Molecular characterization of nonstructural protein NS38 of grass carp reovirus

Viral nonstructural proteins in both enveloped and non-enveloped viruses play important roles in viral replication. Protein NS38 of Grass carp reovirus (GCRV), has been deduced to be a non-structural protein, and, consistent with other reoviruses, is considered to cooperate with the NS80 protein in viral particle assembly. To investigate the molecular basis of the role of NS38, a complete protein was expressed in E.coli for the first time. It was found that there is a better expression of NS38 induced with IPTG at 28 ℃ rather than 37 ℃. In addition, the antiserum of NS38 prepared with purified fusion protein and injected into rabbit could be used for detecting NS38 protein expression in GCRV infected cell lysate, while there is not any reaction crossed with purified virus particle, confirming NS38 is not a component of the viral structural protein. The result reported in this study will provide evidence for further viral protein-protein and protein-RNA interaction in dsRNA viruses replication.     

Effect of low temperature on stability of θ-type plasmids in Carnobacterium maltaromaticum

The heterologous production of useful peptides such as bacteriocins by lactic acid bacteria (LAB) has been studied for use in the biopreservation of foods. Recombinant plasmids can suffer drawbacks such as segregational instability affecting the production of these peptides in certain environments such as absence of selective pressure or low temperature. The link between growth temperature characteristics of parental strains and stability of θ-type plasmids at a low temperature was investigated. The growth of four parental strains at 4 °C and stability of five derivative θ-type plasmids transformed into Carnobacterium maltaromaticum UAL26 at 25 and 4 °C were determined. Two plasmids (pCD11 and pCaT) derived from psychrotrophic LAB and plasmid, pHW800, from Enterococcus faecium 226 with unknown growth temperature characteristics, had excellent stability when strains were grown at 4 °C. Plasmids (pTRKH2 and pUCB820) derived from LAB that did not grow at refrigeration temperatures were not stable at 4 °C. When a DNA fragment from pCD11 containing 22-bp repeats, a putative replication initiation site, and the gene for the RepA protein was inserted into pTRKH2, the resulting derivative plasmid was 100% stable at 4 °C.     

Synchronous replication initiation in novel Mycobacterium tuberculosis dnaA cold-sensitive mutants

The genetic aspects of ori C replication initiation in Mycobacterium tuberculosis are largely unknown. A two-step genetic screen was utilized for isolating M. tuberculosis dna A cold-sensitive (cos) mutants. First, a resident plasmid expressing functional dna A integrated at the att B locus in dna A null background was exchanged with an incoming plasmid bearing a mutagenized dna A gene. Next, the mutants that were defective for growth at 30°C, a non-permissive temperature, but resumed growth and DNA synthesis when shifted to 37°C, a permissive temperature, were subsequently selected. Nucleotide sequencing analysis located mutations to different regions of the dna A gene. Modulation of the growth temperatures led to synchronized DNA synthesis. The dna A expression under synchronized DNA replication conditions continued to increase during the replication period, but decreased thereafter reflecting autoregulation. The dna Acos mutants at 30°C were elongated suggesting that they may possibly be blocked during the cell division. The DnaA115 protein is defective in its ability to interact with ATP at 30°C, but not at 37°C. Our results suggest that the optimal cell cycle progression and replication initiation in M. tuberculosis requires that the dna A promoter remains active during the replication period and that the DnaA protein is able to interact with ATP.     

