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Canine host range and a specific epitope map along with variant sequences in the capsid protein gene of canine parvovirus and related feline, mink, and raccoon parvoviruses.

Canine parvovirus (CPV) is a recently recognized pathogen of dogs that is similar to the long-recognized feline, mink, and raccoon parvoviruses. Relationships between the viruses determined from DNA sequences of the capsid protein genes of 10 virus isolates showed the CPV isolates to be closely related to the other viruses, although comprising a distinct group. No immediate ancestor of CPV was observed amongst the mink, cat, or raccoon viruses examined. Three different directly repeated sequences were present within the noncoding region downstream from the capsid protein genes. Analysis of recombinants between CPV and feline panleukopenia virus at restriction sites within the capsid protein genes mapped a CPV-specific neutralization epitope on the virus capsid, differences in the pH dependence of hemagglutination, and part of the determinant of canine host range between 59 and 64 genome map units (m.u.). Those differences were therefore the result of up to three nucleotide or predicted amino acid sequence differences in that region. A second region between 64 and 73 m.u., which may affect the viability of certain recombinant viruses, contained four nucleotide differences, one of which was a coding change.

Animals↗

Generation of high-titer pseudotyped retroviral vectors with very broad host range.

Encapsidation of the VSV G protein into the virions of MoMLV-derived retroviral vectors in the absence of other VSV-encoded proteins is shown to be an efficient process, although the exact mechanism for this process is currently unclear. Unlike the conventional retroviral vectors bearing the amphotropic envelope protein, the pseudotyped virus has the ability to withstand the shearing forces encountered during ultracentrifugation. This property of the pseudotyped virus enables the generation of high-titer retroviral vector stocks and has potential application for in vivo gene therapy studies. We have found as many as four copies of a pseudotyped vector to integrate into the genome of a single cell when a high multiplicity of infection was used to infect the cells. Multiple integration events were not observed with amphotropic retroviral vectors, probably because of their low virus titers. In addition, when retroviral vectors are pseudotyped with the VSV G protein, they acquire the host range of VSV and are able to infect nonmammalian cells derived from fish, Xenopus, mosquito, and Lepidoptera. Since techniques for efficient gene transfer in some of these nonmammalian systems are not currently available, retrovirus-mediated gene transfer described here should be useful for transgenic and other genetic studies in lower vertebrate species. The inability to establish a stable cell line expressing the VSV G protein, however, limits large-scale production of the pseudotyped retroviral vectors. Generation of stable packaging cell lines for the pseudotyped retroviral vectors is a major challenge for the future.

3T3 Cells↗

Expansion of Viral Host Range through Complementation and Recombination in Transgenic Plants.

We have shown previously that gene VI of cauliflower mosaic virus (CaMV) strain D4 governs systemic infection of Nicotiana bigelovii and that transgenic N. bigelovii expressing the D4 gene VI product can complement at least one CaMV isolate for long-distance transport. We have now found that DNA of two other isolates of CaMV recombine with the gene VI coding sequence present in the transgenic plants. The formation of recombinant viruses occurs as a consequence of CaMV replication, involving two template switches during reverse transcription of the CaMV RNA to DNA. The first template switch occurs at the 5[prime] end of the 35S RNA to the gene VI mRNA produced by the transgenic plants. A second switch occurs at the 5[prime] end of the gene VI mRNA back to the 35S RNA. We also demonstrate that CaMV can acquire sequences from transgenic plants that alter the symptomatology and host range of the virus, an observation that may have important risk assessment implications for strategies using pathogen-derived resistance to protect plants against virus diseases.

Journal Article↗

Molecular cloning and expression of Rhizobium fredii USDA 193 nodulation genes: extension of host range for nodulation.

