Isolation, transmission and purification of the High Plains virus

Journal of Virological Methods 135 (2006) 214–222

Isolation, transmission and purification of the High Plains virus Raymond Louie a,b , Dallas L. Seifers c,∗ , Oscar E. Bradfute b a

USDA-ARS, Corn and Soybean Research, Ohio Agriculture Research and Development Center, 1680 Madison Ave., Wooster, OH 44691, USA b Department of Plant Pathology, The Ohio State University, Wooster, OH 44691, USA c Kansas State University, Agricultural Research Center, Hays, KS 67601, USA Received 27 October 2005; received in revised form 13 March 2006; accepted 14 March 2006 Available online 2 May 2006

Abstract The wheat curl mite (Aceria tosichella Keifer) often simultaneously transmits the High Plains virus and Wheat streak mosaic virus under field conditions, resulting in doubly infected plants. In this study, a pure culture of the High Plains virus (isolate HPV95ID), which was infected with both High Plains virus and Wheat streak mosaic virus, was mechanically transmitted from barley (Hordeum vulg´are L.) to maize (Zea mays L.) by vascular puncture inoculation. Different water temperatures and durations for soaking kernels at pre-inoculation and different incubation temperatures and durations at post-inoculation on transmission of High Plains virus were studied. Transmissions of the High Plains virus were significantly different for post-inoculation incubations at 11, 21, or 30 ◦ C after a 2 h pre-inoculation soaking at 30 ◦ C and post-inoculation incubations of kernels for 1 day versus 2 days. Use of Cs2 SO4 in a partial purification protocol resulted in infectious final fractions. Bioassays, serological assays, analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and examinations by electron microscopy confirmed isolation of a pure culture of High Plains virus from infectious final partially purified fractions. We demonstrate infectivity of the final fractions and associate it with the High Plains disease symptoms, the 32 kDa protein and double membrane bodies and discuss this evidence to support the viral nature of High Plains virus. © 2006 Elsevier B.V. All rights reserved. Keywords: High Plains disease; Vascular puncture inoculation; Maize

1. Introduction A disease of maize (Zea mays L.) and wheat (Triticum aestivum L.), named the High Plains disease, was first reported in the United States in 1993–1994 from Texas and Kansas and later from Colorado, Nebraska, Idaho, and New Mexico (Jensen et al., 1996). The wheat curl mite (Aceria tosichella Keifer) is the vector of the High Plains virus (Seifers et al., 1997) and it often simultaneously transmits the High Plains virus and Wheat streak mosaic virus, resulting in doubly infected plants (Jensen et al., 1996; Marcon et al., 1997). Plants doubly infected have a 32 kDa protein (Jensen et al., 1996; Seifers et al., 2002), mosaic leaf symptoms (Jensen et al., 1996) and double membrane bound bodies (Ahn et al., 1998; Jensen et al., 1996). Maintaining a pure culture of High Plains virus by the wheat curl mite has been demonstrated (Skare et al., 2003), but this can be problematic since differential transmission rates of High Plains virus isolates occur with different wheat curl mite sources (Seifers

∗

Corresponding author. Tel.: +1 785 625 3425x217; fax: +1 785 623 4369. E-mail address: [email protected] (D.L. Seifers).

0166-0934/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2006.03.023

et al., 2002). Although Wheat streak mosaic virus is mechanically transmitted by leaf rub-inoculation, High Plains virus is not. However, High Plains virus was recently mechanically transmitted by vascular puncture inoculation (Louie and Seifers, 1996). In two studies, the 32 kDa protein was present in plants infected only with High Plains virus (Jensen et al., 1996; Ahn, 1998). High Plains disease is associated with viral-like symptoms, obligate transmission by an eriophyid mite (Seifers et al., 1997), a 32 kDa protein (Jensen et al., 1996; Seifers et al., 2002), and the occurrence of double membrane bound bodies (Ahn et al., 1998; Jensen et al., 1996) in tissues of infected plants. These traits are similar to those of other diseases, e.g. fig mosaic (Appiano, 1982; Martelli et al., 1993), rose rosette (Gergerich and Kim, 1983), thistle mosaic (Ahn et al., 1996), wheat spot chlorosis (Bradfute et al., 1970), and yellow ringspot of redbud (Kim and Martin, 1978), where an etiological agent has not been demonstrated. In the case of High Plains disease however, thread-like particles from symptomatic maize leaves have been observed by electron microscopy in preferentially labeled in immunogold labeling experiments and taken as evidence for the etiology and viral nature of High Plains virus (Ahn et al., 1998).

