336
September 1988
Family: Alphaflexiviridae
Genus: Potexvirus
Species: Nerine virus X
Acronym: NVX


Nerine virus X

Sue Phillips
AFRC Institute of Horticultural Research, Littlehampton, West Sussex, England, BN17 6LP

A. A. Brunt
AFRC Institute of Horticultural Research, Littlehampton, West Sussex, England, BN17 6LP

Contents

Introduction
Main Diseases
Geographical Distribution
Host Range and Symptomatology
Strains
Transmission by Vectors
Transmission through Seed
Transmission by Grafting
Transmission by Dodder
Serology
Nucleic Acid Hybridization
Relationships
Stability in Sap
Purification
Properties of Particles
Particle Structure
Particle Composition
Properties of Infective Nucleic Acid
Molecular Structure
Genome Properties
Satellites
Relations with Cells and Tissues
Ecology and Control
Notes
References
Acknowledgements
Figures

Introduction

First detected by Brunt et al. (1970) and later described by Hakkaart et al. (1975), Maat (1976) and Phillips & Brunt (1980).

A virus with filamentous particles c. 540 x 11 nm occurring naturally in Nerine sarniensis (Amaryllidaceae) and Agapanthus praecox (Liliaceae). Isolates from A. praecox are readily sap-transmissible to herbaceous indicator species. Reported to occur in Britain, the Netherlands and New Zealand.

Main Diseases

In Nerine sarniensis the virus often occurs together with one or more other viruses, so its effect alone is unknown (Hakkaart et al., 1975). In Agapanthus praecox subsp. orientalis, however, it induces either symptomless infection or, occasionally, very faint leaf chlorosis (Phillips & Brunt, 1978, 1980).

Geographical Distribution

Reported only from Britain (Brunt et al., 1970), the Netherlands (Hakkaart et al., 1975) and New Zealand (K. Milne, personal communication), but is probably much more widespread.

Host Range and Symptomatology

Although reported to occur naturally only in Nerine sarniensis and Agapanthus praecox ssp. orientalis, isolates from the latter also readily infected 12 of 37 species from 6 of 10 other families (Phillips & Brunt, 1980).

Diagnostic species

Chenopodium amaranticolor and C. murale. Chlorotic lesions in inoculated leaves after 12 days (Fig.1). No systemic infection.

Chenopodium quinoa. Chlorotic and necrotic lesions in inoculated leaves after 10-15 days (Fig.2), followed by faint to severe chlorosis of systemically infected leaves.

Gomphrena globosa. A few pink to white chlorotic lesions in inoculated leaves after 18-21 days. No systemic infection.

Tetragonia expansa. Small (1 mm) chlorotic etched lesions in inoculated leaves after 16-20 days (Fig.3). No systemic infection.

Propagation species

Chenopodium quinoa is a good source of virus for purification and is a convenient host for maintaining cultures.

Assay species

Chenopodium murale and C. quinoa are useful local lesion assay hosts.

Strains

Isolates from the two natural host species are serologically indistinguishable. However, unlike isolates from Nerine sarniensis (Maat, 1976), those from Agapanthus praecox are readily sap-transmissible, attain a high concentration in indicator hosts, and are easily extracted and purified (Phillips & Brunt, 1980).

Transmission by Vectors

No information.

Transmission through Seed

Not seed-borne in Chenopodium quinoa (Phillips & Brunt, 1980).

Serology

The virus is a good immunogen. An antiserum to an isolate from Agapanthus praecox, with a homologous titre of 1/16,384 in tube precipitin tests, was produced by injecting a rabbit intramuscularly on each of two occasions, 21 days apart, with 2-3 mg virus emulsified with Freund’s complete adjuvant (Phillips & Brunt, 1980). An antiserum to an isolate from Nerine sarniensis, with a titre of 1/1024 in microprecipitin tests, was obtained by injecting a rabbit intravenously, followed 2 weeks later by an intramuscular injection of virus emulsified with Freund’s incomplete adjuvant (Maat, 1976).

Relationships

Isolates of the virus from Agapanthus praecox and Nerine sarniensis are serologically indistinguishable (Phillips & Brunt, 1980). The virus was found to be serologically distantly related (serological differentiation indices of 5-9) to six potexviruses (clover yellow mosaic, commelina X, hydrangea ringspot, narcissus mosaic, potato X and viola mottle viruses), but unrelated to seven other potexviruses, namely bamboo mosaic, cactus X, cymbidium mosaic, lily X, papaya mosaic (papaya and ullucus strains), potato aucuba mosaic and white clover mosaic viruses (Maat, 1976; Phillips & Brunt, 1980).

Stability in Sap

Isolates of the virus from Agapanthus praecox are very stable in vitro. Sap from systemically infected Chenopodium quinoa leaves is usually infective after dilution to 10-6 but not 10-7, after heating for 10 min at 95 but not 98°C and after at least 12 months at 20°C. Lyophilized sap remains infective for at least 32 months at 20°C.

