Sampling Natural Viral Communities from Soil for Culture-Independent Analyses

Williamson, Kurt E.; Wommack, K. Eric; Radosevich, Mark

doi:10.1128/aem.69.11.6628-6633.2003

Cited by 156 publications

(135 citation statements)

References 47 publications

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“…SYBR Gold has so far been used to stain viruses in samples from the water column (McDaniel et al 2001, Yager et al 2001, Williamson et al 2002, Anesio et al 2004, Breitbart et al 2004, Brussard 2004, Lisle & Priscu 2004, Wen et al 2004) and sediment (Fischer et al 2003(Fischer et al , 2004 of aquatic systems, in soil samples (Williamson et al 2003) and human feces (Breitbart et al 2003). Direct comparison of staining viruses with SYBR Gold and the commonly used dye SYBR Green I has been made in 4 studies: (1) Chen et al (2001) reported that even when no antifading-solution was used, the fluorescence of SYBR Gold-stained viruses in marine water samples was stable for more than 2 min under EFM, while the SYBR Green I signal faded within 30 s. Using flow cytometry to enumerate Cyanophage P49, the mean fluorescence per P49 virus determined with SYBR Gold was about 2 times higher than that with SYBR Green I.…”

Section: Comparison Of Sybr Green I and Sybr Gold Viral Countsmentioning

confidence: 99%

Optimization of extraction and estimation of viruses in silty freshwater sediments

Fischer¹,

Kirschner²,

Velimirov

2005

Aquat. Microb. Ecol.

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The present study focused on the optimization of procedures for the extraction of viruses from silty freshwater sediments for subsequent enumeration. Viral abundance in 2 different shallow backwater systems of the River Danube (Austria) ranged from 1.45 × 10 9 to 9.58 × 10 9 particles ml -1 wet sediment. The highest virus yields from the bulk of the sediments were obtained by 1 min sonication (3 × 20 s intervals, with 10 s interruptions). An increase in sonication time of up to 5 min decreased viral counts by an average of 15%. Since dissolved DNA within sediment samples could bind to the nucleic acid stain and thereby inflate viral estimates, sediment samples are often treated with DNase before the staining procedure. Moreover, they are usually centrifuged and diluted to a high extent in order to avoid interference of particulate material with virus counting. Centrifugation led to a reduction of viral numbers by 2 to 36% compared to untreated samples and did not reduce the background fluorescence; thus counting of viruses was not facilitated. Diluting 2000 × with Milli-Q water always provided an average of 19% lower viral numbers than diluting 4000 ×. Treatment with DNase had no significant effect on virus counting, with viral numbers in untreated samples being on average 96% of those in DNase-containing samples. Additionally, 2 different nucleic acid stains were compared -viruses stained with SYBR Gold fluoresced brighter than those stained with SYBR Green I and fluorescence lasted longer, while background fluorescence was reduced sufficiently, thus facilitating virus counting. Viral numbers using SYBR Gold were on average twice of those obtained with SYBR Green I. The mean efficiency of virus extraction was 88.8% using the protocol outlined in this paper, and was thus slightly higher than that obtained in previous sediment investigations. KEY WORDS: Virus extraction · Virus counting · Epifluorescence microscopy · Silty freshwater sedimentResale or republication not permitted without written consent of the publisher Aquat Microb Ecol 40: 207-216, 2005 Moreover, EFM yields higher and more precise viral counts than TEM (Hennes & Suttle 1995, Weinbauer & Suttle 1997, Noble & Fuhrman 1998. Enumeration of viruses by EFM is based on using highly fluorescent nucleic acid dyes such as DAPI (Hara et al. 1991, Weinbauer & Suttle 1997, Yo-Pro I (Hennes & Suttle 1995, Xenopoulos & Bird 1997, SYBR Green I (Noble & Fuhrman 1998) or SYBR Gold (Chen et al. 2001). Viral abundance in sediment systems is 10-to 1000-fold higher than in the overlying water column (e.g. Maranger & Bird 1996, Steward et al. 1996, Drake et al. 1998, Hewson et al. 2001, Lawrence et al. 2002, Fischer et al. 2003. Studies using EFM to estimate viruses in sediments applied a variety of protocols, without testing whether the procedures carried out before enumeration (e.g. virus dislodgement from particles, centrifugation, dilution) affect viral counts (Danovaro & Serresi 2000, RicciardiRigault et al. 2000, Danovaro et al. 2001, Fischer et a...

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Section: Comparison Of Sybr Green I and Sybr Gold Viral Countsmentioning

confidence: 99%

Optimization of extraction and estimation of viruses in silty freshwater sediments

Fischer¹,

Kirschner²,

Velimirov

2005

Aquat. Microb. Ecol.