R. Louie et al. / Journal of Virological Methods 135 (2006) 214–222

Studies on High Plains disease and resistance to High Plains virus in maize have emphasized the importance of a long-term maintenance of a pure isolate of High Plains virus to discriminate the effects of High Plains virus from those of Wheat streak mosaic virus (Jensen et al., 1996; Marcon et al., 1997). Moreover, a capability to assay for infectivity is critical for any pathogen characterization inquiry (Mathews, 1991). The objectives of this report were: (1) to describe the use of vascular inoculation for the isolation of High Plains virus in pure culture from a source infected with both Wheat streak mosaic virus and High Plains virus, (2) to determine the effects of soaking on kernels and durations of different temperatures during vascular puncture inoculation on transmission efficiency of High Plains virus, and (3) to describe a purification protocol to produce an infectious preparation. 2. Materials and methods 2.1. Virus source, isolation, and culture of High Plains virus The isolate HPV95ID was determined to be High Plains virus by enzyme-linked immunosorbent assay, also known as ELISA. This assay is described later and uses antiserum produced by Jensen et al. (1996) that was cross-absorbed for Wheat streak mosaic virus (Seifers et al., 1997). Originally, the isolate was wheat curl mite transmitted from an infected sweet corn sample from Idaho to barley (Hordeum vulg´are L.) in Kansas. A 1.5 g leaf sample from infected barley was sent to Ohio for vascular puncture inoculation studies. In this test, 600 kernels of ‘Spirit’ sweet corn were soaked in water for 2 h at room temperature prior to vascular puncture inoculation. After soaking, kernels were blotted dry and placed embryo side up in a 2 l Pyrex dish on eight layers of paper towels moistened with 40 ml of tap water. The infected barley leaf tissue for inoculum was ground with a pestle

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in a mortar in buffer (1:5, w/v). The brei was placed in a 1.5 ml microfuge tube, and centrifuged for ca. 5 s at ca. 15,339 × g in an Eppendorf Centrifuge 544 (Brinkman Inst. Inc., Westbury, NY). Buffers used were either 0.01 M potassium phosphate buffer, pH 7.0 (Buffer A); 0.05 M sodium phosphate, 0.005 M ethylenediaminetetraacetic acid, 0.01 M sodium sulfite buffer, pH 7.8 (Buffer B); or 0.01 M ethylenediaminetetraacetic acid, 5% ␤-mercaptoethanol, 200 ␮g/ml bentonite, 0.01 M potassium phosphate buffer, pH 7.0 (Buffer C). A 3 ␮l of inoculum was placed on the surface of each kernel alongside the embryo axis. For inoculation, five minute pins (0.2 mm diameter) attached to an engraving tool (Cat. no. 11–111, Ideal Industries Inc., Sycamore, IL 60178) were used to pierce the seed coat and scutellum along a 1 mm distance from the embryo axis. The pins were held at ca. 45◦ angle from vertical towards the vascular bundles and penetrated to a depth of ca. 1 mm (Louie, 1995). Each kernel was inoculated three times. After inoculation, the dish containing the kernels was covered with a plastic wrap and placed in a 30 ◦ C chamber for 48 h. The kernels were then planted in pots of autoclaved soil and germinated in the greenhouse. Each buffer treatment consisting of 100 kernels was replicated twice. Subsequent to this transmission test, the High Plains virus isolate was tested by wheat curl mite transmission, ELISA (Table 1), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 1A), and Western blot (Fig. 1B) for High Plains virus as noted below. Two other isolates from infected corn from Kansas, HPV96KS5-PI, and HPV96KS6-PI, were similarly confirmed as High Plains virus were subcultured by vascular puncture inoculation in Spirit sweet corn. Isolate HPVKS96KS5-PI was used in transmission and purification studies and various fractions from the purification studies were evaluated for the etiologic agent by negative staining electron microscopy. Tissue infected with isolate HPV96KS6-PI was thin-sectioned for cytopathological studies by transmission electron microscopy studies.

Table 1 Enzyme-linked immunosorbent assay of sweetcorn leaf samples for High Plains virus or Wheat streak mosaic virus from kernels inoculated by vascular puncture inoculation Sample number

1 2 3 4 5 6 7 8 HPV WSMV Healthy maize a

Sample identitya

10–14 10–41 10–52 10–62 10–106 11–34 11–47 11–58

Lane no.b

2 3 4 5 6 7 8 10 9

ELISA valuesc HPVd

GHVd

WSMVd

GHV

0.470 0.091 0.309 0.415 0.050 0.030 0.057 0.023 0.256 0.056 0.045

10.44 2.02 6.86 9.22 1.11 0.66 1.26 0.51 5.68 1.24 1.00

0.867 0.062 0.044 0.058 0.267 0.049 0.799 0.078 0.029 0.851 0.061

14.21 1.01 0.72 0.95 4.37 0.80 13.09 1.27 0.47 13.95 1.00

Sample identity coded in Ohio for test in Kansas; 11–34, 11–47, and 11–58 were Maize dwarf mosaic-strain A virus-infected, Wheat streak mosaic virus-infected and non-inoculated healthy control maize leaves, respectively. b Lane numbers correspond with lanes on Fig. 1A and B. c The absorbance value at 405 nm for enzyme-linked immunosorbent assay (ELISA). d The number of times greater than the equivalent healthy control value (GHV) of each ELISA value against antiserum to High Plains virus (HPV) and Wheat streak mosaic virus (WSMV).