DNA hybridization with the cloned nodulation region of Rhizobium meliloti as a probe revealed DNA homology with four HindIII fragments, 12.5, 6.8, 5.2, and 0.3 kilobases (kb) in size, of the symbiotic plasmid pRjaUSDA193. Both hybridization and complementation studies suggest that the common nodulation genes nodABC and nodD of R. fredii USDA 193 are present on the 5.2-kb HindIII and 2.8-kb EcoRI fragments, respectively, of the Sym plasmid. Both fragments together could confer nodulation ability on soybeans when present in Sym plasmid-cured (Sym-) and wild-type (Sym+) Rhizobium strains or in a Ti plasmid-cured Agrobacterium tumefaciens strain. Furthermore, the 2.8-kb EcoRI fragment alone was able to form nodulelike structures on Glycine max L. cv. "Peking" (soybean). Microscopic examination of these nodules revealed bacterial invasion of the cells, probably via root hair penetration. Bacterial strains harboring plasmids carrying the 5.2- and 2.8-kb nod fragments elicited root-hair-curling responses on infection. These data suggest that the genes responsible for host range determination and some of the early events of nodulation may be coded for by the 5.2-kb HindIII and 2.8-kb EcoRI fragments.

Cloning, Molecular↗

Structural and functional analysis of the origin of conjugal transfer of the broad-host-range IncW plasmid R388 and comparison with the related IncN plasmid R46.

We cloned and sequenced a 402 bp DNA segment containing the origin of conjugal transfer (oriT) of the IncW plasmid R388. Progressive deletions from each end of the sequence were assayed for oriT activity. Stepwise reductions in mobilization frequencies, representing the loss of functional elements, correlated with deletion of structural motifs in the sequence. A sequence of 330 bp of oriT was sufficient for efficient mobilization. The first 86 bp of the sequence contains five tandemly repeated DNA sequences of 11 bp, followed by a 10 bp perfect inverted repeat. Deletion of the first 95 bp reduced the frequency of transfer by a hundred-fold. The sequence between bp 183 and 218 was necessary and sufficient for low frequency mobilization and, thus, it was assumed to contain the nick site. This basis core was cloned as a 60 bp segment (from bp 176-236) that could be mobilized at low frequency. It includes two inverted repeats and a perfect integration host factor (IHF) consensus binding site. A third functionally important segment in oriT was located between bp 260 and 330. The DNA sequence of the oriT of R388 could be aligned with that of the broad-host-range IncN plasmid R46. Moreover, the relative positions of the three inverted repeats are also conserved. Overall sequence similarity was 52%, but was significantly higher in particular regions, which coincided with the functionally important segments mapped by deletion analysis. Conservation of these segments provided independent support for their essential role in oriT function.

Base Sequence↗

NodZ of Bradyrhizobium extends the nodulation host range of Rhizobium by adding a fucosyl residue to nodulation signals.

The nodulation genes of rhizobia are involved in the production of the lipo-chitin oligosaccharides (LCO), which are signal molecules required for nodule formation. A mutation in nodZ of Bradyrhizobium japonicum results in the synthesis of nodulation signals lacking the wild-type 2-O-methylfucose residue at the reducting-terminal N-acetylglucosamine. This phenotype is correlated with a defective nodulation of siratro (Macroptilium atropurpureum). Here we show that transfer of nodZ to Rhizobium leguminosarum blovar (bv) viciae, which produces LCOs that are not modified at the reducing-terminal N-acetylglucosamine, results in production of LCOs with a fucosyl residue on C-6 of the reducing-terminal N-acetylglucosamine. This finding, together with in vitro enzymatic assays, indicates that the product of nodZ functions as a fucosyltransferase. The transconjugant R. leguminosarum strain producing fucosylated LCOs acquires the capacity to nodulate M. atropurpureum, Glycine soja, Vigna unguiculate and Leucaena leucocephala. Therefore, nodZ extends the narrow host range of R. leguminosarum bv. viciae to include various tropical legumes. However, microscopic analysis of nodules induced on siratro shows that these nodules do not contain bacteroids, showing that transfer of nodZ does not allow R. leguminosarum to engage in a nitrogen-fixing symbiosis with this plant.

Amino Acid Sequence↗

A rabbitpox virus serpin gene controls host range by inhibiting apoptosis in restrictive cells.

