to inoculation with mycelium and conidia was examined in glasshouse conditions on the basis of respiration intensity and heat production. The leaf respiration was measured using Clark-type electrode, while heat emission was evaluated by means of isotermic microcalorimeter. The measurements were performed after 1, 3, 6, 10, 24, 48, 72, 168 and 240 hours since the inoculation moment. Leaves of meadow fescue were characterized by the most intense respiration at the 6^th hour, while barley leaves at the 24^th and 72^nd hour after inoculation. In the case of meadow fescue the greatest heat production was noted in the period between 24 and 168 hours after inoculation. Simultaneously, at the 48^th hour the smallest rate of respiration was observed. Barley leaves emitted the greatest amount of heat only in the first 3 hours of the pathogenesis. In these hours the smallest respiration rate was noted. The observed, opposing reaction of respiration intensity and heat emission in the infected seedlings of both species may illustrate a disorder in metabolic processes in plants during pathogenesis. The plants studied differed in the time of their reaction to pathogen attack: barley responded earlier in heat production, while fescue extended respiration rate in the first hours after inoculation. This is clearly observable, when coefficients of metabolic inefficiency (heat rates per mole O₂) are compared. In the case of barley the highest rates were noticed just after inoculation, whereas in fescue at the 48^th hour. In both species attack of pathogen caused high metabolic efficiency.     

Effects of temperature on infection of French bean leaves (Phaseolus vulgaris L.) by lucerne mosaic virus

The effect of temperature on the number of lesions and the time of their appearance was studied by inoculating French bean leaves (Phaseolus vulgaris L. cv. Perli?ka) with lucerne mosaic virus either 24 or 48 h before or, 24 or 48 h after they were exposed to various temperatures. The temperatures tested were 23, 25, 27, 30, 33 and 36° C. Before and after such exposures the plants were kept in a constant temperature of 25° C. By increasing the temperature before inoculation the number of lesions increased in comparison with the control. The optimal temperature for the maximum number of lesions is between 27° and 30° C. There is no significant difference between those experiments when the exposure time was 24 h or 48 h before inoculation. The same temperatures applied for 24 or 48 h after inoculation have a decreasing effect upon the number of lesions formed by LMV on French bean leaves. The decrease is 30 to 75%. In this case the first necrotic local lesions appeared 42 h after inoculation when exposed to higher temperatures above 27° C for 24 h, and 60 h after inoculation when exposed to these temperatures for 48 h. The shape of lesions varied a little in both cases as the pictures show.     

ELECTRON MICROSCOPIC STUDIES ON THE DEVELOPMENT OF VESICULAR STOMATITIS VIRUS IN KB CELLS

The development of vesicular stomatitis virus in KB cells was studied by electron microscopy. Sections of infected cells were made 1, 4, 7, 10, and 20 hours after inoculation of the cell cultures, and at the same intervals the supernatant fluid was assayed for virus titer by the plaque test in chick embryo cells. At 10, 14, and 20 hours after inoculation, virus rods were observed attached to cytoplasmic membranes, inside cytoplasmic vacuoles, and attached to the membranes delimiting these vacuoles; they were also found on the surface membrane of the cells. Besides the rods, spherical particles of different sizes and shapes were seen. The possibility that these structures are related to the development of virus rods is discussed. A similarity was noted between the site of maturation of vesicular stomatitis virus rods and that of some other arbor viruses.     

单纯疱疹病毒致病模型的研究

对单纯疱疹病毒（HSV）感染小白鼠致病特点进行了观察。小白鼠感染HSV第4天后开始发病,感染后2h血液内可分离出病毒,第48小时病毒血症水平和病毒检出率较高。不同组织病毒分布不同,脑、神经节在感染后第72小时病毒滴度较高,心、肝组织在第5天达到高峰。结果说明所建立的HSV致病模型可用于评价抗HSV药物。     

Gene expression in Brassica campestris showing a hypersensitive response to the incompatible pathogen Xanthomonas campestris pv. vitians