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2.2. Effect of soaking durations and pre- and post-inoculation temperature treatments on kernels for High Plains virus transmission In preparation for inoculation, Spirit sweet corn kernels were soaked in tap water at 11, 21, or 30 ◦ C for 1, 2, or 4 h (preinoculation soak temperatures and durations) and placed in a 2 l Pyrex dish on eight layers of paper towels moistened with 40 ml of tap water. Isolate HPV96KS5-PI inoculum was prepared from ca. 21-day-old infected leaf tissues (1:5, w/v) that was ground with a pestle in a mortar in Buffer B. The brei was placed in a 1.5 ml microfuge tube, and centrifuged for ca. 15 s at ca. 15,339 × g in an Eppendorf Centrifuge 544. A 3 ␮l aliquot of inoculum was placed on the surface alongside the embryo axis of the kernel and the kernel was inoculated as described above. After inoculation, the kernels were incubated at 11, 21, or 30 ◦ C for 1 or 2 days (post-inoculation incubation temperatures and durations) in the same dish covered with a plastic wrap and then planted in pots of autoclaved soil and placed in the greenhouse for symptom development. Each treatment consisted of 50 kernels and was replicated five times in a split, split, split block design. Because of the numerous combinations of treatments, the experiment was divided into two tests. In the first, a soak duration of 2 h was used for all treatments. Soak temperatures were main blocks and incubation temperatures and durations were split blocks. In the second, the incubation temperature at 30 ◦ C was used for all treatments. Soak durations were main blocks and soak temperatures and incubation durations were split blocks. The data were analyzed by an ANOVA and the means separated by a LSD at 0.05. 2.3. Purification protocols for isolation and detection of High Plains virus Proteins from High Plains virus-infected sweet corn leaf tissues arising from kernels inoculated by vascular puncture inoculation (isolate HPV95ID) were extracted, partially purified and concentrated by minipurification, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and identified by Western blotting as previously reported (Seifers et al., 1996). Partial purification of High Plains virus (HPV96KS5-PI) was based on modifications of the Figwort mosaic caulimovirus protocol, which involved differential and sucrose density gradient centrifugation (Shepherd et al., 1987). For this modified procedure, hereafter noted as the High Plains virus-cesium protocol, infected tissue (ca. 45 g, ca. 21-day old) was ground in a mortar with a pestle in a buffer [1:1.5, w/v, in 50 mM 1-piperazineethane sulfonic acid, Fig. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses of sweet corn leaf samples for the High Plains virus (isolate HPV95ID). (A) Lanes 1 and 11, molecular weight markers (BioRad Hercules, CA 94547); lanes 2–6, leaf samples 1–5 (see Table 1) tested for High Plains virus infection; lane 7–8, leaf samples infected with Maize dwarf mosaic virus or Wheat streak mosaic virus; lane 9, healthy maize; lane 10, High Plains virus control. Arrows indicate the 32 kDa protein adjacent to soybean trypsin inhibitor molecular marker, 31.3 kDa. (B) Western blot of the same leaf samples and controls as in (A) except lane 1 has pre-stained markers (BioRad). Arrows indicate reactions of the 32 kDa protein in lanes 2–5 to the High Plains virus antibody

probe. (C) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis: lanes 1, 3, and 4 are molecular weight markers (N.E.B. 7702S); lane 2, Cs2 SO4 partial purification of High Plains virus-infected leaf sample. Arrow indicates the 32 kDa protein just below molecular marker lactate dehydrogenase M, 36.5 kDa. The additional band in lanes 1 and 3 (adjacent to the 32 kDa band in lane 2) and absent in lane 4 was most likely due to the overloading of lane 2.