Poxviruses are unique among viruses in encoding members of the serine proteinase inhibitor (serpin) superfamily. Orthopoxviruses contain three serpins, designated SPI-1, SPI-2, and SPI-3. SPI-1 encodes a 40-kDa protein that is required for the replication of rabbitpox virus (RPV) in PK-15 or A549 cells in culture (A. N. Ali, P. C. Turner, M. A. Brooks, and R. W. Moyer, Virology 202:305-314, 1994). Examination of nonpermissive human A549 cells infected with an RPV mutant disrupted in the SPI-1 gene (RPV delta SPI-1) suggests there are no gross defects in protein or DNA synthesis. The proteolytic processing of late viral structural proteins, a feature of orthopoxvirus infections associated with the maturation of virus particles, also appears relatively normal. However, very few mature virus particles of any kind are produced compared with the level found in infections with wild-type RPV. Morphological examination of RPV delta SPI-1-infected A549 cells, together with an observed fragmentation of cellular DNA, suggests that the host range defect is associated with the onset of apoptosis. Apoptosis is seen only in RPV delta SPI-1 infection of nonpermissive (A549 or PK-15) cells and is absent in all wild-type RPV infections and RPV delta SPI-2 mutant infections examined to date. Although the SPI-1 gene is expressed early, before DNA replication, the triggering apoptotic event occurs late in the infection, as RPV delta SPI-1-infected A549 cells do not undergo apoptosis when infections are carried out in the presence of cytosine arabinoside. While the SPI-2 (crmA) gene, when transfected into cells, has been shown to inhibit apoptosis, our experiments provide the first indication that a poxvirus serpin protein can inhibit apoptosis during a poxvirus infection.

Animals↗

Bacterial stalk rot of Maize, its symptoms and host-range.

Stalk rot of maize, caused by Erwinia carotovora f. sp. zeae Sabet (re-designated as Pectobacterium chrysanthemi pathovar. zeae by KELMAN 1974) showed first premature withering and drying up of the uppermost leaves which was soon followed by the lower leaves. The rot either extended from the base upwards (basal rot) or from the top downwards (top rot). In the case of basal rot, the leaves become yellow and the infected tissue becomes brown, soft, and water soaked. Internally, the stalk turns into a soft mass of disintegrated tissue. At this stage the plants usually topple over. A foul odour, accompanied with the presence of dipterous larvae on and in decaying tissues, are the characteristic symptoms of this disease. With the advance of the disease the stalk finally dries up into a conglomeration of dry and shredded or disjointed fibrous tissue. The top rot begins with wilting and drying up ot the tips of middle leaves of the whorl. A decay that continues rapidly spreads downwards throughout the stalk and the affected plants soon droop. The host range studies were suggestive of the fact that the maize pathogen, apart from causing the disease on maize, could produce soft rot in potato, carrot, onion, sugarbeet, sweet potato, papaya, cabbage, and many other plants and could also infect Sorghum vulgare, Pennisetum typhoidium, tobacco, and tomato.

Ecology↗

Tree host range and world distribution of the extomycorrhizal fungus Pisolithus tinctorius.

The natural occurrence of Pisolithus tinctorius has been confirmed in 33 countries of the world and in 38 states in the United States. This ectomycorrhizal fungus is found associated with various tree species in nurseries, urban areas, orchards, forests, and strip-mined spoils. Experiments have proved that this fungal symbiont forms ectomycorrhizae with Abies procera, Betula pendula, Carya illnoensis, 11 species of Eucalyptus, 30 species of Pinus, Pseudotsuga menziesii var. meniziesii, 2 species of Quercus, and Tsuga heterophylla. Pisolithus has also been reported growing under natural conditions in association with three additional species of Betula, two species of Eucalyptus, nine species of Pinus, and eight species of Quercus, Populus tremuloides, Pseudotsuga grandidenta, and Salix humilis. This fungal symbiont has great potential in forestation efforts because of (1) the availability of practical techniques for artificially introducing in into nursery soils; (2) its ability to improve tree survival and growth in the nursery and the field; (3) its near worldwide distribution on a variety of sites; and (4) its broad host range encompassing many of the world's most important tree species.

Basidiomycota↗

Poliovirus host range is determined by a short amino acid sequence in neutralization antigenic site I.

