Use of protein A to improve sensitisation of latex particles with antibodies to plant viruses

Protein A-coated latex (PAL) was compared with uncoated latex (L) for sensitisation with antibodies to five plant viruses: apple mosaic virus (ApMV), arabis mosaic virus (AMV), plum pox virus (PPV), potato virus Y ordinary strain (PVY°) and prunus necrotic ringspot virus (NRS V). A range of globulin concentrations was used with each antiserum and detection end points determined in serial dilutions of infective sap. When sensitised with antibodies to ApMV, PAL detected ApMV readily, whereas L did not. When sensitized with antibodies to PVY° and AMV, PAL gave higher detection end points than L. However, PAL gave little increase in sensitivity with the antisera to PPV and NRSV. Non-specific aggregation of latex, which sometimes occurred in very dilute sap with PAL, could be dispersed by adding 0.02% Tween-20 to the extraction buffer. Globulins of PVY° and AMV could be used at higher dilutions with PAL than with L, giving a saving in antiserum. Both types of latex sensitised with PVY° antibody globulins readily detected the tobacco veinal necrosis and C strains of this virus.     

The primary structure of the coat protein of alfalfa mosaic virus strain VRU. A hypothesis on the occurrence of two conformations in the assembly of the protein shell

The complete primary structure of the coat protein of strain VRU of alfalfa mosaic virus (AMV) is reported. The strain is morphologically different from all other AMV strains as it contains large amounts of unusually long virus particles. This is caused by structural differences in the coat protein chain. The amino acid sequence has mainly been established by the characterization of peptides obtained after cleavage with cyanogen bromide and digestion with trypsin, chymotrypsin, thermolysin or Staphylococcus aureus protease. The major sequencing technique used was the dansyl-Edman procedure. The VRU coat protein consists of 219 amino acid residues corresponding to a molecular weight of 24056. Compared to the coat protein of strain 425 [Van Beynum et al. (1977) Eur. J. Biochem. 72, 63-78], 15 amino acid substitutions were localized. Most of them have a conservative character and may be explained by single-point mutations. A correction is given for the AMV 425 coat protein: Asn-216 was shown to be Asp-216. The prediction of the secondary structure for the two viral coat proteins was not significantly influenced by the various amino acid substitutions except for the region containing residues 65-100. This led us to the hypothesis that the AMV coat protein may occur in two different conformations favouring its incorporation into either a pentagonal or hexagonal quasi-equivalent position in the lattice of the protein shell. The substitutions in the above-mentioned region of the VRU coat protein may have caused a strong preference for the hexagonal lattice conformation. The model is supported by preliminary sequence data of the same coat protein region in AMV 15/64, a strain morphologically intermediate between 425 and VRU.     

Virus diseases of garden lupin in Great Britain

Two viruses occur widely in lupins in Britain. Alfalfa mosaic virus (AMV), of which two strains were isolated, was found mainly in named Russell varieties. Lupin mottle virus (LMV), a previously undescribed strain of the bean yellow mosaic virus (BYMV) common pea mosaic virus (CPMV) complex, was found more commonly in seedling lupins. Cucumber mosaic virus (CMV) was isolated once. The AMV strains were differentiated by their reaction in Phaseolus vulgaris; they were serologically closely related. Both AMV and LMV were aphid transmitted but not transmitted in lupin seed. LMV was distantly serologically related to both BYMV and CPMV. It cross-protected against BYMV but not against CPMV and it differed from both these viruses in some host reactions. The CMV isolate from lupins was similar to type CMV. It was transmitted both mechanically and by aphid, easily from cucumber to cucumber, but with difficulty from cucumber to lupin.     