R. Louie et al. / Journal of Virological Methods 135 (2006) 214–222

4-(2-hydroxyethyl)-monosodium salt, 10 mM KCl, 2 mM ethylenediaminetetraacetic acid, 10 mM sodium ascorbate, 20 mM dithiothreitol, 0.1% bovine serum albumin, 400 mM sucrose, pH 7.8) (Morr´e and Anderson, 1994)]. The brei was filtered through Miracloth (Fraction A) and again after the first of two cycles of centrifugation at 6000 × g for 20 min at 4 ◦ C (post second centrifugation, Fraction B). The supernatant was centrifuged at 25,000 × g for 30 min at 4 ◦ C. The pellet was resuspended by gently stirring in a Tris hydroxymethylaminoethane–ethylenediaminetetraacetic acid buffer (10 mM Tris hydroxymethylaminoethane–base, 1 mM ethylenediaminetetraacetic acid, pH 7.8) on an oscillating platform shaker for 4 h at 4 ◦ C (Fraction C). The suspended preparation volume was adjusted to 10 ml with Tris hydroxymethylaminoethane–ethylenediaminetetraacetic acid buffer, 3.5 g of Cs2 SO4 was added and the preparation was centrifuged at 6000 × g for 20 min at 4 ◦ C. After filtration through Miracloth the supernatant was placed in an Ultralok (Nalge Company, P.O. Box 20365, Rochester, NY) or Easy-seal tube (Seton Scientific, P.O. Box 33034, Los Gatos, CA) and centrifuged at 135,000 × g for 24–40 h at 10 ◦ C. A light scattering band was collected and dialyzed in 2 l of Tris hydroxymethylaminoethane–ethylenediaminetetraacetic acid buffer for ca. 4 h. The dialyzed preparation was then centrifuged at 25,000 × g for 30 min at 4 ◦ C. The pellet was resuspended in Tris hydroxymethylaminoethane–ethylenediaminetetraacetic acid buffer and this final fraction (Fraction D) was tested for infectivity and the 32 kDa protein. 2.4. Assays 2.4.1. ELISA, sodium dodecyl sulfate-polyacrylamide gel electrophorsis, and Western blot Serological assays for Wheat streak mosaic virus and High Plains virus were by F(ab )2 (Gingery and Nault, 1990) and a modified indirect ELISA (Seifers et al., 1996). Briefly, for indirect ELISA the leaf tissue was ground 1:30 (w/v) in 0.05 M carbonate buffer, pH 9.6 (coating buffer) (Clark and Adams, 1977). The antiserum to High Plains virus was used at a 1:50,000 (v/v) dilution and the anti-rabbit antibody alkaline phosphatase conjugate (1:3000, v/v) (Sigma Chemical Co., St. Louis, MO) was diluted using ELISA blocking buffer (5% non-fat dry milk, 0.01% antifoam A, 0.02% sodium azide, in phosphate buffer saline, pH 7.4). All samples, antibody solutions, and substrate were used at a 200 ␮l volume. Substrate (p-nitrophenyl phosphate, 0.714 mg/ml) in substrate buffer (Clark and Adams, 1977) was added to the wells of the plates, incubated at room temperature, and the absorbance (405 nm) was measured after 30 min (Titertek Multiskan Microelisa Plate Reader, Flow Laboratories Inc., McLean, VA). For sodium dodecyl sulfate-polyacrylamide analyses, the proteins from High Plains virus-infected plants were extracted and minipurified following the procedure of Lane (1986). The method of Laemmli (1970) was used for sodium dodecyl sulfatepolyacrylamide analyses. Electrophoresis was carried out in 10% gels, 0.75 mm thick using a Hoefer SE 600 apparatus for 2.5 h (60 mA). Proteins were stained with Coomassie blue

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(0.125% Coomassie blue R-250, 30% methanol, and 10% acetic acid). Western blotting analyses were performed as described previously (Seifers et al., 1999). The alkaline phosphatase goat anti-rabbit conjugate (1:3000 dilution) (Fisher Scientific, Denver, CO) was used for the immunoblotting protocol as described by (Bollig and Edelstein, 1991). 2.4.2. Infectivity Leaf-rub inoculations of Oh28 maize, ‘Monon’ wheat and ‘Atlas’ sorghum (Louie, 1986) were used to assay plant samples for Wheat streak mosaic virus infection. Infectivity of partially purified fractions of isolates HPV96KS5-PI and HPV96KS6PI was tested by vascular puncture inoculation of 50 kernels of ‘Spirit’ sweet corn as described above. 2.4.3. Electron microscopy Samples of the final resuspended pellet from 23 different purifications involving Cs2 SO4 of isolate HPV96KS5-PI were negatively stained with phosphotungstic acid and examined in a Phillip 201-C electron microscope. Symptomatic maize leaf samples for thin-section electron microscopy were randomly selected from three plants from kernels inoculated by vascular puncture inoculation with a pure culture of isolate HPV96KS6-PI. Leaf samples (1 mm × 3 mm) were fixed in 2% glutaraldehyde–1% formaldehyde (in 0.05 M potassium phosphate buffer, pH 7.4) at room temperature for 4 h, rinsed in the same phosphate buffer overnight at 4 ◦ C, and post-fixed in 2% OsO4 (in 0.05 M potassium phosphate buffer, pH 7.4) for 2 h at 4 ◦ C. Specimens were stained in 1% aqueous uranyl acetate overnight at 4 ◦ C, dehydrated in an ethanol series followed by 100% acetone and then embedded in Spurr’s resin. Sections were cut peridermally with a diamond knife, post-stained with 0.5% uranyl acetated followed by 0.4% lead citrate, and then examined in a Phillips 400 T electron microscope. 3. Results 3.1. Virus source, isolation and assays Over a 3-week period, chlorotic streak and spot symptoms developed in leaves of seedlings from Spirit sweet corn kernels inoculated by vascular puncture inoculation with inoculum prepared from the original infected barley leaves (HPV95ID). Virus infection occurred in 13/197, 19/194, and 23/199 of the plants (symptomatic/total germinated) using inoculum prepared in Buffers A–C, respectively. The numbers of High Plains virus versus Wheat streak mosaic virus-infected plants from inoculum prepared in Buffers A–C were 1 versus 13, 7 versus 12, and 0 versus 23, respectively. Thereafter, Buffer B was used for High Plains virus propagation. The one High Plains virusinfected plant from Buffer A was also infected with Wheat streak mosaic virus. Regardless of the buffer used for inoculum preparation, by 8 days after inoculation a generalized mosaic symptom usually occurred on the first two leaves in most Wheat streak mosaic virus-infected plants (59%). However, some Wheat streak mosaic virus-infected plants (41%)

