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The 33.6-kDa nonstructural (NSM) protein gene, located on the ambisense M RNA segment of tomato spotted wilt virus (TSWV), was cloned and expressed using the Escherichia coli pET-11t expression system. The protein thus produced was purified and used for the production of a polyclonal antiserum. Western immunoblot analyses of TSWV-infected Nicotiana rustica plants showed NSM synthesis only during a short period early in systemic infection. Although NSM was found associated with cytoplasmic nucleocapsid preparations, it was absent from purified virus particles. Analyses of subcellular fractions from young, systemically infected leaves showed the presence of NSM in fractions enriched for cell walls and cytoplasmic membranes, respectively. Furthermore, immunogold labeling of tissue sections of TSWV-infected N. rustica plants showed that this protein was found associated with nucleocapsid aggregates in the cytoplasm and in close association with plasmodesmata. The data obtained provide evidence that NSM represents the viral movement protein of TSWV, involved in cell-to-cell movement of nonenveloped ribonucleocapsid structures.

The open reading frame located in the viral strand of the ambisense S RNA of tomato spotted wilt virus (TSWV), was cloned into transfer vector pAc33DZ1 and inserted downstream of the polyhedrin promoter in the Autographa californica nuclear polyhedrosis virus genome. Recombinant baculoviruses were obtained that showed a high-level expression of a 52.4-kDa protein corresponding to the inserted TSWV gene. The viral protein thus produced was purified and injected into rabbits to raise antibodies. Western immunoblot analyses of extracts from TSWV-infected plants demonstrated that the 52.4-kDa TSWV-specific polypeptide represents a nonstructural protein (denoted NSs), being absent in purified virus particles. Immunogold labeling of tissue sections of TSWV-infected Nicotiana rustica plants showed that this protein was, depending on the virus isolate, either found dispersed throughout the cytoplasm or associated with fibers which appeared as elongated flexible filaments or paracrystalline rods.

The 5'-terminal and 3'-terminal parts of the single open reading frame (ORF) in the L RNA of tomato spotted wilt virus (TSWV) were expressed using a prokaryotic expression system. Using antibodies raised against the translational products obtained a 330-kDa protein could be specifically detected in preparations of purified virions and in nucleocapsid preparations from TSWV-infected leaf tissue. The results obtained indicate that the L protein of TSWV, though much larger than that of the animal-infecting bunyaviruses, is present in virus particles in an unprocessed, intact form.

Basipetal transport of various substances through half-inch stem sections of healthy and aster yellows virus-infected Nicotiana rustica L. var. humilis was determined. The effects of infection by three strains of the virus, dwarf, severe, and Tulelake were observed on the transport of 14 C-labeled β-indoleacetic acid, malic acid, succinic acid, sucrose, glucose, mannose, and the ions sulfate, phosphate, and rubidium. Transport of all these substances except phosphate ion was more rapid through diseased than through healthy stem sections. In the dark, and with supplementary auxin, transport of phosphate ion was also more rapid through diseased than through healthy stem sections. Transport of β-indoleacetic acid and of sugars through healthy stem sections was more rapid in the dark than in the light, but transport of these compounds through diseased stem sections was unaffected by light. No appreciable chemical conversion of the substances tested was observed during the transport time used.

Purified barley stripe mosaic virus was more stable between pH 6.0 and 6.5 than at higher and lower pH levels. The stability decreased rapidly below pH 5.5 and above pH 7.0. The virus was more stable at pH 7.0 in tris(hydroxymethyl)aminomethane buffer than in phosphate buffer. A detergent, Igepon T-73 (sodium N -methyl N -oleoyl taurate), decreased the stability of purified virus, whereas it had no effect on the stability of unpurified virus. The virus was more stable at ionic strengths of about 0.1 to 0.2 than at higher or lower ionic strengths. The effect of any one of these factors on the stability of the virus depended on the level of the other factors and on the degree of purity of the virus. In 0.1% Igepon T-73 or at an ionic strength of 0.06, purified virus was more stable when highly concentrated than when dilute. This fact suggested that the virus combined with a second component in the purified preparation. Since the diluted purified virus was stabilized by addition of an extract from healthy barley plants, this second component may have been a component of the host plant. The component from healthy barley plants that stabilized the purified virus in 0.05% Igepon T-73 would not pass through dialysis tubing, was heat stable, was precipitated by ethanol and by ammonium sulfate, and was not soluble in acetone, ether, or chloroform. Extracts of plants of Phaseolus vulgaris L., Cucurbita pepo L., Triticum aestivum L., Nicotiana rustica L., and N. glutinosa L., also stabilized barley stripe mosaic virus. Several methods of clarification for removal of host components before purification were tested.

When Nicotiana glutinosa leaves, immediately after inoculation with tobacco mosaic virus (TMV) were floated on 0.1–20 mg/l of kinetin (K), kinetin riboside (KR), N 6 -benzyladenine (BAD), N 6 -benzylaminopurine (BAP), or N 6 -isopentenyladenosine (IPA), lesion formation was inhibited but virus production was stimulated. In Phaseolus vulgaris L. var. Pinto (another local-lesion host), however, K, KR, and IPA severely, and BAP or BAD to a lesser extent, inhibited lesion formation and virus production. In addition, BAD caused a substantial increase in lesion size whereas IPA and BAP reduced the lesion size. When TMV-inoculated leaves of Nicotiana tabacum L. var. Wisconsin 38 and of Nicotiana rustica (two systemic hosts) were treated with the cytokinins, virus production was stimulated with all cytokinins except KR, which inhibited virus production in N. rustica . No consistent effect of cytokinins on chlorophyll retention in healthy or virus-infected leaves from the plants was noted. These results show that the effect of cytokinins on viruses appears to be very specific and may depend on particular host, virus system, cytokinin and its concentration, and other factors.