Role of the alfalfa mosaic virus movement protein and coat protein in virus transport

The movement protein (MP) and coat protein (CP) encoded by Alfalfa mosaic virus (AMV) RNA 3 are both required for virus transport. RNA 3 vectors that expressed nonfused green fluorescent protein (GFP), MP:GPF fusions, or GFP:CP fusions were used to study the functioning of mutant MP and CP in protoplasts and plants. C-terminal deletions of up to 21 amino acids did not interfere with the function of the CP in cell-to-cell movement, although some of these mutations interfered with virion assembly. Deletion of the N-terminal 11 or C-terminal 45 amino acids did not interfere with the ability of MP to assemble into tubular structures on the protoplast surface. Additionally, N- or C-terminal deletions disrupted tubule formation. A GFP:CP fusion was targeted specifically into tubules consisting of a wild-type MP. All MP deletion mutants that showed cell-to-cell and systemic movement in plants were able to form tubular structures on the surface of protoplasts. Brome mosaic virus (BMV) MP did not support AMV transport. When the C-terminal 48 amino acids were replaced by the C-terminal 44 amino acids of the AMV MP, however, the BMV/AMV chimeric protein permitted wild-type levels of AMV transport. Apparently, the C terminus of the AMV MP, although dispensable for cell-to-cell movement, confers specificity to the transport process.     

Transmission of the hop strain of arabis mosaic virus by Xiphinema diversicaudatum*

Hop plants became infected with the hop strain of arabis mosaic virus (AMV(H)) when grown in hopfield and woodland soil in which infected plants had been growing. Infection occurred in soil infested with the dagger nematode Xiphinema diversicaudatum, but neither in uninfested soil nor in soil previously heated to kill nematodes. X. diversicaudatum transferred direct from hop soils transmitted AMV(H) to young herbaceous plants and to hop seedlings; some of the hop seedlings developed nettlehead disease. A larger proportion of plants was infected using X. diversicaudatum obtained from a woodland soil and then given access to the roots of hop or herbaceous plants infected with AMV(H). AMV(H) was transmitted by adults and by larvae, in which the virus persisted for at least 36 and 29 wk, respectively. Difficulties were encountered in detecting AMV(H) in infected hop plants, due partly to the delay in virus movement from roots to shoots. Infection of hop shoots was seldom detected until the year after the roots were infested and sometimes nettlehead symptoms did not appear until the third year. Isolates of arabis mosiac virus from strawberry did not infect hop. The results are discussed in relation to the etiology and control of nettlehead and related diseases of hop.     

Arabis mosaic and Prunus necrotic ringspot viruses in hop (Humulus lupulus L.)

Purified virus preparations made from nettlehead-diseased hop plants, or from Chenopodium quinoa, to which the virus was transmitted by inoculation of sap, contained polyhedral virus particles of 30 mμ diameter which were identified serologically as arabis mosaic virus (AMV). There were serological differences between AMV isolates from hop and from strawberry, and also differences in host range and in symptoms caused in C. quinoa and C. amaranticolor. AMV was always associated with nettlehead disease. The nematode Xiphinema diversicaudatum occurred in small numbers in most hop gardens, but was numerous where nettlehead disease was spreading rapidly. Preparations from nettlehead-affected hops also contained a second virus, serologically related to Prunus necrotic ringspot virus (NRSV), in mild and virulent forms which infected the same range of test plants but showed some serological differences. Mild isolates did not protect C. quinoa plants against infection by virulent isolates. Hop seedlings inoculated with virulent isolates of NRSV developed symptoms indistinguishable from those of split leaf blotch disease. Latent infection with NRSV was prevalent in symptomless hop plants. Nettlehead disease is apparently associated with dual infection of AMV and virulent isolates of NRSV. An unnamed virus with rod-shaped particles 650 mμ long was common in hop and was transmitted by inoculation of sap to herbaceous plants. Cucumber mosaic virus was obtained from a single plant of Humulus scandens Merr.     

Elimination in vitro of two Grapevine Nepoviruses by an Alternating Temperature Regime*

A 40 day in vitro treatment with 6 h at 39°C followed by 18 h at 22°C was effective in eliminating both grapevine fanleaf virus (GFLV) and arabis mosaic virus (AMV) from the developing shoot tips (2 mm) of grapevine shoot tip cultures. Longer treatment durations with consecutive 12 h periods at 35°C and 22°C eliminated GFLV in some cases, but did not eliminate AMV.     