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took as long as 21–26 days after inoculation to develop symptoms but these plants only had limited chlorotic streak symptoms on the fifth or later developing leaves. In contrast, the eight plants infected with High Plains virus from inoculum prepared from Buffers A or B all developed a generalized mottle or mosaic symptom, but at more than 11 days after inoculation. ELISA was used to test leaf samples from five plants that developed symptoms 21 days after inoculation (sample numbers 1–5, Table 1). Sample numbers 1–4 tested positive for High Plains virus, sample 1 tested positive for both Wheat streak mosaic virus and High Plains virus and sample 5 tested positive for only Wheat streak mosaic virus. Bioassays of these samples by rub-inoculations of Oh28 corn, ‘Atlas’ sorghum, and ‘Monon’ wheat host differentials confirmed the ELISA findings that sample numbers 1 and 5, but not 2–4 tested positive for Wheat streak mosaic virus. After an initial appearance of chlorotic spots and streaks primarily over the veins, plants infected with only High Plains virus developed a mottle or mosaic symptom in the upper leaves. Later, necrosis but not reddening, occurred on the lower leaves. Infection with Wheat streak mosaic virus, High Plains virus, or both, was not lethal to the seedlings. In the doubly infected plant, symptoms of Wheat streak mosaic virus infection were obscured by the greater color contrast of symptoms of High Plains disease. Symptoms of High Plains disease alone were not noticeably different from symptoms caused by coinfections. Assays of samples of maize plants inoculated with inoculum prepared from the infected barley tissue (HPV95ID) that were positive in ELISA for High Plains virus and the High Plains virus control (lane 10) yielded a band in sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis (Fig. 1A, lanes 2–5) at approximately 32 kDa. The healthy maize control negative in ELISA for High Plains virus and Wheat streak mosaic virus (Fig. 1A, lane 9) yielded no bands. The sample positive in ELISA for Wheat streak mosaic virus only (Fig. 1A, lane 8) yielded two faint bands not present in the High Plains virus positive samples or in the High Plains virus control. The faint band present in lane

6 close to the position of the High Plains virus bands in lanes 2–5 did not react in the Western blot to the High Plains virus antibody probe, nor did any other bands except the 32 kDa band in lanes 2–5 and 10 (Fig. 1B). Isolates HPV96KS5-PI and HPV96KS6-PI, which tested positive for High Plains virus by ELISA, were associated with the 32 kDa protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses (Fig. 1C for HPV96KS5-PI) and tested negative for Wheat streak mosaic virus by bioassays (data not shown). In thin sections examined in a Phillips 400 T electron microscope, numerous double membrane bound bodies were found in the cytoplasm of every cell of mesophyll and vascular tissue of leaves of the three plants infected by HPV96KS6-PI (Fig. 2A). Double membrane bound bodies were round to ovoid in section, 0.1–0.2 ␮m across, and they consisted of unconsolidated fibrils surrounded by two unit membranes (Fig. 2B). The outer membrane appears to be acquired by budding from the lumen of the endoplasmic reticulum to the cytoplasm. Double membrane bound bodies did not appear to be associated with any other cytoplasmic organelles or structures. Except for the presence of double membrane bound bodies, cell ultrastructure was not obviously altered. No evidence of other intracellular pathogens was observed including: virus particles, phytoplasmas, spiroplasmas, bacteria, fungi, or protozoa, or of viroplasms or other virus associated inclusions. Double membrane bound bodies were not found in samples of similarly prepared healthy sweet corn leaves (data not shown). Fibrils do not appear dense and organized, as are the nucleoprotein particles of many viruses. Fibril concentration appears to vary with section thickness and position of the double membrane bound bodies within the section. Fibrils are usually concentrated peripherally and were more dispersed at the center. Inoculum from leaf tissue or from partially purified preparations for serial propagation of High Plains virus was prepared with Buffer B or with Tris hydroxymethylaminoethane–ethylenediaminetetraacetic acid buffer, respectively.

Fig. 2 Electron micrographs showing double membrane bodies in sections of leaves from sweet corn plants inoculated with the High Plains virus (isolate HPV96KS6PI). (A) Part of a cell showing numerous double membrane bound bodies among normal cytoplasmic components. Scale bar = 1 ␮m. (B) A higher magnification shows double membrane bound bodies are composed of unconsolidated fibrils bond by two unit membranes. Scale bar = 0.1 ␮m.