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“…While these model systems have provided valuable insights into phage-host interactions in soils, culture-independent detection and analyses will be critical in obtaining a more complete understanding of the ecological impacts of viruses in soils. As recently as 2003, the abundance of autochthonous viruses in soil was unknown (5,57). Previous assessments of viral abundance in soils were based on determination of PFU by using susceptible indicator strains (64), microscopic enumeration of optically active viruses such as baculoviruses (53), or PCR amplification of viral nucleic acids by using specific primers (43).…”

mentioning

confidence: 99%

Abundance and Diversity of Viruses in Six Delaware Soils

Williamson

Radosevich

Wommack

2005

Appl Environ Microbiol

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259

199

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The importance of viruses in marine microbial ecology has been established over the past decade. Specifically, viruses influence bacterial abundance and community composition through lysis and alter bacterial genetic diversity through transduction and lysogenic conversion. By contrast, the abundance and distribution of viruses in soils are almost completely unknown. This study describes the abundance and diversity of autochthonous viruses in six Delaware soils: two agricultural soils, two coastal plain forest soils, and two piedmont forest soils. Viral abundance was measured using epifluorescence microscopy, while viral diversity was assessed from morphological data obtained through transmission electron microscopy. Extracted soil virus communities were dominated by bacteriophages that demonstrated a wide range of capsid diameters (20 nm to 160 nm) and morphologies, including filamentous forms and phages with elongated capsids. The reciprocal Simpson's index suggests that forest soils harbor more diverse assemblages of viruses, particularly in terms of morphological distribution. Repeated extractions of virus-like particles (VLPs) from soils indicated that the initial round of extraction removes approximately 70% of extractable viruses. Higher VLP abundances were observed in forest soils (1.31 ؋ 10 9 to 4.17 ؋ 10 9 g ؊1 dry weight) than in agricultural soils (8.7 ؋ 10 8 to 1.1 ؋ 10 9 g ؊1 dry weight). Soil VLP abundance was significantly correlated to moisture content (r ‫؍‬ 0.988) but not to soil texture. Land use (agricultural or forested) was significantly correlated to both bacterial (r ‫؍‬ 0.885) and viral (r ‫؍‬ 0.812) abundances, as were soil organic matter and water content. Thus, land use is a significant factor influencing viral abundance and diversity in soils.

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“…The recovery rate was 37% of enterovirus according to Monpoeho et al (2001). Williamson et al (2003) proposed four elution buffers (10% beef extract, 250 mM glycine buffer, 10 mM sodium pyrophosphate, and 1% potassium citrate). Beef extract and glycine buffer were the most effective in eluting viable phages inoculated into soils, with up to 29% recovery.…”

Section: Virus Elution Methodsmentioning

confidence: 99%

“…TEM is the only method that provides data on both the abundance and morphology of virus-like particles (Borsheim et al 1990). However, the costs involved and technical aspects can lead to variable and inaccurate estimates of the total abundance (Sambrook et al 1989, Williamson et al 2003.…”

Section: Other Detection Techniquesmentioning

confidence: 99%

Methods of Virus Detection in Soils and Sediments

Staggemeier¹,

Almeida²,

Spilki³

2011

VR&R

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Anthropogenic impacts on soils have become one of the major environmental concerns nowadays. Soils and sediments under water bodies may contain viruses and bacteria on higher loads than those identified in contaminated waters. Thesecontaminants may interfere with the whole environment of the affected area, not only damaging the soil and the water but also affecting wildlife. Viruses can travel across the ground contaminating groundwater. The focus of research concerning viruses in soil matrices has been the destination, transport, and detection of pathogenic viruses exogenous to soils. Many methods have been suggested in the last few years for viral detection in soil and sediments. Viruses can be extracted from soil by elution, concentrated and finally detected by molecular techniques cell culture. There are many elution methods described in the literature and 11 techniques are cited in this review. Molecular detection can be performed by Polymerase Chain Reaction (PCR) and its modifications. New assays for viral quantification are emerging, such as adaptation of plaque assays (PAs), most-probable number assays (MPNs), transmission electron microscopy (TEM), epifluorescence microscopy (EfM), and flow cytometry (FC).

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Sampling Natural Viral Communities from Soil for Culture-Independent Analyses

Cited by 156 publications

References 47 publications

Optimization of extraction and estimation of viruses in silty freshwater sediments

Optimization of extraction and estimation of viruses in silty freshwater sediments

Abundance and Diversity of Viruses in Six Delaware Soils

Methods of Virus Detection in Soils and Sediments

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