Xanthomonas campestris pv. vitians, a pathogen of lettuce, elicits a hypersensitive response within 12 hours of inoculation into Brassica leaves, characterized by tissue collapse, loss of membrane integrity, vein blockage and melanin production. In contrast, the compatible pathogen, X. c. pv. campestris, has no visible effects on leaves for 48 hours, after which inoculated areas show chlorosis which eventually spreads, followed by rotting.mRNA was prepared from leaves inoculated with suspensions of both pathovars or with sterile medium up to 24 hours following inoculation. In vitro translation of total and poly A⁺ RNA in rabbit reticulocyte lysate in the presence of ³⁵S methionine followed by separation of the polypeptide products by 2D-PAGE, allowed comparison of the effects of these treatments on plant gene expression. Major changes in gene expression were observed as a consequence of the inoculation technique. In addition, after inoculation with X. c. vitians, up to fifteen additional major polypeptides appeared or greatly increased by four hours. Some of these had disappeared by nine hours and several more had appeared. No major polypeptides disappeared or decreased greatly in intensity following inoculation with X. c. vitians.     

AN ELECTRON MICROSCOPE STUDY OF THE DEVELOPMENT OF A MOUSE HEPATITIS VIRUS IN TISSUE CULTURE CELLS

Samples taken at different intervals of time from suspension cultures of the NCTC 1469 line of mouse liver—derived (ML) cells infected with a mouse hepatitis virus have been studied with the electron microscope. The experiments revealed that the viruses are incorporated into the cells by viropexis within 1 hour after being added to the culture. An increasing number of particles are found later inside dense cytoplasmic corpuscles similar to lysosomes. In the cytoplasm of the cells from the samples taken 7 hours after inoculation, two organized structures generally associated and never seen in the controls are observed: one consists of dense material arranged in a reticular disposition (reticular inclusion); the other is formed by small tubules organized in a complex pattern (tubular body). No evidence has been found concerning their origin. Their significance is discussed. With the progression of the infection a system of membrane-bounded tubules and cisternae is differentiated in the cytoplasm of the ML cells. In the lumen of these tubules or cisternae, which are occupied by a dense material, numerous virus particles are observed. The virus particles which originate in association with the limiting membranes of tubules and cisternae are released into their lumen by a "budding" process. The virus particles are 75 mµ in diameter and possess a nucleoid constituted of dense particles or rods limiting an electron transparent core. The virus limiting membrane is sometimes covered by an outer layer of a dense material. In the cells from the samples taken 14 to 20 hours after inoculation, larger zones of the cell cytoplasm are occupied by inclusion bodies formed by channels or cisternae with their lumens containing numerous virus particles. In the samples taken 20 hours or more after the inoculation numerous cells show evident signs of degeneration.     

Response of Membrane-bound Mg-activated ATPase of Tobacco Leaves to Tobacco Mosaic Virus

Infectious material was formed at an early stage, and migrated into the mesophyll from the epidermis of tobacco leaves (Nicotiana tabacum cv. Samsun NN) during the period of 1 to 3 hours after inoculation with tobacco mosaic virus (TMV). The activity of membrane-bound Mg²⁺-activated ATPase from the mesophyll was stimulated two to four times within 30 minutes after inoculation with 1.0 microgram per milliliter of TMV. Maximum TMV stimulation of membrane-bound Mg²⁺-activated ATPase activity in epidermis and mesophyll was observed at 0.5 and 3.0 hours after inoculation, respectively. This stimulation was also observed with ultraviolet irradiated TMV (only RNA was destroyed), whereas, the stimulation was not observed with heat-irradiated TMV (both coat and RNA were destroyed). Stimulation equal to that of TMV was observed by inoculation with cucumber green mottle mosaic virus and to a lesser extent with cucumber mosaic virus.

These results illustrate that the stimulus resulting from inoculation with TMV transfers to underlying cells faster than the migration of TMV particles. This stimulus might be closely correlated to the structure of virus, but not to the infectivity of virus.

     

Susceptibility of chickens to avian encephalomyelitis virus. II. Behavior of the virus in day-old chicks.