ANEMONE MOSAIC–A VIRUS DISEASE

The name anemone mosaic is proposed for a previously unrecorded virus disease of Anemone coronaria L.; infected plants have mottled leaves, and broken and distorted flowers. This virus can cause winter browning, and can contribute to crinkle in anemones.
The virus infected forty-seven out of ninety plant species tested; it was transmitted by mechanical inoculation, and by four of the six aphid species tested. Most aphids ceased to be infective within 30 min. when continuing to feed after leaving an infected plant.
Properties in vitro varied according to conditions of the tests; the thermal inactivation point was always below 62°C., the dilution end-point did not exceed 1/2500, and the virus inactivated at 18°C., the fewer than 72 hr.
Intracellular inclusion bodies were produced in all hosts examined.
Anemone mosaic virus is very similar to viruses placed in the turnip virus 1 group of Hoggan & Johnson, and is serologically related to cabbage black ringspot virus, although AMV infection did not protect plants against infection with cabbage black ring-spot virus.
Weeds naturally infected with AMV were found in anemone plantations, and this virus was detected, together with cucumber mosaic and tobacco necrosis viruses, in corms imported into this country.     

Alfalfa mosaic and cucumber mosaic virus infection in chickpea and lentil: incidence and seed transmission

Samples collected in 1994 and 1995 from commercial crops of chickpeas and lentils growing in the agricultural region of south-west Western Australia were tested for infection with alfalfa mosaic (AMV) and cucumber mosaic (CMV) viruses, and for members of the family Potyviridae using enzyme-linked immunosorbent assay (ELISA). In 1994 no virus was detected in the 21 chickpea crops tested but in 1995, out of 42 crops, AMV was found in two and CMV in seven. With lentils, AMV and/or CMV was found in three out of 14 crops in 1994 and 4 out of 13 in 1995, both viruses being detected in two crops in each year. Similar tests on samples from chickpea and lentil crops and plots growing at experimental sites, revealed more frequent infection with both viruses. No potyvirus infection was found in chickpeas or lentils in agricultural areas either in commercial crops or at experimental sites. However, bean yellow mosaic virus (BYMV) was detected along with AMV and CMV in irrigated plots of chickpeas and lentils at a site in Perth. When samples of seed from infected crops or plots of chickpeas and lentils were germinated and leaves or roots of seedlings tested for virus infection by ELISA, AMV and CMV were found to be seed-borne in both while BYMV was seed-borne in lentils. The rates of transmission found through seed of chickpea to seedlings were 0.1–1% with AMV and 0.1–2% with CMV. Seed transmission rates with lentil were 0.1–5% for AMV, 0.1–1% for CMV and 0.8% for BYMV. Individual seed samples of lentil and chickpea sometimes contained both AMV and CMV. With both species, infection with AMV and CMV was sometimes found in commercial seed stocks or seed stocks from multiplication crops of advanced selections nearing release as new cultivars. Seed-borne virus infection has important practical implications, as virus sources can be re-introduced every year to chickpea and lentil crops or plots through sowing infected seed stocks leading to spread of infection by aphid vectors, losses in grain yield and further contamination of seed stocks.     

Distribution of Deoxyribonucleic Acid Complementary to the Ribonucleic Acid of Avian Myeloblastosis Virus in Tissues of Normal and Tumor-Bearing Chickens

(3)H-labeled 70S ribonucleic acid (RNA) from purified avian myeloblastosis virus (AMV) was used as a probe in deoxyribonucleic acid (DNA)-RNA hybridization experiments to detect the presence of DNA complementary to the AMV genome in various tissues from noninfected normal chickens and from chickens infected with AMV. There was a remarkable constancy in the average cellular concentration of virus-specific DNA found in every tissue from the same uninfected chicken, and even in different chickens from the same strain. In contrast, different tissues from chickens bearing AMV-induced kidney tumors (embryonal nephromas) revealed an unequal distribution in the average virus-specific DNA content per cell. The increase was limited to tumor cells and to tissues that contain target cells for AMV, i.e., red blood cells, kidney cells, and possibly leukocytes. The red blood cells from AMV-infected chickens suffering from acute myeloblastic leukemia, although producing no virus, contained as many viral genome equivalents per cell as did leukemic myeloblasts known to produce large quantities of AMV. An increased viral DNA content was observed in the target cells of chickens that did not show any sign of tumor formation 6 months after infection with AMV. This study demonstrates that vertically transmitted viral DNA is uniformly and stably distributed among all tissues of the offspring, but that horizontal infection after hatching results in an increase in viral DNA content only in some dividing, target tissues that may or may not give rise to neoplasias.     