Effect of temperature on life table parameters of Podisus nigrispinus (Het., Pentatomidae) fed with Alabama argillacea (Lep., Noctuidae) larvae

Abstract: The influence of temperature on life table parameters of Podisus nigrispinus (Dallas) (Het., Pentatomidae) fed with Alabama argillacea (Hübner) (Lep., Noctuidae) larvae was studied. This predator was kept at constant temperatures of 20, 23, 25, 28, 30 and 33±0.2°C, at relative humidity of 60±10% and photoperiod of L : D 14 : 10. Gross (GRR) and net ( R ₀) reproductive rates of P. nigrispinus ranged from 1.6 to 366.6 and from 0.02 to 189.5 females/female at temperatures of 33 and 28°C, respectively; generation time ( T  ) ranged from 33.3 (33°C) to 85.5 (20°C) days; doubling time ( D ) from 0.82 (33°C) to 17.8 (20°C) days; intrinsic rate of increase ( r _m ) from −0.13 (33°C) to 0.12 (28°C) per day; and the finite rate of increase ( λ ) from 0.88 (33°C) to 1.12 (28°C) females/female added to the population per day. The ideal age to release P. nigrispinus should be when this predator presents higher reproductive values (VR _x ); that is, its adults are about 7 days old, independent of prevailing temperature. Population growth of P. nigrispinus was affected by temperature with maximum numerical response between 28 and 30°C. The negative population growth shown at 33°C may not occur in natural conditions due to milder microclimate in the cotton agroecosystem and due to oscillations of temperature in the course of the day.     

Suppression of PmRab7 by dsRNA Inhibits WSSV or YHV Infection in Shrimp

Viral entry into host cells requires endocytosis machineries of the host for viral replication. PmRab7, a Penaeus monodon small GTPase protein, was investigated for its function in vesicular transport during viral infection. The double-stranded RNA of Rab7 was injected into a juvenile shrimp before challenging with white spot syndrome virus (WSSV) or yellow head virus (YHV). PmRab7 mRNA was specifically decreased at 48 h after dsRNA-Rab7 injection. Silencing of PmRab7 dramatically inhibited WSSV-VP28 mRNA and protein expression. Unexpectedly, the silencing of PmRab7 also inhibited YHV replication in the YHV-infected shrimp. These results suggested that PmRab7 is a common cellular factor required for WSSV or YHV replication in shrimp. Because PmRab7 should function in the endosomal trafficking pathway, its silencing prevents successful viral trafficking necessary for replication. Silencing of PmRab7 could be a novel approach to prevent both DNA virus (WSSV) and RNA virus (YHV) infection of shrimp.     

Thermal dependence of enzyme function and inhibition; implications for, herbicide efficacy and tolerance

Environmental temperature is a critical factor in the lives of almost all organisms. Plants experience periods of thermal stress related to seasonal patterns of temperature and periodic water deficits. Within the range of non-lethal temperatures, there are a number of thermal effects on metabolism that are a result of the thermal dependence of enzymes. The thermal dependence of enzyme kinetic parameters was used to predict that the efficacy of the herbicide pyrithiobac on Palmer amaranth would be reduced at temperatures outside a 20–34°C thermal application range. This prediction is validated in a controlled environment study described in this paper. Palmer amaranth was grown for 16 days in growth chambers with 34/18°C day/night temperature regime. Pyrithiobac was applied to plants at 18, 27 or 40°C. After 1 h at the application temperatures the plants were returned to the 34/18°C regime for 14 days and post-application biomass accumulation (efficacy) was determined. Dry weight accumulation, as a percentage of untreated controls, was 25, 2.5 and 70% for 18, 27 and 40°C application temperatures. Pyrithiobac efficacy was highest for the application within the thermal application range and significantly reduced at temperatures above and below. The validation of the earlier prediction suggests that temperature-related kinetic limitations on herbicide efficacy may also occur in plants with bioengineered herbicide resistance based on herbicide metabolism. The theoretical aspects of such thermal limitations on herbicide resistance mechanisms are discussed.     

New phytoviral vector for superexpression of target proteins in plants

Previously, we obtained a new viral vector based on the Alternanthera mosaic virus strain MU (AltMV-MU). The gene of interest was placed under control of two subgenomic promoters (sgp). Viral vector provided the superexpression of target proteins in Nicotiana benthamiana. In the present work, to increase the level of protein expression, this viral vector was modified and the coat protein gene was placed under control of three sgp. The new viral vector provided superexpression of the protein of interest in plants, and its amount was more than 50% of the total soluble protein in cells. The efficiency of the target protein expression in the plants transformed with viral vectors containing two and three sgp was compared.     

