Structural changes associated with poliovirus inactivation in soil.

The loss of infectivity of poliovirus in moist and dried soils was a result of irreversible damage to the virus particles. The damage included (i) dissociation of viral genomes and capsids and (ii) degradation of viral ribonucleic acid (RNA) in the soil environment. Under drying conditions, capsid components could not be recovered from the soils. Further studies in sterile soils indicated that, under moist conditions, the viral RNA was probably damaged before dissociation from the capsid. However, in sterile, dried soil, RNA genomes were recovered largely intact from the soil. These results suggest that polioviruses are inactivated by different mechanisms in moist and drying soils.     

Persistence of Denitrifying Enzyme Activity in Dried Soils

The effects of air drying soil on denitrifying enzyme activity, denitrifier numbers, and rates of N gas loss from soil cores were measured. Only 29 and 16% of the initial denitrifying enzyme activity in fresh, near field capacity samples of Maury and Donerail soils, respectively, were lost after 7 days of air drying. The denitrifying activity of bacteria added to soil and activity recently formed in situ were not stable during drying. When dried and moist soil cores were irrigated, evolution of N gas began, and it maximized sooner in the dried cores. This suggests that the persistence of denitrifying enzymes permits accelerated denitrification when dried soils are remoistened. Enzyme activity increased significantly in these waterlogged cores, but fluctuations in enzyme activity were small compared with fluctuations in actual denitrification rate, and enzyme activities were always greater than denitrification rates. Apparent numbers of isolatable denitrifiers (most-probable-number counts) decreased more than enzyme activity as the soils were dried, but after the soils were rewetted, the extent of apparent growth was not consistently related to the magnitude of N loss. We hypothesize that activation-inactivation of existing enzymes by soil O₂ is of greater significance in transient denitrification events than is growth of denitrifiers or synthesis of new enzymes.     

The effect of soil drying on millet

Summary Two tropical soils were collected when moist. Part of the soil was kept moist under aerobic conditions for one week (Treatment A) and the other part air dried over the same period and then remostened (Treatment B). The soils were then sown to millet. Both the yields and percentage nitrogen of the millet were very significantly increased by Treatment B and also the amount of mineral soil nitrogen produced. Additional treatments showed that yield increases were due to increased nitrogen mineralisation and that pathogenic conditions in the moist soil (Treatment A) were not responsible for the low yields.     

Hydrologic Regime Controls Soil Phosphorus Fluxes in Restoration and Undisturbed Wetlands

Many wetland restoration projects occur on former agricultural soils that have a history of disturbance and fertilization, making them prone to phosphorus (P) release upon flooding. To study the relationship between P release and hydrologic regime, we collected soil cores from three restoration wetlands and three undisturbed wetlands around Upper Klamath Lake in southern Oregon, U.S.A. Soil cores were subjected to one of three hydrologic regimes—flooded, moist, and dry—for 7.5 weeks, and P fluxes were measured upon reflooding. Soils from restoration wetlands released P upon reflooding regardless of the hydrologic regime, with the greatest releases coming from soils that had been flooded or dried. Undisturbed wetland soils released P only after drying. Patterns in P release can be explained by a combination of physical and biological processes, including the release of iron‐bound P due to anoxia in the flooded treatment and the mineralization of organic P under aerobic conditions in the dry treatment. Higher rates of soil P release from restoration wetland soils, particularly under flooded conditions, were associated with higher total P concentrations compared with undisturbed wetland soils. We conclude that maintaining moist soil is the means to minimize P release from recently flooded wetland soils. Alternatively, prolonged flooding provides a means of liberating excess labile P from former agricultural soils while minimizing continued organic P mineralization and soil subsidence.     