The VR strain of avian encephalomyelitis virus, which had been adapted to embryonated hen's eggs, was inoculated into 2-day-old chicks by the subcutaneous route (10(2.5) approximately 10(3.0) EID50) or by the oral route (10(4.8) EID50). The chicks were examined chronologically for the distribution of the virus in the body. As a result, minute amounts of the virus were detected from the liver, spleen, pancreas, and muscle at the site of inoculation one day after inoculation and various amounts from almost all the organs 3 days and more after inoculation. The virus titer could nearly reach a maximum 7 to 9 days after inoculation. Above all, such high virus titers as ranging from 10(4.3) to 10(5.8) EID50/0.1 g were demonstrated in the brain, heart, liver, spleen, and pancreas. After that, there was a tendency for virus titer to decrease in most organs and for virus to multiply persistently in the pancreas, brain, and eyeball. Virus titer was maintained at a level of 10(2.3) approximately 10(2.8) EID50/0.1 g in these three organs even 21 days after inoculation. In the group of subcutaneous inoculation, all the chicks manifested clinical signs of infection 5 to 10 days after inoculation. On the other hand, no chicks were involved in clinical infection in the group of oral inoculation. Multiplication of the virus was delayed in the body of these chicks. Small amounts of the virus were detected from the spleen and pancreas 11 days after inoculation. Low titers (10(2.7) EID50/0.1 g at the highest) of the virus were only detected from the brain, spinal cord, spleen, pancreas, esophagus, and other organs 14 and 21 days after inoculation.     

Crystal formation of swine vesicular disease virus in porcine kidney cells (IB-RS-2 cells)

Crystal formation of swine vesicular disease virus (SVDV) in IB-RS-2 cells was studied by electron microscopy. Cells were harvested 0, 3, 3.5, 4, 4.5, 5, 6 and 7 hours after inoculation. Crystalline arrays of SVDV was first observed in the cytoplasm of a few cells 4.5 hours after inoculation. In the cytoplasm of many cells harvested at 5 hours, 1 to 3 crystalline arrays of SVDV were observed. After that, a small number of cells had crystalline arrays in the cytoplasm. The cells with crystalline arrays were rich in ribosome and polysome with dilated mitochondria and many tiny vesicles. An individual virus particle was ca. 18 nm in diameter, and the center-to-center space ca. 22 nm. Crystalline arrays varied in size depending on the plane of section.     

Growth Characteristics of Cytomegalovirus in Human Fibroblasts with Demonstration of Protein Synthesis Early in Viral Replication

In high-multiplicity infection of human fibroblasts, human cytomegalovirus of WI-38 human diploid cells produced early cell rounding 6 to 24 h after inoculation. This early cell rounding was caused only by inoculation with infectious virions. Inhibitors of protein synthesis, but not DNA inhibitors, prevented this cytopathic effect. Apparently, a new protein is synthesized in infected fibroblasts from about 2 h postinoculation. Infectivity of cell-associated and supernatant infectious virus reached maximal levels at 5 to 7 and 10 days postinoculation, respectively. Synthesis of DNA, infectious virus, complement-fixing antigen, and precipitin antigen all began between 24 and 48 h, with the bulk of synthesis occurring 48 to 96 h postinoculation.     

PSEUDOMONAS AERUGINOSA INFECTIONS OF THE EYE

Pseudomonas aeruginosa seldom invades the body except in persons or in organs lacking natural defenses, and usually the infection is chronic rather than acute, evoking little systemic response. When introduced into the cornea, however, as in penetration by a foreign body or in contaminated medicines, it acts with extreme virulence, in many cases causing blindness and even necessitating enucleation.Although many attempts at control of Ps. aeruginosa, even with powerful antibiotics, have been unsuccessful, polymyxin B appeared to have good effect and was tested in experimental infection of the cornea in rabbits.It was demonstrated by preliminary studies in vitro that polymyxin B was effective against nine strains of Ps. aeruginosa which on inoculation caused rapidly progressive ulcers in the corneas of rabbits.A strain of proved virulence was introduced into both eyes of each of 18 rabbits. The left eyes only were treated with subconjunctival injections at 48-hour intervals of a solution of polymyxin B, to which epinephrine was added as a vasoconstrictor to prevent rapid dispersion. The right eyes remained untreated as controls.In five of the six rabbits treated immediately after inoculation, the treated eyes remained clear, while moderate infiltration developed in the sixth. In the six rabbits not treated for 24 hours after inoculation, ulcers developed but remained localized during therapy. In those not treated for 48 hours after inoculation, ulcers developed before treatment began but did not spread as rapidly as in the controls.Hyaluronidase was added to the preparation for half the rabbits in each group but had no perceptible beneficial effect.     