R. Louie et al. / Journal of Virological Methods 135 (2006) 214–222

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Table 2 Average percentages of infections in ‘Spirit’ sweetcorn plants inoculated with High Plains Virus (HPV95KS5-PI) by vascular puncture inoculation at 2 h soaking and different soaking temperatures at pre-inoculation and different post-inoculation incubation durations and temperatures Soak temperature (◦ C)

Incubation duration (day) and temperature 1 Day

11 21 30 Means

2 Days

11 ◦ C

21 ◦ C

30 ◦ C

Means

11 ◦ C

21 ◦ C

30 ◦ C

Means

20.2 20.0 15.2

39.0 39.4 36.2

61.6 65.8 61.8

40.3 41.7 37.7

21.6 15.8 16.4

65.2 58.8 56.0

64.2 66.4 63.6

50.3 47.0 45.3

18.5

38.2

63.1

17.9

60.0

64.7

39.9A 18.2A

47.6B 49.1B

63.9C

totala

Means Incubation day Incubation temperature Source of variation

d.f.

F-value

Pr > F

Incubation day Soak temperature × incubation day Incubation temperature × incubation day Soak temperature × incubation temperature × incubation day Soak temperature Incubation temperature Soak temperature × incubation temperature

1 2 2 4 2 2 4

16.99 0.56 14.68 0.14 1.07 150.83 0.35

0.0002 0.5771 <0.0001 0.9682 0.3558 <0.0001 0.8390

a

Means total with the same letter are not significantly different.

After 19 serial propagations of isolate HPV96K5S-PI in almost 13 months, a 32 kDa band was still associated with inoculum that was partially purified by Cs2 SO4 gradient centrifugation of High Plains virus-infected plants (Fig. 1C, lane 2). When this preparation was used as inoculum for vascular puncture inoculation, 5% transmission of High Plains virus occurred and without contamination from Wheat streak mosaic

virus. After 2.25 years and 43 serial propagations, HPV96K5SPI was stored in liquid nitrogen. The 15 serial propagations of HPV96KS6-PI in almost 12 months during the electron microscopy studies also remained free from Wheat streak mosaic virus contamination. After 40 months and a total of 38 serial propagations, HPV96KS6-PI also was stored in liquid nitrogen.

Table 3 Average percentages of infection in ‘Spirit’ sweet corn plants inoculated with High Plains virus (HPV96KS5-PI) by vascular puncture inoculation at different soaking durations and temperatures at pre-inoculation and at 30 ◦ C incubation for different durations at post-inoculation Soak duration (h)

Incubation duration (day) and soak temperatures 1 Day

2 Days

11 ◦ C

21 ◦ C

30 ◦ C

Means

11 ◦ C

21 ◦ C

30 ◦ C

Means

1 2 4

45.4 45.5 48.8

55.8 52.8 57.0

61.4 57.3 52.6

54.2 51.9 52.8

71.3 68.4 69.6

64.8 63.0 68.0

70.5 66.1 47.0

68.9 65.8 61.5

Means

46.6

54.9

55.0

69.0

65.5

56.6

61.5A

58.8A 53.0A 59.6A

56.5A 65.1B 59.2A

Means totala Soak duration Incubation day Soak temperature

58.2A

Source of variation

d.f.

F-value

Pr > F

Incubation day Soak duration × incubation day Soak temperature × incubation day Soak duration × soak temperature × incubation day Soak duration Soak temperature Soak duration × soak temperature

1 2 2 4 2 2 4

57.22 1.27 11.98 0.93 0.82 0.18 1.76

<0.0001 0.2939 0.0001 0.4557 0.4499 0.8371 0.1624

a

Means total with the same letter are not significantly different.

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Table 4 Percent transmission of partially purified fractions of High Plains virus (HPV96KS5-PI) by vascular puncture inoculation of ‘Spirit’ sweet corn kernels Experiments

Percent transmission of purified fractionsa A

B

C

D

64.0 62.8 100.0 80.6 81.6 90.6 76.7

60.0 64.4 100.0 89.5 91.4 78.1 78.1

79.0 51.1 66.0 82.1 0 0 84.6

1.2 1.0 79.1 90.0 0 15.4 2.9

Mean

79.5

80.2

51.8

27.1

S.D.

13.4

14.6

37.2

39.7

1 2 3 4 5 6 7

a

The homogenate was filtered through Miracloth (Fraction A) and again after the first of two cycles of centrifugation at 6000 × g for 20 min (post second centrifugation is Fraction B). The supernatant was centrifuged at 25,000 × g for 30 min. The pellet was resuspended by gently stirring in a Tris hydroxymethylaminoethane–ethylenediaminetetraacetic acid buffer on an oscillating platform shaker (Fraction C). After Fraction C was dialyzed, the preparation was centrifuged at 25,000 × g for 30 min at 4 ◦ C. The pellet was resuspended in Tris hydroxymethylaminoethane–ethylenediaminetetraacetic acid buffer (Fraction D).