Capsid protein synthesis from replicating RNA directs specific packaging of the genome of a multipartite, positive-strand RNA virus

Flock house virus (FHV) is a bipartite, positive-strand RNA insect virus that encapsidates its two genomic RNAs in a single virion. It provides a convenient model system for studying the principles underlying the copackaging of multipartite viral RNA genomes. In this study, we used a baculovirus expression system to determine if the uncoupling of viral protein synthesis from RNA replication affected the packaging of FHV RNAs. We found that neither RNA1 (which encodes the viral replicase) nor RNA2 (which encodes the capsid protein) were packaged efficiently when capsid protein was supplied in trans from nonreplicating RNA. However, capsid protein synthesized in cis from replicating RNA2 packaged RNA2 efficiently in the presence and absence of RNA1. These results demonstrated that capsid protein translation from replicating RNA2 is required for specific packaging of the FHV genome. This type of coupling between genome replication and translation and RNA packaging has not been observed previously. We hypothesize that RNA2 replication and translation must be spatially coordinated in FHV-infected cells to facilitate retrieval of the viral RNAs for encapsidation by newly synthesized capsid protein. Spatial coordination of RNA and capsid protein synthesis may be key to specific genome packaging and assembly in other RNA viruses.     

Infectivity variation of Discaria trinervis-nodulating Frankia in Patagonian soil according to season and storage conditions

Changes in the infectious capacity of soilborne Frankia from the same site may depend on environmental conditions. To test this, we examined the effect of season of sampling, sample storage protocol and storage time. The nodulation capacity of Frankia from rhizospheric soils of Discaria trinervis (Hook et Arn.) Reiche (Rhamnaceae) growing in northwest Patagonia (Argentina) was measured using the most probable number method. Soil samples were collected seasonally and either stored moist at 4°C or air dried at room temperature for few days. Old (air-dried) soil samples were also assayed. All soils nodulated D. trinervis seedlings. Nodulation units (NU) ranged from 44 (spring, moist storage) to about 1 ml⁻¹ of soil (summer moist, and summer and autumn, air-dried storage), with intermediate values in other samples. Soils stored for 12 years, 6 months or 1 week had similar NU. Frankia NU positively correlated with soil water content ( r = 0.6, P < 0.05); therefore, it is likely that soil moisture is a relevant factor regulating soilborne Frankia nodulation ability in Patagonian soils. We suggest that Frankia can remain as spores or grow saprophytically in Patagonian soils.     

Enterovirus inactivation in soil.

The inactivation of radioactively labeled poliovirus type 1 and coxsackievirus B 1 in soils saturated with surface water, groundwater, and septic tank liquor was directly proportional to temperature. Virus persistence was also related to soil type and the liquid amendment in which viruses were suspended. At 37 degrees C, no infectivity was recovered from saturated soil after 12 days; at 4 degrees C, viruses persisted for at least 180 days. No infectivity was recovered from dried soil regardless of temperature, soil type, or liquid amendment. Additional experiments showed that evaporation of soil water was largely responsible for the decreased recovery of infectivity from drying soil. Increased rates of virus inactivation at low soil moisture levels were also demonstrated.     

Enterovirus inactivation in soil.

The inactivation of radioactively labeled poliovirus type 1 and coxsackievirus B 1 in soils saturated with surface water, groundwater, and septic tank liquor was directly proportional to temperature. Virus persistence was also related to soil type and the liquid amendment in which viruses were suspended. At 37 degrees C, no infectivity was recovered from saturated soil after 12 days; at 4 degrees C, viruses persisted for at least 180 days. No infectivity was recovered from dried soil regardless of temperature, soil type, or liquid amendment. Additional experiments showed that evaporation of soil water was largely responsible for the decreased recovery of infectivity from drying soil. Increased rates of virus inactivation at low soil moisture levels were also demonstrated.     

Preliminary characterization of coxsackievirus B3 temperature-sensitive mutants.