Improved purification procedure and some serological and physica properties of cassava common mosaic virus from South America

Large quantities of cassava common mosaic virus (CCMV) were purified from systemically infected Nicotiana benthamiana plants. A polyclonal antiserum, with a titre of 1/128 in the tube precipitin test, was produced by immunising rabbits with purified virus. Viral antigens were detected in cassava, using both the double-antibody sandwich or plate-trapped antigen forms of enzyme-linked immunosorbent assay (ELISA). The virus reacted with antisera to the potexviruses potato virus X and tulip virus X in F(ab')₂ ELISA. As determined by ELISA, isolates of CCMV from cassava and chaya are closely serologically related to each other. Leaf extracts from infected N. benthamiana plants were infective to a dilution of 10^--⁴ but not 10^--⁵; after heating for 10 min at 65 °C but not 70 °C; and after storage at room temperature for 14 days. The virus has a sedimentation coefficient of 126 S_20,w, a single coat protein molecule of c . mol. wt 21 000, and a single-stranded RNA genome of c . mol. wt 2.0 ± 10⁶. Several dsRNA species, including the putative viral replicative form of c . mol. wt 4.1 ± 10⁶, were isolated from virus-infected cassava and N. benthamiana .     

Influence of temperature on egg hatching, growth and survival of larvae of Heterobranchus longifilis Val. 1840 (Teleostei: Clariidae)

Eggs of Heterobranchus longifilis Val. 1840 were artificially fertilized and incubated at a range of temperatures (20, 23, 25, 27, 29 and 32°C). The time from fertilization to hatching decreased with increasing temperature. No eggs survived to hatch at 20 and 32°C incubation temperatures, while at 23 and 29°C hatching was only minimal. Optimum hatching was obtained at 25 and 27°C, which corresponds to the ambient temperature range during the breeding season. Larvae of H. longifilis were reared for 11 days post-hatching at 20, 25, 27, 29 and 32°C. Growth increased with temperature (P < 0.05), whereas survival depicted an inverse relationship. Growth was minimal at 20°C and larvae rarely survived to the end of the experiment. Optimum temperature for the primary nursing of H. longifilis larvae was within the 25–27°C temperature range.     

Balancing photosynthetic light-harvesting and light-utilization capacities in potato leaf tissue during acclimation to different growth temperatures

We investigated the effect of temperature during growth and development on the relationship between light-harvesting capacity, indicated by chlorophyll concentration, and light-utilization potential, indicated by light- and bicarbonate-saturated photosynthetic oxygen evolution, in Solanum tuberosum L. cv. Norland. Conal plantles were transplanted and grown at 20°C for 2 weeks before transfer to 12, 16, 20, 24 and 28°C for 6 weeks. After 4 weeks of the temperature treatments, leaf tissue fresh weights per area were one-third higher in plants grown at 12°C vs those grown at 28°C. Conversely, chlorophyll content per area in tissue grown at 12°C was less than one-half of that of tissue grown at 28°C at 4 weeks. Photosynthetic capacity measured at a common temperature of 20°C and expressed on a chlorophyll basis was inversely proportional to growth temperature. Leaf tissue from plants grown at 12°C for 4 weeks had photosynthetic rates that were 3-fold higher on a chlorophyll basis than comparable tissue from plants grown at 28°C. These results suggest that the relationship between light-harvesting capacity and light-utilization potential varies 3-fold in response to the growth temperatures examined. The role of this response in avoidance of photoinhibition is discussed.     

