Robert G. Milne

Cryptic plant viruses.

Publisher Summary It is interesting that at this relatively late stage in virus research, new kinds of viruses continue to come to light. Cryptic viruses successfully evaded detection until recently because they induce no or, perhaps in some cases, very slight disease symptoms, are not transmissible in the ordinary way, and have particles present in such low concentrations as to escape casual discovery. The fact that cryptic viruses appear to cause no economically important disease has tended to give them low research priority. Nevertheless they are of interest as they contain double-stranded (ds) RNA, present only in another very different group of plant viruses, and appear unable to pass from carrier to non-carrier cells across a graft union. The latter is a decidedly non-virus like property. They are also of practical importance as they may be responsible for misleading results with methods for the detection of single-stranded RNA viruses based on the presence in plants of the double-stranded replicative forms.

Transmission by Olpidium brassicae of Mirafiori lettuce virus and Lettuce big-vein virus, and Their Roles in Lettuce Big-Vein Etiology

ABSTRACT Big-vein disease occurs on lettuce worldwide in temperate conditions; the causal agent has been presumed to be Lettuce big-vein virus (LBVV), genus Varicosavirus, vectored by the soilborne fungus Olpidium brassicae. Recently, the role of LBVV in the etiology of big-vein disease has been questioned because a second soilborne virus, Mirafiori lettuce virus (MiLV), genus Ophiovirus, has been found frequently in big-vein-affected lettuce. LBVV and MiLV, detectable and distinguishable by enzyme-linked immunosorbent assay using specific antisera, were tested for their ability to be transmitted from lettuce to lettuce by mechanical inoculation of sap extracts, or by zoospores of O. brassicae, and to cause big-vein disease. Both viruses were mechanically transmissible from lettuce to herbaceous hosts and to lettuce, but very erratically. LBVV was transmitted by O. brassicae but lettuce infected with only this virus never showed symptoms. MiLV was transmitted in the same manner, and lettuce infected with this virus alone consistently developed big-vein symptoms regardless of the presence or absence of LBVV. With repeated mechanical transmission, isolates of both viruses appeared to lose the ability to be vectored, and MiLV appeared to lose the ability to cause big-vein symptoms. The recovery of MiLV (Mendocino isolate, from Cali-fornia) from stored O. brassicae resting spores puts the earliest directly demonstrable existence of MiLV at 1990.

An Ophiovirus isolated from lettuce with big-vein symptoms

Summary. Big-vein is a widespread and damaging disease of lettuce, transmitted through soil by the chytrid fungus Olpidium brassicae, and generally supposed to be caused by Lettuce big-vein virus (LBVV; genus Varicosavirus). This virus is reported to have rigid rod-shaped particles, a divided double-stranded RNA genome, and one capsid protein of 48 kD, but has not been isolated or rigorously shown to cause the disease. We provide evidence that a totally different virus, here named Mirafiori lettuce virus (MiLV), is also very frequently associated with lettuce showing big-vein symptoms. MiLV was mechanically transmissible from lettuce to Chenopodium quinoa and to several other herbaceous test plants. The virus was partially purified, and an antiserum prepared, which did not react with LBVV particles in decoration tests. As reported for LBVV, MiLV was labile, soil-transmitted and had a single capsid protein of 48 kD, but the particles morphologically resembled those of ophioviruses, and like these, MiLV had a genome of three RNA segments approximately 8.5, 1.9 and 1.7 kb in size. MiLV preparations reacted strongly in Western blots and in ISEM with antiserum to Tulip mild mottle mosaic virus, an ophiovirus from Japan also apparently Olpidium-transmitted. They reacted weakly but clearly in Western blots with antiserum to Ranunculus white mottle virus, another ophiovirus. When lettuce seedlings were mechanically inoculated with crude or partially purified extracts from MiLV-infected test plants, many became systemically infected with MiLV and some developed big-vein symptoms. Such plants did not react in ELISA using an LBVV antiserum or an antiserum to tobacco stunt virus, and varicosavirus-like particles were never seen in them in the EM after negative staining. We conclude that MiLV is a hitherto undescribed virus assignable to the genus Ophiovirus. The cause or causes of lettuce big-vein disease and the properties of LBVV may need to be re-evaluated in light of our results.

Rapid high-resolution immune electron microscopy of plant viruses

Abstract Two immune electron microscopic methods are described that allow rapid demonstration of (a) specific antibody coating of virus particles or (b) specific clumping and a tenfold increase in the number of particles visible on the grid. The operations take only 20 min and produce high-resolution images not only with purified preparations but also with crude preparations or those containing salts, buffers or sucrose.

Multiplication of tobacco mosaic virus in tobacco leaf palisade cells

Abstract Sections of palisade cells of Turkish tobacco leaves were examined by electron microscopy at various times after inoculation of the upper epidermis with tobacco mosaic virus (TMV). Virus particles were first seen in 15-hour samples, and by 48 hours after inoculation very many particles were present as individuals or in semicrystalline aggregates in the cytoplasm. Nuclei appeared normal and no virus particles were seen in or particularly associated with them. Occasionally particles were seen in invaginations or vacuoles in chloroplasts, but this is not thought to imply an active association. Filamentous regions first appearing in the cytoplasm at 15 hours were always associated with infection, but they did not appear to be direct TMV precursors.