Prototype temperature-sensitive (ts) mutants of a coxsackievirus B3 parent virus capable of replication to similar levels at 34 or 39.5 degrees C were examined for the nature of the temperature-sensitive event restricting replication in HeLa cells at 39.5 degrees C. The ts mutant prototypes represented three different non-overlapping complementation groups. The ts1 mutant (complementation group III) synthesized less than 1% of the infectious genomic RNA synthesized by the coxsackievirus B3 parent virus at 39.5 degrees C and was designated an RNA- mutant. Agarose gel analysis of glyoxal-treated RNA from cells inoculated with ts1 virus revealed that cell RNA synthesis continued in the presence of synthesis of the small amount of viral RNA. This mutant was comparatively ineffective in inducing cell cytopathology and in directing synthesis of viral polypeptides, likely due to the paucity of nascent genomes for translation. The ts5 mutant (complementation group II) directed synthesis of appreciable quantities of both viral genomes (RNA+) and capsid polypeptides; however, assembly of these products into virions occurred at a low frequency, and virions assembled at 39.5 degrees C were highly unstable at that temperature. Shift-down experiments with ts5-inoculated cells showed that capsid precursor materials synthesized at 39.5 degrees C can, after shift to 34 degrees C, be incorporated into ts5 virions. We suggest that the temperature-sensitive defect in this prototype is in the synthesis of one of the capsid polypeptides that cannot renature into the correct configuration required for stability in the capsid at 39.5 degrees C. The ts11 mutant (complementation group I) also synthesized appreciable amounts of viral genomes (RNA+) and viral polypeptides at 39.5 degrees C. Assembly of ts11 virions at 39.5 degrees C occurred at a low frequency, and the stability of these virions at 39.5 degrees C was similar to that of the parent coxsackievirus B3 virions. The temperature-sensitive defect in the ts11 prototype is apparently in assembly. The differences in biochemical properties of the three prototype ts mutants at temperatures above 34 degrees C may ultimately offer insight into the differences in pathogenicity observed in neonatal mice for the three prototype ts mutants.     

Effects of forest soil desiccation on the growth of Eucalyptus regnans F. Muell. seedlings

Abstract. The growth rate of Eucalyptus regnans seedlings in their first year can be much increased if the soil is first dried and then rewetted. The ratio of growth on predried soil to growth on undried soil (the Growth Ratio or GR) reaches a maximum at air-dryness (pF 6.0–6.4). In E. regnans forest soil, GR is greatest in humus-rich topsoil and declines with depth. The effect of air-drying persists for several months after rewetting when soil is stored under glasshouse conditions. It is largely unaffected by repeated drying and wetting, by the rate of drying or by the season of collection. The mixing of dried and undried soil or the placement of a layer of dried soil above undried soil produces an enhancement of growth proportional to the amount of dried soil added. Firing of a litter layer above soil at wilting point increases subsequent seedling growth to that in air-dried soil. The addition of ash from a litter fire to undried soil produces an increase in growth approximately equal to that caused by air-drying The drying effect is most pronounced in soils from mature E. regnans forest and nearby brackenland and is less in dense younger forest, frost-hollow grasslands and old grassy gaps in the mature forest. The effect is restored by the inoculation of E. regnans mycorrhizal roots from both dried and undried soil. The effect varies along an gradient from 500 to 1500 m a.s.l. and is a maximum in the wet E. regnans climatic zone and a minimum in zones or local aspects where forests are normally subject to frequent drying. The stimulatory effect on seedling growth in soils of the E. regnans zone may have an effect on the outcome of competition during regeneration in large gaps. Part of the growth responses previously ascribed to the ‘ash-bed’ effect may be due to the desiccation effect in these soils.     