The mouse-adapted strain of poliovirus type 2 (Lansing) induces fatal poliomyelitis in mice after intracerebral inoculation, whereas mice inoculated with poliovirus type 1 (Mahoney) show no signs of disease. Previous work indicated that the adaptation to mouse virulence is associated with the viral capsid proteins and that mutations in neutralization antigenic site I of poliovirus reduce neurovirulence of the Lansing strain in mice. The role of antigenic site I in mouse neurovirulence was further explored by constructing an antigenic hybrid virus. Six amino acids in antigenic site I of the Mahoney strain were replaced with a sequence specific for the Lansing strain by using a mutagenesis cartridge. The hybrid virus was neutralized by polyclonal antisera elicited by the type 1 and type 2 strains of poliovirus and by neutralizing monoclonal antibodies directed against antigenic site I of type 2 virus. The hybrid virus induced paralytic disease in mice, an observation demonstrating that a short sequence of amino acids in antigenic site I is an important determinant of poliovirus host range. Antigenic site I may be involved in attachment of poliovirus to cells of the mouse central nervous system.

Animals↗

Broad-host-range Rhizobium species strain NGR234 secretes a family of carbamoylated, and fucosylated, nodulation signals that are O-acetylated or sulphated.

Rhizobium species strain NGR234 is the most promiscuous known rhizobium. In addition to the non-legume Parasponia andersonii, it nodulates at least 70 genera of legumes. Here we show that the nodulation genes of this bacterium determine the production of a large family of Nod-factors which are N-acylated chitin pentamers carrying a variety of substituents. The terminal non-reducing glucosamine is N-acylated with vaccenic or palmitic acids, is N-methylated, and carries varying numbers of carbamoyl groups. The reducing N-acetyl-glucosamine residue is substituted on position 6 with 2-O-methyl-L-fucose which may be acetylated or sulphated or non-substituted. All three internal residues are N-acetylated. At pico- to nanomolar concentrations, these signal molecules exhibit biological activities on the tropical legumes Macroptilium and Vigna (Phaseoleae), as well as on both the temperate genera Medicago (Trifoliae) and Vicia (Viciae). These data strongly suggest that the uniquely broad host range of NGR234 is mediated by the synthesis of a family of varied sulphated and non-sulphated lipo-oligosaccharide signals.

Acetylation↗

Unusual amino acid determinants of host range in the Mtx2 family of mosquitocidal toxins.

Five different mosquitocidal toxin (mtx2) gene homologs have been cloned from eight Bacillus sphaericus strains. Pairwise comparisons of the predicted amino acid sequences show between four and eight substitutions compared with the prototype Mtx2 from B. sphaericus strain SSII-1. Mtx2 from strain SSII-1 was approximately 7-fold more toxic to Culex mosquito larvae than the Mtx2 homolog from B. sphaericus strain 31-2. Conversely, Mtx2 from strain 31-2 was approximately 100-fold more toxic to Aedes mosquito larvae than Mtx2 from strain SSII-1. Lys224 in Mtx2 was found to be the most important amino acid for toxicity to Culex larvae, and substitution of Lys224 with threonine abolished the toxicity of Mtx2 from strain SSII-1 to these larvae. In complete contrast, Thr224 was found to be crucial for the toxicity of Mtx2 from strain 31-2 to Aedes larvae, and substitution of Thr224 with lysine caused a approximately 100-fold drop in toxicity to these larvae. Thus, amino acid 224 in the Mtx2 family of mosquitocidal toxins is an unusual and important determinant of mosquito larvicidal activity and host range.

Aedes↗

virF, the host-range-determining virulence gene of Agrobacterium tumefaciens, affects T-DNA transfer to Zea mays.

The monocotyledonous plant Zea mays does not develop tumors after inoculation with Agrobacterium tumefaciens and is thus defined as nonhost. Agroinfection, Agrobacterium-mediated delivery of maize streak virus, demonstrates that transferred DNA (T-DNA) transfer to the plant does occur. Nopaline-type Agrobacterium strains such as C58 are efficient in the transfer process whereas the octopine-type strain A6 is unable to transfer T-DNA to maize. This phenotypic difference maps to the tumor-inducing (Ti) plasmid but not to the T-DNA. Steps preceding T-DNA transfer, such as attachment and induction of the virulence genes, were shown to take place in the octopine strain. The nopaline-plasmid-specific locus tzs and the octopine-plasmid-specific locus pinF (virH) are not involved in the strain specificity. However, mutations in the virF locus rendered the octopine strain agroinfectious on maize, whereas such virF-defective octopine strains, when complemented by virF on a plasmid, completely lost their agroinfectivity. We propose that VirF, known to increase the host range of the bacteria in other systems, acts as an inhibitor of T-DNA transfer to maize.