Results from inoculation of two vector-specialized forms of potato yellow dwarf virus (PYDV) on cell monolayers derived from their vectors and related nonvectors indicate that the effect of pH on inoculation is mediated through the virus, not through the host. The optimum pH for sanguinolenta yellow dwarf virus (SYDV) inoculation on cell monolayers is near 5.9; the optimum pH for inoculation of constricta yellow dwarf virus (CYDV) on cell monolayers is near 5.3. Forty-five minutes is sufficient for inoculation of PYDV on leafhopper cell monolayers. A pH of 7.0 is better than 6.5 or 7.5 for inoculating leaves of Nicotiana rustica with SYDV. Use of histidine-MgCl2 (His-MgCl2) solution for inoculation of SYDV on N. rustica gave better results than did phosphate buffer at the same pH. In His-MgCl2 buffer SYDV is most stable near pH 6.4 at 0° and pH 6.5 at 30°; CYDV is most stable near pH 6.9 at 0° and pH 5.9 at 30°. Near the optimal pH for inoculation of cell monolayers with SYDV at 30°, namely pH 5.9, the half-life of the virus in the His-MgCl2 solution is 21.4 min. The corresponding half-life for CYDV near its optimal pH for inoculation, namely 5.3, is 7.7 min. Both SYDV and CYDV reached peak concentrations in N. rustica about 10 days after the appearance of the first systemic symptom. After reaching such a peak about 30% of the infectious SYDV was lost by day 12, but thereafter the concentration remained almost constant up to day 24. Similarly, about 70% of peak CYDV infectivity was lost by day 12, and more than 80% by day 14 and day 16. Purified sterile infectious inocula were obtained by harvesting infected N. rustica plant tissue at the time of peak virus concentration. SYDV can be stored at −80° for as long as 3 years without appreciable loss of infectivity. CYDV, similarly prepared and stored, seriously deteriorated in infectivity between 6 and 12 months after storage.

Three isolates of aster yellows virus, collected in California, were differentiated on the basis of symptoms on common plantain, Plantago major L., Nicotiana rustica L. var. humilis, aster,1 and periwinkle and were designated as the Severe, Dwarf, and Tulelake strains. Leafhopper transmission tests conducted on 20 species of plants indicated only minor host range differences between the three strains. Cross protection experiments on plantain and N. rustica provided evidence that plants infected with either Dwarf or Severe strains were protected from infection by Tulelake strain. Protection was reciprocal between the Dwarf and Severe strains. In contrast, in plantain plants infected with Tulelake strain and challenged by the Dwarf or Severe strain the original strain was replaced and symptoms of the challenging strain developed. The most unexpected results were obtained when N. rustica plants infected with Tulelake strain were challenged with either Dwarf or Severe strain. This antagonistic interaction resulted in mutual suppression of both the Tulelake and challenging strain in each combination and the development of symptomless plants. Leafhoppers acquired virus from 14 of 23 such plants. The Tulelake strain was recovered from 7 plants, and both the Tulelake strain and the challenging strain were acquired from the seven other plants.

Electron microscopy of tissues of Nicotiana rustica and Trifolium incarnatum infected with potato yellow dwarf virus indicated that the virus particles were bacilliform with average dimensions of 380 mμ by 75 mμ. A striking association of virus particles with the nuclei of infected cells was apparent from sections which showed numerous virus particles at the nuclear periphery and in what appeared to be intranuclear virus inclusions. Careful examination of these apparent inclusions revealed the presence of the nuclear envelope surrounding them in addition to cytoplasmic organelles within them. Such profiles were interpreted as having arisen when the sections passed through invaginations of the cytoplasm into the nucleus. In all the sections showing virus particles associated with the nucleus, large numbers of virus particles were found to be present in expanded areas between the two lamellae of the nuclear envelope. This location is suggested as a possible site of virus assembly. Several micrographs of particles found in this location suggested incorporation of the inner lamella of the nuclear envelope into the viral envelope. The viral envelope was seen to be composed of three lamellae, approximately 35 A in thickness and spaced about 50 A apart. The individual lamellae had the appearance of being constructed of laterally united subunits, about 35 A in diameter. Various micrographs indicated a possible helical arrangement of certain components present in the virus core. The significance of these findings in elucidating relationships between potato yellow dwarf virus and some other plant and animal viruses is discussed.

In order to investigate whether Myzus persicae (Sulz.) takes up tobacco mosaic virus, droplets emerging from stylets cut while aphids were feeding, were tested for the virus by (1) inoculation of Nicotiana rustica and N. tabacum var. White Burley, (2) examination under the electron microscope, and (3) a microserological method. All gave negative results. Consequently the authors are of the opinion that M. persicae does not imbibe tobacco mosaic virus from tobacco.