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Nod factor-treated Medicago truncatula roots and seeds show an increased number of nodules when inoculated with a limiting population of Sinorhizobium meliloti.

Medicago truncatula is a model legume plant that interacts symbiotically with Sinorhizobium meliloti, the alfalfa symbiont. This process involves a molecular dialogue between the bacterium and the plant. Legume roots exude flavonoids that induce the expression of a set of rhizobial genes, the nod genes, which are essential for nodulation and determination of the host range. In turn, nod genes control the synthesis of lipo-chito-oligosaccharides (LCOs), Nod factors, which are bacteria-to-plant signal molecules mediating recognition and nodule organogenesis. M. truncatula roots or seeds have been treated with Nod factors and hydroponically growing seedlings have been inoculated with a limiting population of S. meliloti. It has been shown that submicromolar concentrations of Nod factors increase the number of nodules per plant on M. truncatula. Compared with roots, this increase is more noticeable when seeds are treated. M. truncatula seeds are receptive to submicromolar concentrations of Nod factors, suggesting the possibility of a high affinity LCO perception system in seeds or embryos as well.

Medicago truncatula↗

Canine parvovirus: relationship to wild-type and vaccine strains of feline panleukopenia virus and mink enteritis virus.

Canine parvovirus (CPV), feline panleukopenia virus (FPLV) and mink enteritis virus (MEV) were compared serologically, by determination of their host range in cell cultures, as well as by restriction enzyme analysis. Maps of the virus genomes were established using seven different restriction enzymes cutting at a total of 56 sites. MEV and FPLV gave maps which were identical except for one restriction site. The map of CPV is closely related to those of FPLV/MEV since their DNAs share about 80% of the restriction sites tested. However, CPV is clearly distinct from FPLV/MEV since either eight (German isolate) or nine (Belgian, Swiss and American isolates) restriction sites are different. The DNAs of six vaccine strains of FPLV and MEV were also analysed. They gave maps which closely resembled those of the respective wild-type strains. CPV and FPLV/MEV also differed with respect to antigenicity, as well as to host range in cell cultures.

Animals↗

The life cycle of Henneguya nuesslini Schuberg & Schroder, 1905 (Myxozoa) involves a triactinomyxon-type actinospore.

The life cycle of the histozoic myxozoan parasite Henneguya nuesslini was investigated in two salmonid host species. Naive brown trout, Salmo trutta, and brook trout, Salvelinus fontinalis, were experimentally infected in two trials by triactinomyxon type actinospores from naturally infected Tubifex tubifex. In exposed common carp, Cyprinus carpio, no myxospore production was detected. The parasite formed cysts with mature myxospores in the connective tissue of the fish 102 days post-exposure. The morphology of both actinosporean and myxosporean stages was described by light microscopy and a 1417-bp fragment of the 18S rDNA gene was sequenced. Sequence analysis confirmed the absolute congruence of the two developmental stages and assisted in determining species identity. Host range, tissue specificity and myxospore measurements provided sufficiently distinctive features to confirm species validity and were thus crucial for identification. The triactinomyxon spores had 16 secondary germ cells, unique dimensions, a very opaque sporoplasm matrix and three conspicuously protruding, pyriform polar capsules. This is the first record of a Henneguya sp. life cycle with a triactinomyxon-type actinospore, which suggests a close relationship with the Myxobolus group and a polyphyletic origin of the genus Henneguya.

Animals↗

Multiplicity of delta-endotoxin genes with different insecticidal specificities in Bacillus thuringiensis aizawai 7.29.

The hypothesis according to which multiple and different delta-endotoxin genes could determine the host-range specificity of the lepidopteran strains of Bacillus thuringiensis is being checked in the case of strains aizawai 7.29 (serotype 7) and entomocidus 601 (serotype 6). From these strains, several crystal protein genes, belonging to different structural types, have been isolated. One of the cloned genes that is not present in strain entomocidus 601 is duplicated in strain aizawai 7.29. This gene belongs to a previously characterized type of crystal protein gene and encodes a protein preferentially active against Pieris brassicae. Two other genes, of presumed chromosomal location, are present in both strains and each displays a unique physical map. In both strains the two genes are in close proximity and in the same orientation. The first, which belongs to a new type of crystal protein gene, encodes a 130-140 kD protein that is not significantly active against the two insect species tested. The other new type of crystal protein gene directs the synthesis of a polypeptide preferentially active against Spodoptera littoralis.

Animals↗

Effect of Lactose Concentration on the Efficiency of Plating of Bacteriophages on Streptococcus cremoris.