Response of hemopoietic cells to avian acute leukemia viruses: effects on the differentiation of the target cells.

Chicken bone marrow cells were infected with three avian acute leukemia viruses (ALV)--avian myeloblastosis virus (AMV), myelocytomatosis virus strain MC29 and Mill Hill 2 virus (MH2)--and then cultured in agar in the presence of conditioned medium. Under these conditions, it was found that very few cells served as target cells for these three viruses. Density gradient separation showed that ALV target cells were found primarily in the light density fractions and might be represented by cells committed to the mononuclear phagocyte pathway. Separation of bone marrow cells on the basis of their sedimentation velocity at unit gravity suggested that MC29 and AMV did not share the same target cells. In addition, the analysis of surface receptors and functional markers characteristic of macrophages (Fc and complement receptors, phagocytosis and immune phagocytosis) indicated that the ALV-transformed cells were blocked during their differentiation. These results indicate that the transforming ability of ALV interferes with the differentiation of their target cells.     

Alfalfa mosaic virus isolates from lucerne in South Australia: biological variability and antigenic similarity

Alfalfa mosaic virus (AMV) was isolated from lucerne (Medicago sativa) plants with a variety of disease symptoms in eight of 13 sites in South Australia indicating that the virus is widespread in the state. The host ranges and symptomatology of the virus isolates varied considerably. Twelve selected local lesion isolates were shown to be distinct when mechanically inoculated to a range of plant species and cultivars. However, agar-gel diffusion and enzyme-linked immunoassay tests with polyclonal antisera prepared against glutaraldehyde-fixed virus preparations of the five most readily distinguishable AMV isolates, failed to reveal significant antigenic differences between the 12 virus isolates. This indicates that serological tests with polyclonal antisera can detect a wide range of AMV variants but would be unlikely to differentiate between strains. The wide host range and variability of AMV precluded the grouping of isolates into strains of the virus.     

Structural Studies of Avian Myeloblastosis Virus: Comparison of Polypeptides in Virion and Core Component by Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

Two different systems of dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in separate laboratories detected analogous patterns of dye bands in virions of avian myeloblastosis virus (AMV). At least 11 of the dye bands co-migrated with the major polypeptides reported in Rous sarcoma virus. Particles with the morphology of the AMV core component, obtained after exposure of AMV to the nonionic surfactant Sterox SL, contained major polypeptides p12, p27, p60, p64, p91, and p98. The polypeptide p12 has been previously shown to be the major constituent of the inner ribonucleoprotein (RNP) of the AMV core, and has been designated p12(N). Two RNP polypeptides, p64 and p91, co-electrophoresed with purified AMV DNA polymerase and have now been designated p64(P) and p91(P). The polypeptide p27 has been identified as a probable constituent of the core shell, and has accordingly now been designated p27(C). In comparison to virions of AMV, the AMV core component contained a greatly reduced amount of polypeptide p15 and appeared to lack a major polypeptide, p19. Consequently, these polypeptides may be associated either with the exterior of the core shell or the interior of the viral envelope. Glycopeptides were not detected in AMV cores, in agreement with earlier reports that they reside in external projections from the viral envelope.     