Should Tomato Spotted Wilt Virus Be Considered as a Possible Member of the Family Bunyaviridae

From a comparison of published data on the properties of tomato spotted wilt virus and viruses in the family Bunyaviridae, it is concluded that tomato spotted wilt virus should be considered as a possible member of the family.

Partial purification, structure and infectivity of complete maize rough dwarf virus particles

Abstract Maize rough dwarf virus, a reolike virus, was purified from roots of maize by clarification with the fluorocarbon 1,1,2-trifluoro-1,2,2-trichloroethane (Freon 113) or with carbon tetrachloride followed by sucrose density gradient centrifugation. The product consisted mostly of complete particles, but some subviral particles were present. The virus was spherical, varying from 63 to 70 nm in diameter under different staining conditions; it had a double capsid and typical reovirus structure with, probably, 92 morphological units in the outer capsid. In addition, each particle possessed 12 projecting spikes (A spikes) each some 11 nm long, one at each 5-fold symmetry axis. Various physical or chemical treatments stripped off the A spikes and part of the outer capsid to give a spherical 50–57 nm subviral particle possessing 12 previously hidden spikes (B spikes) each about 8 nm long, coaxial with the A spikes. Each B spike was implanted on a differentiated part of the inner capsid here called a baseplate. Treatment of whole virus or subviral particles with aqueous neutral potassium phosphotungstate produced smooth subviral particles without B spikes, and longer treatment with phosphotungstate or brief treatment with n-butanol also removed the nucleic acid to give ghosts exhibiting internal structures. Detached B spikes were composed of 5 morphological subunits surrounding a central hole. Infectivity of preparations was tested by injecting them into adult females of the planthopper vector Laodelphax striatellus, which were later screened for production duction of virus symptoms on maize and barley. Crude and purified preparations containing the large virus particles were infectious; Freon-treated preparations possessed the highest infectivity. Subviral preparations made by chloroform treatment were not or very slightly infectious and smooth subviral particles made by treatment with phosphotungstate were not infectious.

Comparative analysis of recombinant Human Papillomavirus 8 L1 production in plants by a variety of expression systems and purification methods

Human papillomavirus 8 (HPV-8), one of the high-risk cutaneous papillomaviruses (cHPVs), is associated with epidermodysplasia verruciformis and nonmelanoma skin cancer in immuno-compromised individuals. Currently, no vaccines against cHPVs have been reported; however, recent studies on cross-neutralizing properties of their capsid proteins (CP) have fostered an interest in vaccine production against these viruses. We examined the potential of producing HPV-8 major CP L1 in Nicotiana benthamiana by agroinfiltration of different transient expression vectors: (i) the binary vector pBIN19 with or without silencing suppressor constructs, (ii) the nonreplicating Cowpea mosaic virus-derived expression vector pEAQ-HT and (iii) a replicating Tobacco mosaic virus (TMV)-based vector alone or with signal peptides. Although HPV-8 L1 was successfully expressed using pEAQ-HT and TMV, a 15-fold increase was obtained with pEAQ-HT. In contrast, no L1 protein could be immune detected using pBIN19 irrespective of whether silencing suppressors were coexpressed, although such constructs were required for identifying L1-specific transcripts. A fourfold yield increase in L1 expression was obtained when 22 C-terminal amino acids were deleted (L1ΔC22), possibly eliminating a nuclear localization signal. Electron microscopy showed that plant-made HPV-8 L1 proteins assembled in appropriate virus-like particles (VLPs) of T = 1 or T = 7 symmetry. Ultrathin sections of L1ΔC22-expressing cells revealed their accumulation in the cytoplasm in the form of VLPs or paracrystalline arrays. These results show for the first time the production and localization of HPV-8 L1 protein in planta and its assembly into VLPs representing promising candidate for potential vaccine production.

Electron microscopy of leaves infected with Sowbane mosaic virus and other small polyhedral viruses.

Leaves of Chenopodium amaranticolor were infected with sowbane mosaic virus (SMV), sectioned and examined by electron microscopy. Leaves of Brassica pekinensis and C. amaranticolor were infected with turnip yellow mosaic virus and cowpea mosaic virus, respectively, and similarly studied. With all three viruses it was difficult, in sections, to distinguish the small isometric virus particles from ribosomes though sometimes this was possible, especially when the viruses crystallized. Pretreatment of tissue with permanganate or EDTA appeared to destroy the ribosomes but resulted in excessive disorganization of the tissue. Although SMV did not normally crystallize, wilting the infected leaves caused it to do so. All three viruses were found free in the cytoplasm and were absent from nuclei, chloroplasts, and mitochondria. Some abnormal structures found in the infected tissues are described.

Electron microscopy of tobacco mosaic virus in leaves of Nicotiana glutinosa

Abstract Electron microscopic examination of lesions produced in Nicotiana glutinosa by infection with tobacco mosaic virus (TMV) showed clearly that virus particles were present. Only about one-fiftieth of the sections of cells in the infected regions exhibited virus particles and even in these the numbers of particles were not large. Virus was found only in the cytoplasm. Careful search failed to reveal any regions containing the filamentous bodies that have been reported as associated with TMV infections in other hosts.