The efficiency of plating of phages derived by ultraviolet induction of, or by lytic growth on, certain strains of Streptococcus cremoris was found to vary by as much as 10 depending on the lactose concentration of the medium in which the indicator bacteria were grown and the length of time the stationary-phase indicator cultures were aged. This effect was noted only when the culture was used as an indicator for phages that had previously grown on an apparently unrelated strain of bacteria. Conditions of culturing and aging had no detectable effect upon the ability of a strain to serve as an indicator for phage that had previously been cultured on the same strain. These observations suggest the presence of some kind of physiologically labile restriction system in strains of S. cremoris. The implications of this finding for increasing the sensitivity of the host range test in determining phage susceptibility, whether from induced lysates, whey, or lytic phage stocks, are discussed. It is recommended that, for all such testing, the concentration of lactose in buffered media be increased to such levels as required to obtain a final pH similar to that of a freshly coagulated milk culture, namely, below pH 5.0.

Journal Article↗

Cin-mediated recombination at secondary crossover sites on the Escherichia coli chromosome.

The Cin recombinase is known to mediate DNA inversion between two wild-type cix sites flanking genetic determinants for the host range of bacteriophage P1. Cin can also act with low frequency at secondary (or quasi) sites (designated cixQ) that have lower homology to either wild-type site. An inversion tester sequence able to reveal novel operon fusions was integrated into the Escherichia coli chromosome, and the Cin recombinase was provided in trans. Among a total of 13 Cin-mediated inversions studied, three different cixQ sites had been used. In two rearranged chromosomes, the breakpoints of the inversions were mapped to cixQ sites in supB and ompA, representing inversions of 109 and 210 kb, respectively. In the third case, a 2.1-kb inversion was identified at a cixQ site within the integrated sequences. This derivative itself was a substrate for a second inversion of 1.5 kb between the remaining wild-type cix and still another cixQ site, thus resembling a reversion. In analogy to that which is known from DNA inversion on plasmids, homology of secondary cix sites to wild-type recombination sites is not a strict requirement for inversion to occur on the chromosome. The chromosomal rearrangements which resulted from these Cin-mediated inversions were quite stable and suffered no growth disadvantage compared with the noninverted parental strain. The mechanistic implications and evolutionary relevance of these findings are discussed.

Bacterial Outer Membrane Proteins↗

Host restriction of Salmonella enterica serotype Typhimurium pigeon isolates does not correlate with loss of discrete genes.

The definitive phage types (DT) 2 and 99 of Salmonella enterica serotype Typhimurium are epidemiologically correlated with a host range restricted to pigeons, in contrast to phage types with broader host ranges such as epidemic cattle isolates (DT104 and DT204). To determine whether phage types with broad host range possess genetic islands absent from host-restricted phage types, we compared the genomes of four pigeon isolates to serotype Typhimurium strain LT2 using a DNA microarray. Three of the four isolates tested caused fluid accumulation in bovine ligated ileal loops, but they had reduced colonization of liver and spleen in susceptible BALB/c mice and were defective for intestinal persistence in Salmonella-resistant CBA mice. The genomes of the DT99 and DT2 isolates were extremely similar to the LT2 genome, with few notable differences on the level of complete individual genes. Two large groups of genes representing the Fels-1 and Fels-2 prophages were missing from the DT2 and DT99 phage types we analyzed. One of the DT99 isolates examined was lacking a third cluster of five chromosomal genes (STM1555 to -1559). Results of the microarray analysis were extended using Southern analysis to a collection of 75 serotype Typhimurium clinical isolates of 24 different phage types. This analysis revealed no correlation between the presence of Fels-1, Fels-2, or STM1555 to -1559 and the association of phage types with different host reservoirs. We conclude that serotype Typhimurium phage types with broad host range do not possess genetic islands influencing host restriction, which are absent from the host-restricted pigeon isolates.

Animals↗

Diverse CD81 proteins support hepatitis C virus infection.

Hepatitis C virus (HCV) entry is dependent on CD81. To investigate whether the CD81 sequence is a determinant of HCV host range, we expressed a panel of diverse CD81 proteins and tested their ability to interact with HCV. CD81 large extracellular loop (LEL) sequences were expressed as recombinant proteins; the human and, to a low level, the African green monkey sequences bound soluble HCV E2 (sE2) and inhibited infection by retrovirus pseudotype particles bearing HCV glycoproteins (HCVpp). In contrast, mouse or rat CD81 proteins failed to bind sE2 or to inhibit HCVpp infection. However, CD81 proteins from all species, when expressed in HepG2 cells, conferred susceptibility to infection by HCVpp and cell culture-grown HCV to various levels, with the rat sequence being the least efficient. Recombinant human CD81 LEL inhibited HCVpp infectivity only if present during the virus-cell incubation, consistent with a role for CD81 after virus attachment. Amino acid changes that abrogate sE2 binding (I182F, N184Y, and F186S, alone or in combination) were introduced into human CD81. All three amino acid changes in human CD81 resulted in a molecule that still supported HCVpp infection, albeit with reduced efficiency. In summary, there is a remarkable plasticity in the range of CD81 sequences that can support HCV entry, suggesting that CD81 polymorphism may contribute to, but alone does not define, the HCV susceptibility of a species. In addition, the capacity to support viral entry is only partially reflected by assays measuring sE2 interaction with recombinant or full-length CD81 proteins.