Recovery of soil respiration after drying

Soils are frequently exposed to drying and wetting events and previous studies have shown that rewetting results in a strong but short-lived flush of microbial activity. The aim of this study was to determine the effect of the water content during the dry period on the size and duration of the flush and on the rate of recovery. Two soils (a sand and a sandy loam) were maintained at different water contents (WC) 30, 28 and 25 g water kg^?1 soil (sand) and 130, 105 and 95 g water kg^?1 soil (sandy loam) for 14 days, then rewet to the water content at which respiration was optimal [WC 35 (sand), WC200 (sandy loam)] and maintained at this level until day 68. Ground pea straw (C/N 26) was added and incorporated on day 1. The controls were maintained at the optimal water content throughout the 68 days. Respiration rates during the dry phase (days 1?C14) decreased with decreasing water content. The flush of respiration after rewetting peaked on day 15 in the sandy loam and on day 16 in the sand; it was greatest in the soils that had been maintained at the lowest water content [WC25 (sand) and WC95 (sandy loam)]. Cumulative respiration during the remainder of the incubation period in which all soils were maintained at optimal water content increased more strongly in the soils that had been dry compared to the constantly moist control. On the final day of the dry period (day 14), cumulative respiration in the dry soils was 29?C65% (sand) and 67?C94% (sandy loam) of the constantly moist control whereas on day 68 it was 80?C84% (sand) and 86?C96% (sandy loam). The greater increase in cumulative respiration in the previously dry soils can be explained by the reduced decomposition rates during the dry period which resulted in higher substrate availability on day 14 compared to the constantly moist control. Microbial community structure assessed by phospholipid fatty acid analyses changed over time in all treatments but was less affected by water content than respiration; it differed only between the highest and the lowest water content. These differences were maintained throughout the incubation period in the sandy loam and transiently in the sand. It can be concluded that the soil water content during the dry phase affects the size of the flush in microbial activity upon rewetting and that microbial activity in previously dried soils may not be fully restored even after 54 days of moist incubation, suggesting that drying of soil can have a significant and long-lasting impact on microbial functioning.     

Mechanisms of infection with Epstein-Barr virus. I. Viral DNA replication and formation of noninfectious virus particles in superinfected Raji cells.

Human lymphoblastoid Raji cells, which do not produce virus, supported replication of Epstein-Barr virus (EBV) upon superinfection. Early antigen, viral capsid antigen, and virions were produced in Raji cells superinfected with EBV. Viral DNA replicated under complete inhibition of host cell DNA synthesis to the extent that a few micrograms of EBV DNA were recovered from 107 superinfected Raji cells, corresponding to 5,000 viral genomes/cell. Homology of the synthesized viral DNA to parental EBV DNA was more than 90%. Virions produced by the Raji cells contained a 55S DNA but failed to induce early antigen, viral capsid antigen, and viral DNA synthesis after a second superinfection of Raji cells.     

Development of DNA vaccines for foot-and-mouth disease, evaluation of vaccines encoding replicating and non-replicating nucleic acids in swine.

We have developed naked DNA vaccine candidates for foot-and-mouth disease (FMD), an important disease of domestic animals. The virus that causes this disease, FMDV, is a member of the picornavirus family, which includes many important human pathogens, such as poliovirus, hepatitis A virus, and rhinovirus. Picornaviruses are characterized by a small (7-9000 nucleotide) RNA genome that encodes capsid proteins, processing proteinases, and enzymes required for RNA replication. We have developed two different types of DNA vaccines for FMD. The first DNA vaccine, pP12X3C, encodes the viral capsid gene (P1) and the processing proteinase (3C). Cells transfected with this DNA produce processed viral antigen, and animals inoculated with this DNA using a gene gun produced detectable antiviral immune responses. Mouse inoculations with this plasmid, and with a derivative containing a mutation in the 3C proteinase, indicated that capsid assembly was essential for induction of neutralizing antibody responses. The second DNA vaccine candidate, pWRMHX, encodes the entire FMDV genome, including the RNA-dependent RNA polymerase, permitting the plasmid-encoded viral genomes to undergo amplification in susceptible cells. pWRMHX encodes a mutation at the cell binding site, preventing the replicated genomes from causing disease. Swine inoculated with this vaccine candidate produce viral particles lacking the cell binding site, and neutralizing antibodies that recognize the virus. Comparison of the immune responses elicited by pP12X3C and pWRMHX in swine indicate that the plasmid encoding the replicating genome stimulated a stronger immune response, and swine inoculated with pWRMHX by the intramuscular, intradermal, or gene gun routes were partially protected from a highly virulent FMD challenge.     