The Use of <Emphasis Type="Italic">Bradyrhizobium</Emphasis> to Enhance Growth and Yield of Soybean in Calcareous Soil in Uzbekistan

In this work the effect of inoculation with Bradyrhizobium japonicum S2492 on soybean (Glycine max (L) Merr) growth, nodulation and yield in nitrogen-deficient soil of Uzbekistan was studied. The field experiments were carried out in Tashkent Province of Uzbekistan in a randomized complete block design with four replicates of each treatment. The results revealed positive effects on growth, nodule number and yields of soybean after inoculation with B. japonicum S2492. The yield of soybean varieties was 48% higher for inoculated than for uninoculated plants. The effect of the inoculation was specific for variety but not for growth type. The protein and oil contents of seeds also increased after inoculation. It was concluded that B. japonicum S2492 can be considered as a biofertilizer for increasing the productivity of soybean in nitrogen-deficient soils in Uzbekistan.     

Interaction between the nematophagous fungus Duddingtonia flagrans and infective larvae of Haemonchus contortus (Nematoda: Trichostrongyloidea)

The interaction between Duddingtonia flagrans and infective larvae of Haemonchus contortus was studied in vitro under optical and scanning electron microscopy. Trap formation by the fungus started 9 hours after inoculation and first larvae were found 11 hours after larval inoculation on colonies grown on the surface of dialysis membranes. Scanning electron micrographs were taken 12, 24, 36 and 48 h after larval predation. Details of predation structures and fungus-larvae interaction are described. A mucilaginous substance occurred at the points of adherence of traps to nematode cuticle. Bacteria were also found at some points of interaction between fungus and larval cuticle. Cuticle penetration by fungus hyphae occurred only 48 h after predation.     

Effect of fatty acids on growth of Japanese encephalitis virus cultivated in BHK-21 cells and phospholipid metabolism of the infected cells.

Growth of Japanese encephalitis virus (JEV) in BHK-21 cells was stimulated in the presence of 20 to 40 mug of the sodium salt of oleic acid (cis-9-octadecenoic acid, 9-18:1) per ml supplemented in Waymouth medium. The stimulatory effect of the salt was highest when 9-18:1 was added after adsorption of the virus. Study of the effect of other fatty acids on growth of JEV showed the following results: the longer the chain length of the saturated fatty acid salt, the higher the stimulatory effect on viral growth. In contrast, polyunsaturated fatty acids had an inhibitory effect on viral growth. The effect of isomeric cis-octadecenoic acids on viral growth was variable, depending upon the position of the double bond. The cis-6-octadecenoic acid had the highest inhibitory effect on growth of JEV compared to other isomeric octadecenoic acids. The sodium salt of (1-14C) cis-9-octadecenoic acid (9-18:1, 20 mug/ml) was rapidly incorporated into control and JEV-infected cells. Specific radioactivity in phosphatidylcholine dropped 12 to 24 h after virus inoculation, whereas synthesis of phosphatidylethanolamine increased 12 to 24 h after virus inoculation in infected cells compared to uninfected cells. Results from these studies suggest that phospholipid metabolism of infected cells is markedly changed, which can be associated with altered fatty acid metabolism when using labeled 9-18:1 fatty acid as a marker.     

An Adenovirus Isolated from the Feces of Mice

Experimental infection of an inbred strain of DK1 mice was carried out with a mouse adenovirus strain K87, isolated from the feces of apparently healthy DK1 mice. Strain K87 was orally administered to four-week-old mice and the virus was recovered from their feces for at least 3 weeks. The highest virus titers in the feces were observed between 1 and 2 weeks after inoculation. In 7-week-old mice the period of virus excretion was about one week shorter. When neonatal or 2-week-old mice were administered virus orally, somewhat irregular results but similar to those from the 4 or 7-week-old mice were obtained. In these infected mice, virus growth was detected in the intestinal tract but not in oropharyngeal washings, nasal tissue, lung, spleen or urine. After inoculation through several parenteral routes, the virus was also detected in the feces and virus growth seemed to be limited mainly to the intestinal tract. No clinical manifestations were observed in any infected mice. When the virus was almost undetectable in the mice infected either orally or parenterally, the virus was readministered orally, but no further virus was recovered in the feces. Neutralizing antibody in the serum was detected 3 weeks after primary inoculation. Induction of immunological tolerance was examined by inoculating mice in utero 2–4 days before birth, but no evidence of induced tolerance was obtained. The use of the adenovirus strain K87-mouse system as a model system for the study of the infectious process and immune mechanisms is suggested because strain K87 has a tissue tropism analogous to many human adenoviruses.