Journal Article↗

Coexpression of exogenous and endogenous mouse mammary tumor virus RNA in vivo results in viral recombination and broadens the virus host range.

Mouse mammary tumor virus is a replication-competent B-type murine retrovirus responsible for mammary gland tumorigenesis in some strains of laboratory mice. Mouse mammary tumor virus is transmitted horizontally through the milk (exogenous or milk-borne virus) to susceptible offspring or vertically through the germ line (endogenous provirus). Exogenously acquired and some endogenous mouse mammary tumor viruses are expressed at high levels in lactating mammary glands. We show here that there is packaging of the endogenous Mtv-1 virus, which is expressed at high levels in the lactating mammary glands of C3H/HeN mice, by the virions of exogenous C3H mouse mammary tumor virus [MMTV(C3H)]. The mammary tumors induced in C3H/HeN mice infected with exogenous MMTV (C3H) virus contained integrated copies of recombinant virus containing a region of the env gene from an endogenous virus. This finding indicates that there was copackaging of the Mtv-1 and MMTV(C3H) RNAs in the same virions. Moreover, because Mtv-1 encodes a superantigen protein with a V beta specificity different from that encoded by the exogenous virus, the packaging of Mtv-1 results in an infectious virus with a broader host range than MMTV(C3H).

Animals↗

pIJ101, a multi-copy broad host-range Streptomyces plasmid: functional analysis and development of DNA cloning vectors.

Streptomyces lividans ISP 5434 contains four small high copy number plasmids: pIJ101 (8.9 kb), pIJ102 (4.0 kb), pIJ103 (3.9 kb) and pIJ104 (4.9 kb). The three smaller species appear to be naturally occurring deletion variants of pIJ101. pIJ101 and its in vivo and in vitro derivatives were studied after transformation into S. lividans 66. pIJ101 was found to be self-transmissible by conjugation, to elicit "lethal zygosis" and to promote chromosomal recombination at high frequency in both S. lividans 66 and S. coelicolor A3(2). A restriction endonuclease cleavage map of pIJ101 was constructed for 11 endonucleases; sites for five others were lacking. Many variants of pIJ101 were constructed in vitro by inserting DNA fragments determining resistance to neomycin, thiostrepton or viomycin, and having BamHI termini, into MboI or BclI sites on the plasmid, sometimes with deletion of segments of plasmid DNA. The physical maps of these plasmids were related to their phenotypes in respect of lethal zygosis and transfer properties. In vivo recombination tests between pairs of variant plasmids were also done. These physical and genetic studies indicated that determinants of conjugal transfer occupy less than 2.1 kb of the plasmid. A second segment is required for spread of the plasmid within a plasmid-free culture to produce the normal lethal zygosis phenotype: insertion of foreign DNA in this region caused a marked reduction in the diameter of lethal zygosis zones. The minimum replicon was deduced to be 2.1 kb or less in size; adjacent to this region is a 0.5 kb segment which may be required for stable inheritance of the plasmid. The copy number of several derivatives of pIJ101 in S. lividans 66 was between 40 and 300 per chromosome and appeared to vary with the age or physiological state of the culture. pIJ101 derivatives have a wide host range within the genus Streptomyces: 13 out of 18 strains, of diverse species, were successfully transformed. Knowledge of dispensable DNA segments and the availability of restriction sites for the insertion of DNA, deduced from the properties of plasmids carrying the E. coli plasmid pACYC184 introduced at various sites, was used in the construction of several derivatives of pIJ101 suitable as DNA cloning vectors. These were mostly designed to be non-conjugative and to carry pairs of resistance genes for selection. They include a bifunctional shuttle vector for E. coli and Streptomyces; a Streptomyces viomycin resistance gene of this plasmid is expressed in both hosts.