Effect of Temperature on the Formation of Microsclerotia of Verticillium dahliae

Microsclerotium formation by six isolates of Verticillium dahliae was studied at different temperatures both in vitro and in Arabidopsis thaliana . In vitro mycelial growth was optimal at 25°C, but microsclerotium formation was greatest at 20°C (two isolates) or 15–20°C (one isolate). Seedlings of A. thaliana were root-dipped in a conidial suspension, planted, and either placed at 5, 10, 15, or 25°C, or left at 20°C until the onset of senescence, after which some of the plants were placed at 5, 10, 15, or 25°C. The amount of microsclerotia per unit of shoot weight was assessed in relation to isolate and temperature. The optimal temperature for production of microsclerotia was 15–25°C. Two isolates each produced about 10 times more microsclerotia than each of the other four isolates. For these isolates, high R ²_adj.-values of 0.77 and 0.66 were obtained, with temperature and its square as highly significant (P   < 0.001) independent variables. R ²_adj.-values for the other isolates varied between 0.28 and 0.39. Moving plants to different temperatures at the onset of senescence led to microsclerotial densities that were intermediate between densities on plants that had grown at constantly 20°C and plants grown at other temperatures. This suggests that vascular colonization rate and rate of microsclerotium formation are similarly affected by temperature. The senescence rate of plants appeared unimportant except for plants grown at 25°C, which showed the highest amounts of microsclerotia per unit of plant weight in the most rapidly senescing plants.     

Effects of febrile temperature on adenoviral infection and replication: implications for viral therapy of cancer

We previously conducted a phase I/II study using arterial infusions of ONYX-015 (dl1520), a replication-selective adenoviral vector, with E1b deleted, for patients with metastatic colorectal cancer. No dose-limiting toxicities occurred, but >90% of the patients experienced fever. The effects of temperature on the replication of dl1520 in normal and transformed cells had not been studied. Therefore, replication and cell viability assays were performed with a panel of nontransformed and transformed cell lines cultured at 37 and 39.5 degrees C and treated with adenovirus type 5 (Ad5) or dl1520. Ad5-mediated cytolytic effects were inhibited and production of infectious particles decreased by >1,000-fold in the nontransformed cells at 39.5 degrees C. Seven of nine of the tumor cell lines retained significant cell-killing effects when treated with Ad5 at 39.5 degrees C. When dl1520 was used, no cytolytic effects were observed at 39.5 degrees C in the nontransformed cell lines; however, cytolytic effects occurred in six of nine tumor cell lines at 39.5 degrees C. Notably, a subset of the tumor cell lines demonstrated increased dl1520-mediated cytolytic effect and replication at 39.5 degrees C. Suppression of Ad5 and dl1520 replication at 39.5 degrees C was not related to p53 status or HSP70 expression. Also, at 39.5 degrees C, E1a expression was inhibited in nontransformed cells but was still abundant in the transformed cells, indicating that a novel early block in viral replication occurred in the nontransformed cells. Fever may therefore augment the therapeutic index of oncolytic viruses by inhibiting replication in normal cells while permitting or enhancing viral replication in some tumor cells.     

Alteration in the contents of unsaturated fatty acids in dnaA mutants of Escherichia coli

DnaA protein, the initiator of chromosomal DNA replication in Escherichia coli , has a high affinity for acidic phospholipids containing unsaturated fatty acids. We have examined here the fatty acid composition of phospholipids in dnaA mutants. A temperature-sensitive dnaA46 mutant showed a lower level of unsaturation of fatty acids (ratio of unsaturated to saturated fatty acids) at 42°C (non-permissive temperature) and at 37°C (semi-permissive temperature), but not at 28°C (permissive temperature), compared with the wild-type strain. Plasmid complementation analysis revealed that the dnaA46 mutation is responsible for the phenotype. Other temperature-sensitive dnaA mutants showed similar results. On the other hand, a cold-sensitive dnaAcos mutant, in which overinitiation of DNA replication occurs at low temperature (28°C), showed a higher level of unsaturation of fatty acids at 28°C. Based on these observations, we discuss the role of phospholipids in the regulation of the activity of DnaA protein.     