Purification

Isolates from Agapanthus praecox are best purified from infected Chenopodium quinoa plants, the following procedure yielding up to 130 mg virus per kg leaf tissue (Phillips & Brunt, 1980). Homogenise leaves (1 g/3 ml) in 0.07 M phosphate buffer (pH 7.6) containing 0.1% (v/v) thioglycollic acid, clarify extract by adding butan-1-ol to 8.0% (v/v) and, after stirring gently for 30 min, subject the mixture to one or two cycles of differential centrifugation (20 min at 12,000 g; 60 min at 65,000 g); resuspend the final pellets in 0.03 M neutral phosphate buffer (1 ml/25 g leaf material), and further purify the virus by permeation chromatography on controlled-pore glass beads (Barton, 1977).

Isolates from Nerine sarniensis have been purified as follows (Maat, 1976): homogenise N. sarniensis leaves (1 g/3 ml) or Nicotiana clevelandii leaves (1 g/2 ml) in 0.1 M Tris buffer at pH 9.0 containing 0.1% sodium thioglycollate and 0.02 M sodium diethyldithiocarbamate, clarify the extract by adding 25% (v/v) of a 1:1 mixture of diethyl ether and carbon tetrachloride and subjecting the mixture to low speed centrifugation (10 min at 8000 g). Collect the aqueous phase, add polyethylene glycol (M. Wt 6000) to 5% (w/v) and, after 1 h, collect the precipitate by low speed centrifugation (15 min at 10,000 g) and resuspend the virus in 0.03 M neutral phosphate buffer using 50-60% of the starting extract volume. Concentrate the virus particles by high speed centrifugation (1 h at 78,100 g or 1.5 h at 48,700 g) and, after resuspension in a small volume of buffer, further purify them by rate zonal sucrose density gradient centrifugation (3 h at 67,000 g). Centrifuge virus-containing fractions for 1 h at 78,100 g and resuspend the pellets in 0.1 M Tris buffer at pH 9.0 (1.5 ml per 100 g leaf tissue).

Properties of Particles

Sedimentation coefficient: (s°20,w): 120 S. Some virus preparations also contain a minor component, presumably of aggregated virus particles, which sediments at c. 200 S.

Buoyant density in CsCl: (g cm-3): 1.31.

A260/A280 : 1.40; A260/A244: 1.18.

Particle Structure

The virus particles are flexuous filaments (Fig.4), mostly measuring c. 540 x 11 nm when mounted in neutral 2% potassium phosphotungstate (Brunt et al., 1970; Maat, 1976; Phillips & Brunt, 1980).

Particle Composition

Nucleic acid: Single-stranded RNA; c. 5% by weight of the particle (estimated spectrophotometrically). M. Wt 2.5 x 106, estimated by polyacrylamide gel electrophoresis under non-denaturing conditions (Phillips & Brunt, 1980).

Protein: A single polypeptide, M. Wt c. 25,600, estimated by SDS/polyacrylamide gel electrophoresis (Phillips & Brunt, 1980).

Relations with Cells and Tissues

No information.

Notes

Nerine virus X is serologically distantly related to narcissus mosaic virus which, although not reported to infect N. sarniensis, occurs naturally in Nerine manselli and Nerine bowdenii (Maat, 1976; Koenig et al., 1973). In N. sarniensis, nerine virus X usually occurs together with arabis mosaic, cucumber mosaic, nerine latent and/or one or more uncharacterised potyviruses (Hakkaart et al., 1975; Brunt, 1977). It is, however, readily distinguished from these viruses by serological tests and by the morphology of its particles; a cDNA probe is also available in New Zealand for diagnostic use (K. S. Milne, personal communication).

Nerine virus X has been eliminated from some stocks of N. sarniensis by meristem-tip culture (Hakkaart et al., 1975; Stone et al., 1983).

References

  1. Barton, J. gen. Virol. 35: 77, 1977.
  2. Brunt, Rep. Glasshouse Crops Res. Inst., 1976: 123, 1977.
  3. Brunt, Hollings & Stone, Rep. Glasshouse Crops Res. Inst., 1969: 138, 1970.
  4. Hakkaart, Maat & Quak, Acta Hort. 47: 51, 1975.
  5. Koenig, Lesemann, Brunt & Kühne, Intervirology 1: 348, 1973.
  6. Maat, Acta Hort. 59: 81, 1976.
  7. Phillips & Brunt, Rep. Glasshouse Crops Res. Inst., 1977: 131, 1978.
  8. Phillips & Brunt, Acta Hort. 110: 65, 1980.
  9. Stone, Phillips, Brunt & Pawley, Rep. Glasshouse Crops Res. Inst., 1981: 143, 1983.


Figure 1

Lesions in inoculated Chenopodium murale leaf.

Figure 2

Lesions in inoculated C. quinoa leaf.

Figure 3

Lesions in inoculated Tetragonia expansa leaf.

Figure 4

Particles ‘trapped&146; on antiserum-coated grids from desiccated C. quinoa leaf tissue homogenised in phosphate-buffered saline containing 0.05% (v/v) Tween 20. Virus particles mounted in 2% potassium phosphotungstate. Bar represents 250 nm.