The etiology of hop chlorotic disease

Hop chlorotic disease was first described in England in 1930, but it has since been seldom seen and its etiology has remained unknown. In 1983 a patch of plants with the disease occurred in a large area of hops (Humulus lupulus) cv. Bramling Cross planted at Yalding, Kent in 1967. All plants in a rectangular area enclosing the disease outbreak were infected with hop mosaic, hop latent and prunus necrotic ringspot viruses; the diseased plants were additionally infected with arabis mosaic virus (AMV). The disease was also associated with seed-transmitted AMV, and was induced in hop seedlings inoculated with partially purified preparations of AMV originating from chlorotic disease-affected hops prepared from Chenopodium quinoa. The disease appears to be caused by AMV, but AMV isolates from hops with chlorotic disease were serologically indistinguishable from AMV isolates from hops with symptoms of bare-bine and/or nettlehead and showed similar pathogenicity in diagnostic hosts. The basis of the difference between isolates in their pathogenicity in hop remains unknown.     

Ribonucleotide sequence homology among avian oncornaviruses.

RNA sequence relatedness among avian RNA tumor virus genomes was analyzed by inhibition of DNA-RNA hybrid formation between 3H-labeled 35S viral RNA and an excess of leukemic or normal chicken cell DNA with increasing concentrations of unlabeled 35S viral RNA. The avian viruses tested were Rous associated virus (RAV)-3, avian myeloblastosis virus (AMV), RAV-60, RAV-61, and B-77 sarcoma virus. Hybridization of 3H-labeled 35S AMV RNA with DNA from normal chicken cells was inhibited by unlabeled 35S RAV-0 RNA as effeciently (100%) as by unlabeled AMV RNA. Hybridization between 3H-labeled 35S AMV RNA and DNA from leukemic chicken myeloblasts induced by AMV was suppressed 100 and 68% by unlabeled 35S RNA from AMV and RAV-0, respectively. Hybridization between 3H-labeled RAV-0 and leukemic chicken myeloblast DNA was inhibited 100 and 67% by unlabeled 35S RNA from RAV-0 and AMV, respectively. It appears therefore that the AMV and RAV-0 genomes are 67 to 70% homologous and that AMV hybridizes to RAV-0 like sequences in normal chicken DNA. Hybridization between AMV RNA and leukemic chicken DNA was inhibited 40% by RNA from RAV-60 or RAV-61 and 50% by B-77 RNA. Hybridization between RAV-0 RNA and leukemic chicken DNA was inhibited 80% by RAV-60 or RAV-61 and 70% by B-77 RNA. Hybridization between 3H-labeled 35S RNA from RAV-60 or RAV-61 and leukemic chicken myeloblast DNA was reduced equally by RNA from RAV-60, RAV-61, AMV or RAV-0; this suggests that RNA from RAV-60 and RAV-61 hybridizes with virus-specific sequences in leukemic DNA which are shared by AMV, RAV-0, RAV-60, and RAV-61 RNA'S. Hybridization between 3H-labeled 35S RNA from RAV-61 and normal pheasant DNA was inhibited 100% by homologous viral RNA, 22 TO 26% BY RNA from AMV or RAV-0, and 30 to 33% by RNA from RAV-60 or B-77. Nearly complete inhibition of hybricization between RAV-0 RNA and leukemic chicken DNA by a mixture of AMV and B-77 35S RNAs indicates that the RNA sequences shared by B-77 virus and RAV-0. It appears that different avian RNA tumor virus genomes have from 50 to 80% homology in nucleotide sequences and that the degree of hybridization between normal chicken cell DNA and a given viral RNA can be predicted from the homology that exists between the viral RNA tested and RAV-0 RNA.     

VSV Pseudotype Particles with the Coat of Avian Myeloblastosis Virus

PSEUDOTYPES of vesicular stomatitis virus (VSV) with the coat of avian myeloblastosis (AMV) or murine leukaemia viruses—VSV(AMV) and VSV(MLV)—can be produced by growing VSV in chick cells preinfected with AMV or in mouse cells preinfected with MLV¹. The VSV particles carrying their own neutralization antigen and double-neutralizable particles may be inactivated with antiserum against VSV. The surviving pseudotypes possess neutralization, host-range and interference specificities corresponding to the tumour virus donating their coat. It has also been shown that a conditional lethal mutant of VSV in which a structural protein is affected is complemented under restrictive conditions with AMV. This mutant, ts-45, when complemented with AMV again predominantly produces the pseudotype VSV(AMV).