Quantitative 16S rDNA-targeted polymerase chain reaction and oligonucleotide hybridization for the detection of Paenibacillus azotofixans in soil and the wheat rhizosphere

Abstract: A molecular method for the detection of Paenibacillus azotofixans in soil and the wheat rhizosphere was developed. The system consisted of polymerase chain reaction (PCR) amplification of part of the variable V1 to V4 regions of the 16S ribosomal RNA gene, followed by hybridization with a specific oligonucleotide probe homologous to part of the intervening region. In vitro specificity tests showed that the detection system worked specifically for P. azotofixans strains, and did not detect other Paenibacillus species or species of other bacterial genera. Vegetative cells of a rifampicin resistant P. azotofixans derivative were trackable in Flevo silt loam (FSL) soil in 24 h experiments using both selective plating and most probable number (MPN)-PCR combined with probing, and plate counts parallelled MPN-PCR estimations of numbers of specific targets. MPN-PCR allowed for the detection of down to 10² introduced cells per g of dry soil. Introduced P. azotofixans spores did not form colonies on selective plates, but were detectable via PCR. The P. azotofixans populations introduced into the silt loam soil suffered a slow decline of the detectable plate count over a period of 14 days. MPN-PCR revealed a similar decline of the number of specific DNA targets. Greater numbers of targets were found in wheat rhizosphere from Flevo silt loam soil, and these numbers persisted throughout the experiment. Soil drying resulted in enhanced persistence of the target sequences, whereas in a constantly moist soil the numbers of target sequences declined. Rewetting of dried soil resulted in declining target sequence numbers. The MPN-PCR detection method is adequate to assess the impact of stress conditions affecting P. azotofixans in FSL and probably other soils, since it abolishes the need for culturing or specific markers and is direct and unambiguous due to its high specificity.     

Factors affecting the survival of sclerotia of Sclerotium cepivorum in the Fraser Valley of British Columbia

Sclerotia of Sclerotium cepivorum buried in muck soil in the Fraser Valley decayed with time. The rate of decay of sclerotia was influenced by local environmental conditions. A mixture of soil with sclerotia increased their survival but there was no difference in the rates of decay in three different soils. The decay was greatest during winter when Fraser Valley fields are often flooded. Sclerotial decay was also affected by pretreatment of the sclerotia. Dried sclerotia decayed significantly (P < 0.05) faster than sclerotia which had not been dried, a phenomenon which is apparently due to changes in micro-organisms on the sclerotia. Dried sclerotia which had been incubated in moist soil had fewer bacteria and more fungi than sclerotia which had been incubated in soil without being dried. The increase in fungi on the dried sclerotia was due to a dramatic increase in Trichoderma spp.     

Encapsidation of poliovirus replicons encoding the complete human immunodeficiency virus type 1 gag gene by using a complementation system which provides the P1 capsid protein in trans.

Poliovirus genomes which contain small regions of the human immunodeficiency virus type 1 (HIV-1) gag, pol, and env genes substituted in frame for the P1 capsid region replicate and express HIV-1 proteins as fusion proteins with the P1 capsid precursor protein upon transfection into cells (W. S. Choi, R. Pal-Ghosh, and C. D. Morrow, J. Virol. 65:2875-2883, 1991). Since these genomes, referred to as replicons, do not express capsid proteins, a complementation system was developed to encapsidate the genomes by providing P1 capsid proteins in trans from a recombinant vaccinia virus, VV-P1. Virus stocks of encapsidated replicons were generated after serial passage of the replicon genomes into cells previously infected with VV-P1 (D. C. Porter, D. C. Ansardi, W. S. Choi, and C. D. Morrow, J. Virol. 67:3712-3719, 1993). Using this system, we have further defined the role of the P1 region in viral protein expression and RNA encapsidation. In the present study, we constructed poliovirus replicons which contain the complete 1,492-bp gag gene of HIV-1 substituted for the entire P1 region of poliovirus. To investigate whether the VP4 coding region was required for the replication and encapsidation of poliovirus RNA, a second replicon in which the complete gag gene was substituted for the VP2, VP3, and VP1 capsid sequences was constructed. Transfection of replicon RNA with and without the VP4 coding region into cells resulted in similar levels of expression of the HIV-1 Gag protein and poliovirus 3CD protein, as indicated by immunoprecipitation using specific antibodies. Northern (RNA) blot analysis of RNA from transfected cells demonstrated comparable levels of RNA replication for each replicon. Transfection of the replicon genomes into cells infected with VV-P1 resulted in the encapsidation of the genomes; serial passage in the presence of VV-P1 resulted in the generation of virus stocks of encapsidated replicons. Analysis of the levels of protein expression and encapsidated replicon RNA from virus stocks after 21 serial passages of the replicon genomes with VV-P1 indicated that the replicon which contained the VP4 coding region was present at a higher level than the replicon which contained a complete substitution of the P1 capsid sequences. These differences in encapsidation, though, were not detected after only two serial passages of the replicons with VV-P1 or upon coinfection and serial passage with type 1 Sabin poliovirus.(ABSTRACT TRUNCATED AT 400 WORDS)     