Chromosome Deletion↗

Simian virus 40 revertant enhancers exhibit restricted host ranges for enhancer function.

We have assayed the cell-specific activity of a matched set of four enhancers found in viral revertants derived from simian virus 40 (SV40) enhancer mutants. These enhancers all contain 71-base-pair duplications that span identical regions or, in one case, the same region shifted by 2 nucleotides. The four enhancers differ, however, in that each one either carries a different wild-type pair of the genetically defined SV40 enhancer A, B, or C elements, with the other two elements mutated, or carries all three elements mutated. The three enhancers carrying two copies of a wild-type element effectively enhance transcription in CV-1 and HeLa cells, but only the enhancer containing a duplicated wild-type C element exhibits activity in NIH 3T3 cells. These results show that the ability of the A, B, and C elements to compensate for one another is cell specific and that selection for enhancer function in one cell type can generate enhancers with different cell-specific activities. These results are consistent with the hypothesis that tandem duplication of multiple distinct enhancer elements, as in wild-type strains of SV40 (e.g., the 72-base-pair repeat), has the property of expanding the host range of an enhancer.

Animals↗

Host range of small-ruminant lentivirus cytopathic variants determined with a selectable caprine arthritis- encephalitis virus pseudotype system.

The small-ruminant lentiviruses ovine maedi-visna virus (MVV) and caprine arthritis-encephalitis virus (CAEV) cause encephalitis, progressive pneumonia, arthritis, and mastitis in sheep and goats. Icelandic MVV strains, which are lytic in tissue culture, have a wide species distribution of functional receptors, which includes human cells. In contrast, functional receptors for the nonlytic CAEV CO are absent from human cells. To determine if the wide species distribution of functional receptors is a common property of MVV strains or related to cytopathic phenotype, we tested the infectivity of viruses pseudotyped with the envelope glycoproteins of MVV K1514, CAEV CO, and lytic and nonlytic North American MVV strains to cells of different species. Replication-defective CAEV proviral constructs lacking the env, tat, and vif genes and carrying the neomycin phosphotransferase gene in the vif-tat region were developed for the infectivity assays. Cotransfection of human 293T cells with these proviral constructs and plasmids expressing CAEV, MVV, or vesicular stomatitis virus envelope glycoproteins produced infectious pseudotyped virus which induced resistance of infected cells to G418. Using these pseudotypes, we confirmed the wide species distribution of Icelandic MVV receptors and the narrow host range of CAEV. However, functional receptors for the two North American MVV strains tested, unlike the Icelandic MVV and similar to CAEV, were limited to cells of ruminant species, regardless of cytopathic phenotype. The results indicate a differential receptor recognition by MVV strains which is unrelated to cytopathic phenotype.

Amino Acid Sequence↗

African swine fever virus multigene family 360 and 530 genes are novel macrophage host range determinants.

Pathogenic African swine fever virus (ASFV) isolates primarily target cells of the mononuclear-phagocytic system in infected swine and replicate efficiently in primary macrophage cell cultures in vitro. ASFVs can, however, be adapted to grow in monkey cell lines. Characterization of two cell culture-adapted viruses, MS16 and BA71V, revealed that neither virus replicated in macrophage cell cultures. Cell viability experiments and ultrastructural analysis showed that infection with these viruses resulted in early macrophage cell death, which occurred prior to viral progeny production. Genomic cosmid clones from pathogenic ASFV isolate E70 were used in marker rescue experiments to identify sequences capable of restoring MS16 and BA71V growth in macrophage cell cultures. A cosmid clone representing a 38-kbp region at the left terminus of the genome completely restored the growth of both viruses. In subsequent fine-mapping experiments, an 11-kbp subclone from this region was sufficient for complete rescue of BA71V growth. Sequence analysis indicated that both MS16 and BA71V had significant deletions in the region containing members of multigene family 360 (MGF 360) and MGF530. Deletion of this same region from highly pathogenic ASFV isolate Pr4 significantly reduced viral growth in macrophage cell cultures. These findings indicate that ASFV MGF360 and MGF530 genes perform an essential macrophage host range function(s) that involves promotion of infected-cell survival.

African Swine Fever Virus↗