昆津病毒复制子——一种新型病毒载体

病毒复制子 (Replicon) 是指来源于病毒基因组的能够自主复制的RNA分子,保留了病毒非结构蛋白基因,而结构蛋白基因缺失或由外源基因替代。昆津病毒 (Kunjun virus) 为黄病毒科黄病毒属成员,其复制子具有表达效率高、细胞毒性低、遗传稳定等特点,在病毒基因组复制调控机制、外源蛋白表达、新型疫苗和基因治疗等领域得到了广泛应用。以下就昆津病毒复制子系统的构建、特性及应用方面的研究进展作一综述。     

Thermal ecology of western tent caterpillars Malacosoma californicum pluviale and infection by nucleopolyhedrovirus

Abstract 1. Western tent caterpillars hatch in the early spring when temperatures are cool and variable. They compensate for sub-optimal air temperatures by basking in the sun.
2. Tent caterpillars have cyclic population dynamics and infection by nucleopolyhedrovirus (NPV) often occurs in populations at high density.
3. To determine whether climatic variation might influence viral infection, the environmental determinants of larval body temperature and the effects of temperature on growth and development rates and larval susceptibility to NPV were examined.
4. In the field, larval body temperature was determined by ambient temperature, irradiance, and larval stage. The relationship between larval body temperature and ambient temperature was curvilinear, a property consistent with, but not necessarily limited to, behaviourally thermoregulating organisms.
5. Larvae were reared at seven temperatures between 18 and 36 °C. Larval growth and development increased linearly with temperature to 30 °C, increased at a lower rate to 33 °C, then decreased to 36 °C. Pupal weights were highest for larvae reared between 27 and 30 °C.
6. The pathogenicity (LD₅₀) of NPV was not influenced by temperature, but the time to death of infected larvae declined asymptotically as temperature increased.
7. Taking into account larval growth, the theoretical yield of the virus increased significantly between 18 and 21 °C then decreased slightly as temperatures increased to 36 °C.
8. Control and infected larvae showed no difference in temperature preference on a thermal gradient. The modes of temperature preference were similar to those for optimal growth and asymptotic body temperatures measured in the field on sunny days.
9. Warmer temperatures attained by basking may increase the number of infection cycles in sunny springs but do not protect larvae from viral infection.     

Geographical variation in Japan in egg development of the mayfly, Ephoron shigae (Ephemeroptera: Polymitarcyidae)

1. The effect of temperature on embryonic development was compared in four populations, two bisexual and two unisexual, of Ephoron shigae , including one each near the northern and southern periphery of the species range in Japan.
2. Eggs from every population were chilled at 4, 8 or 12 °C for diapause development after 50 days at 20 °C for pre-diapause development (experiment I). Some eggs hatched during chilling at 8 °C or 12 °C, whereas no eggs hatched at 4 °C. The rate of hatching in a given condition of chilling was higher for the eggs from warmer winter environments.
3. Chilling at 4 or 8 °C effectively facilitated diapause development. Chilling at 12 °C was, in general, not so effective, but relatively effective for the eggs from warmer winter environments.
4. Eggs were incubated at 8, 12, 15 or 20 °C after chilling at 4 °C to examine the effect of temperature on post-diapause development (experiment II). The eggs incubated at higher temperature after chilling hatched quicker and more synchronously and had higher hatching success.
5. The relationship between temperature and the days required for hatching after chilling was well described by the power function. There was no significant difference in the slope of the regression lines (i.e. temperature dependency) among local populations. However, a longer time was required for hatching at a given temperature for the population from the colder winter environment.
6. There was no detectable difference in the observed intraspecific variations between unisexual and bisexual populations.     

Constraints to virus infection in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis> plants transformed with a potyvirus amplicon

Background  

Plant genomes have been transformed with full-length cDNA copies of viral genomes, giving rise to what has been called 'amplicon' systems, trying to combine the genetic stability of transgenic plants with the elevated replication rate of plant viruses. However, amplicons' performance has been very variable regardless of the virus on which they are based. This has boosted further interest in understanding the underlying mechanisms that cause this behavior differences, and in developing strategies to control amplicon expression.