Microbial activity and community composition in saline and non-saline soils exposed to multiple drying and rewetting events

Background and aims

The effects of drying and rewetting (DRW) have been studied extensively in non-saline soils, but little is known about the impact of DRW in saline soils. An incubation experiment was conducted to determine the impact of 1?C3 drying and re-wetting events on soil microbial activity and community composition at different levels of electrical conductivity in the saturated soil extract (ECe) (ECe 0.7, 9.3, 17.6 dS m^?1).

Methods

A non-saline sandy loam was amended with NaCl to achieve the three EC levels 21 days prior to the first DRW; wheat straw was added 7 days prior to the first DRW. Each DRW event consisted of 1 week drying and 1 week moist (50% of water holding capacity, WHC). After the last DRW, the soils were maintained moist until the end of the incubation period (63 days after addition of the wheat straw). A control was kept moist (50% of WHC) throughout the incubation period.

Results

Respiration rates on the day after rewetting were similar after the first and the second DRW, but significantly lower after the third DRW. After the first and second DRW, respiration rates were lower at EC17.6 compared to the lower EC levels, whereas salinity had little effect on respiration rates after the third DRW or at the end of the experiment when respiration rates were low. Compared to the continuously moist treatment, respiration rates were about 50% higher on day 15 (d15) and d29. On d44, respiration rates were about 50% higher at EC9.7 than at the other two EC levels. Cumulative respiration was increased by DRW only in the treatment with one DRW and only at the two lower EC levels. Salinity affected microbial biomass and community composition in the moist soils but not in the DRW treatments. At all EC levels and all sampling dates, the community composition in the continuously moist treatment differed from that in the DRW treatments, but there were no differences among the DRW treatments.

Conclusions

Microbes in moderately saline soils may be able to utilise substrates released after multiple DRW events better than microbes in non-saline soil. However, at high EC (EC17.6), the low osmotic potential reduced microbial activity to such an extent that the microbes were not able to utilise substrate released after rewetting of dry soil.     

Functional characterization of cleavage-defective mutants of encephalomyocarditis virus.

We characterized seven temperature-sensitive capsid cleavage (cleavage-defective) mutants of encephalomyocarditis virus. Our experimental approach was to monitor in vitro proteolysis reactions of either wild-type or cleavage-defective mutant capsid precursors mixed with cell-free translation products (containing the viral protease) of either wild-type or mutant viral RNA. The cell-free translation reactions and in vitro proteolysis reactions were done at 38 degrees C, because at this temperature cleavage of the capsid precursors was restricted in reactions containing cleavage-defective mutant viral RNA as the message, relative to those reactions containing wild-type viral RNA as the message. Wild-type or cleavage-defective mutant capsid precursors were prepared by adding cycloheximide to cell-free translation reactions primed with wild-type or mutant viral RNA, respectively, 12 min after the initiation of translation. In vitro proteolysis of wild-type capsid precursors with cell-free translation products of either wild-type or cleavage-defective mutant viral RNA led to similar products at 38 degrees C, indicating that the cleavage-defective mutant viral protease was not temperature sensitive. As a corollary to this, at 38 degrees C cleavage-defective mutant capsid precursors were not cleaved as completely as were wild-type capsid precursors by products of cell-free translation of wild-type viral RNA. The results from these in vitro proteolysis experiments indicate that all seven of the cleavage-defective mutants have capsid precursors with a temperature-sensitive configuration.