3.2. Effect of soaking duration and pre- and post-inoculation temperature treatments on High Plains virus transmission No treatment combination with vascular puncture inoculation significantly affected transmission of High Plains virus. However at post-inoculation, the combined treatments of incubation duration of 1 day versus 2 days that averaged 39.9% versus 47.6% (Table 2) and 53.0% versus 65.1% (Table 3), respectively, were significantly different (P ≤ 0.0002 and 0.0001, respectively). Again at post-inoculation, transmission rates in the combined treatments of 1 and 2 days incubation temperatures at 11, 21, and 30 ◦ C that averaged 18.2, 49.1, and 63.9%, respectively, also were significantly different (P ≤ 0.0001, Table 2). Treatments of soak durations and temperatures at pre-inoculation were not significantly different (Table 3). Hence, the standard vascular puncture inoculation protocol adapted from previous studies (Louie, 1995) was also suitable for transmission of the High Plains virus. 3.3. Purification of High Plains virus In the High Plains virus-cesium purification protocol from seven similar experiments, average transmission rates of isolate HPV96K5S-PI and their standard deviations from the mean of Fractions A–D were 79 ± 13.4, 80 ± 14.6, 52 ± 37.2, and 27 ± 39.7%, respectively (Table 4). Rates of transmission apparently decreased and variations among experiments increased along each step of the purification protocol. However, average rates of transmission of Fractions A and B, which was prepared with inoculum at a 1:1.5 (w/v) dilution of tissue to buffer, appeared to be higher than those rates achieved with inoculum prepared at 1:5 (w/v) dilution of tissue to buffer in the standard

protocol. In electron microscopic examinations, none of 23 final resuspended pellets from the High Plains virus-cesium purification protocol of isolate HPV96KS5-PI negatively stained with phosphotungstic acid revealed the presence of any organelles or virions. 4. Discussion High Plains virus is not transmissible by leaf rub-inoculation (Jensen et al., 1996), but it is transmissible by vascular puncture inoculation (Louie and Seifers, 1996). By inoculating large numbers of kernels using vascular puncture inoculation, the probability of more infected plants was increased and concomitantly, the likelihood of plants infected singly by either Wheat streak mosaic virus or High Plains virus. High Plains virus has been isolated in pure culture from yellow foxtail using wheat curl mites (Seifers et al., 1998; Skare et al., 2003). However, because yellow foxtail is not a host for Wheat streak mosaic virus (Sill and Connin, 1953; Seifers et al., 1998) this differs from our use of vascular puncture inoculation to separate the two viruses from plants with mixed infections. The differential rate of symptom development and the invasiveness of delayed symptoms between High Plains virus and Wheat streak mosaic virus-infected plants were useful aids in discriminating between the two types of infections. Three isolates, free of mixed infections, were produced in this study. Control of the High Plains disease is best achieved with disease resistant germplasm. However, studies on High Plains disease and resistance to High Plains virus in maize have found long-term maintenance of a pure isolate of High Plains virus problematic (Jensen et al., 1996; Marcon et al., 1997). Moreover, an assay for infectivity, critical to pathogen characterization (Matthews, 1991), was lacking. Our ability to transmit High Plains virus by vascular puncture inoculation has facilitated our studies by providing a reliable and convenient source of virusinfected material. Our highest average rate of transmission for isolate HPV96KS5-PI was 71.3%. This rate is higher than transmission rates shown for other High Plains virus isolates (Seifers et al., 2004). Previously, factors affecting the susceptibility of the host at the time of inoculation were critical to the transmission of Maize white line mosaic virus and Maize rough dwarf fijivirus (Louie, 1995; Louie and Abt, 2004). In this study, when the treatment was to soak kernels in tap water at 30 ◦ C for 2 h before inoculations and then incubate them at 30 ◦ C for 2 days prior to planting, the average rates of High Plains virus transmission in each replication ranged from 32 to 83% (S.D. = 17.2). Combined treatments of incubation duration of 1 day versus 2 days (Tables 2 and 3) and of incubation temperatures 30 ◦ C versus 21 ◦ C versus 11 ◦ C (Table 2) were significantly different. However, large variability among replications in these experiments suggested that despite previous successes with Maize white line mosaic virus and Maize rough dwarf fijivirus, we have not accounted for some critical parameters affecting the transmission of High Plains virus. Using a buffer for isolation of organelles and membranous cell components in our purification of isolate HPV96KS5-PI (Morr´e and Anderson, 1994) and using a 1:1.5 dilution (w/v) ratio of infected tissue to buffer,

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100% transmission rates could be obtained from some fractions after centrifugation at 6000 × g (Table 4). Thus, in addition to resolving factors that affect host susceptibility during kernel germination, efforts also must be made concurrently to insure a high titer and viability of High Plains virus for vascular puncture inoculation. In this manner, vascular puncture inoculation currently could be used to screen for resistant germplasm. Several High Plains virus isolates have been maintained but levels of infected plants were low and several isolates, including isolate HPV96ID in this study, were lost after period of time (Seifers et al., 2002). In these experiments, isolates HPV96KS5PI and HPV96KS6-PI were isolated and maintained in pure culture by vascular puncture inoculation and carried through 19 and 15 serial propagations for almost 13 and 12 months, respectively. One reason for the extended propagation time of the current isolates compared to those observed by Seifers (2002) may reside with the use of different buffers. The High Plains virus-cesium purification protocol produced an infectious preparation and allowed us to assay the preparation for the 32 kDa protein, the double membrane bodies, and the thread-like structures. The protocol was developed for purification of Figwort mosaic virus (Shepherd et al., 1987) and the major modification was to use a buffer for isolation of organelles and membranous cell components (Morr´e and Anderson, 1994). We also omitted the 10–40% linear sucrose density gradient centrifugation and used Cs2 SO4 instead of CsCl to achieve a steeper gradient at the same rotor speed. As a result, we were always able to recover infectious fractions from low speed centrifugation (Fractions A and B) or in the case of high-speed centrifugation, from Fraction D in six of seven purifications. Additionally, we usually observe a single discrete band at ca. 42 mm from the top of the Easy-seal tube. Unlike previous antiserum to High Plains virus (Seifers et al., 1997), the antiserum produced from the final preparation of this and subsequent purification protocols was effective for antibody production and did not required crossabsorption with healthy tissue or Wheat streak mosaic virus for use in ELISA (Louie, unpublished results). Assays of Fraction D by sodium dodecyl sulfate-polyacrylamide gel electrophoresis always yielded the 32 kDa protein and suggested that the etiologic agent was associated with the fraction. Assays of leaf tissue infected with our pure isolate of High Plains virus by thinsection electron microscopy showed that that double membrane bound bodies were present (Fig. 2). Double membrane bound bodies have been shown to be present in plants only infected by High Plains virus (Jensen et al., 1996; Ahn et al., 1998). The cytopathic ultrastructure of High Plains disease could not be distinguished from that of wheat spot mosaic disease (previously referred to as Wheat spot mosaic virus) (Bradfute and Nault, 1969, Bradfute et al., 1970). The double membrane bound bodies found in this study also were similar to those previously reported as associated with High Plains disease in mixed infections with Wheat streak mosaic virus (Ahn, 1996, 1998) and a group of other mite-borne virus-like diseases (Martelli et al., 1993). Double membrane bound bodies are not found in maize leaves experimentally infected with other viruses, phytoplasmas, or spiroplasmas (Bradfute and Robertson, 1977, 1981; Bradfute et al., 1979).

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Using High Plains virus-specific antiserum to the 32 kDa protein (Ahn et al., 1998), specifically immunogold labeled thread-like structures have been isolated from 10 to 40% sucrose gradients. The protocol used by Ahn was developed for Maize stripe virus (Falk and Tsai, 1984) and also was used for purification of High Plains virus for the production of the antiserum to the 32 kDa protein (Jensen et al., 1996). In this protocol, the High Plains virus nucleoprotein sedimented in a broad-diffuse zone (Jensen et al., 1996) and material from this zone used for antiserum production gave strong reaction to host proteins and required cross-absorption to remove such reactivity (Seifers et al., 1997). Moreover, neither infectivity of the thread-like structures nor the presence of intact double membrane bound bodies was demonstrated. A capability for infectivity assay of a pathogen is critical to virological studies (Matthew, 1991). We demonstrated by vascular puncture inoculation that fractions partially purified by a High Plains virus-cesium protocol were free of organelles, Wheat streak mosaic virus and any association with its mite vector and were infectious. Transmission of this infectious fraction by vascular puncture inoculation repeatedly produced symptoms of the High Plains disease and the 32 kDa protein and double membrane bodies were always demonstrated in assays of infected plants. The 32 kDa protein and double membrane bodies have been consistently linked with High Plains disease (Jensen et al., 1996; Ahn, 1998). However, we found no double membrane bound bodies by electron microscopy in any of the final resuspended pellets from our partially purified fractions. Our inability to demonstrate an agent by electron microscopic examination of partially purified preparations may have be due to the destruction of double membrane bound bodies (Ahn et al., 1996). We also have not isolated nor demonstrated infectivity of our dsRNA preparations (Louie, unpublished results). However, we may have had viral nucleic acid in our partially purified preparations since transmission of viral RNA and DNA is possible with vascular puncture inoculation (Redinbaugh et al., 2001). Thread-like particles in preferentially labeled in immunogold labeling experiments indicated a viral nature of High Plains virus (Ahn et al., 1998), but we were unable to confirm this finding in our infectious preparations. Presently, association of infectivity to a physical entity continues to be the challenge for characterization of the High Plains virus, as well as for those other diseases with double membrane bodies but an unknown etiological agent. Acknowledgements We thank John Abt, Tom Lanker, Jean Vacha, and Jeff Ackerman (Kansas State University) for their highly skilled technical assistance and Robert Whitmoyer for rendering the digitize photographs. We thank Elke Kretzschmar and Robert Whitmoyer for examination of partially purified preparations by electron microscopy, Bert Bishop for statistical analyses, and Michele Gardiner (Novartis Seeds) for providing the ‘Spirit’ sweet corn seeds. This work is a cooperative investigation of USDA-ARS, OSU and the OARDC, Wooster, Ohio, and Kansas State University. Salaries and support provided by state and federal funds

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