Amino Acid Sequence↗

Activator-dependent and activator-independent defective recombinant retroviruses from bovine leukemia virus.

The replication-competent bovine leukemia virus (BLV) has been modified for use as a vector for foreign genes. The gag, pol, env, and pX regions of the virus were replaced by an exogenous nuclear location signal LacZ (nlsLacZ) or SVnlsLacZ gene. Transfection of the ovine cell line FLK-BLV, which expresses all BLV proteins from a wild-type provirus, with this viral DNA resulted in a viral titer of 10(4) CFU/ml. The inclusion of a large portion of the gag region did not significantly increase the titer. Both activator-dependent and activator-independent retroviruses were constructed. In activator-dependent vectors, the expression of the insert was dependent on the presence of the Tax protein, which activated the BLV long terminal repeat. In activator-independent vectors, the expression of the insert was constitutive because of the presence of an internal promoter. Infections with the recombinant retrovirus were inhibited by specific neutralizing antibodies. The structure of the transduced genetic material was not rearranged. BLV vectors encoding a reporter nlsLacZ gene, the product of which can be detected in single cells, greatly simplified studies of their biological properties. Determination of the host range of BLV vectors established that BLV-based recombinant retroviruses are effective in the transduction of genes in a variety of species and cell types.

Animals↗

The HeLa cell receptor for enterovirus 70 is decay-accelerating factor (CD55).

Enterovirus 70 (EV70) is a recently emerged human pathogen belonging to the family Picornaviridae. The ability of EV70 to infect a wide variety of nonprimate cell lines in vitro is unique among human enteroviruses. The importance of virus receptors as determinants of viral host range and tropism led us to study the host cell receptor for this unusual picornavirus. We produced a monoclonal antibody (MAb), EVR1, which bound to the surface of HeLa cells and protected them against infection by EV70 but not by poliovirus or by coxsackievirus B3. This antibody also inhibited the binding of [35S]EV70 to HeLa cells. MAb EVR1 did not bind to monkey kidney (LLC-MK2) cells, nor did it protect these cells against virus infection. In Western immunoassays and in immunoprecipitations, MAb EVR1 identified a HeLa cell glycoprotein of approximately 75 kDa that is attached to the cell membrane by a glycosyl-phosphatidylinositol (GPI) anchor. Decay-accelerating factor (DAF, CD55) is a 70- to 75-kDa GPI-anchored membrane protein that is involved in the regulation of complement and has also been shown to function as a receptor for several enteroviruses. MAb EVR1 bound to Chinese hamster ovary (CHO) cells constitutively expressing human DAF. Anti-DAF MAbs inhibited EV70 binding to HeLa cells and protected them against EV70 infection. Transient expression of human DAF in murine NIH 3T3 cells resulted in binding of labelled EV70 and stably, transformed NIH 3T3 cells expressing DAF were able to support virus replication. These data indicate that the HeLa cell receptor for EV70 is DAF.

3T3 Cells↗

Retargeting of coronavirus by substitution of the spike glycoprotein ectodomain: crossing the host cell species barrier.

Coronaviruses generally have a narrow host range, infecting one or just a few species. Using targeted RNA recombination, we constructed a mutant of the coronavirus mouse hepatitis virus (MHV) in which the ectodomain of the spike glycoprotein (S) was replaced with the highly divergent ectodomain of the S protein of feline infectious peritonitis virus. The resulting chimeric virus, designated fMHV, acquired the ability to infect feline cells and simultaneously lost the ability to infect murine cells in tissue culture. This reciprocal switch of species specificity strongly supports the notion that coronavirus host cell range is determined primarily at the level of interactions between the S protein and the virus receptor. The isolation of fMHV allowed the localization of the region responsible for S protein incorporation into virions to the carboxy-terminal 64 of the 1,324 residues of this protein. This establishes a basis for further definition of elements involved in virion assembly. In addition, fMHV is potentially the ideal recipient virus for carrying out reverse genetics of MHV by targeted RNA recombination, since it presents the possibility of selecting recombinants, no matter how defective, that have regained the ability to replicate in murine cells.

Amino Acid Sequence↗

Sialic acid functions in enterovirus 70 binding and infection.

The interaction of viruses with host cell receptors is the initial step in viral infection and is an important determinant of virus host range, tissue tropism, and pathogenesis. The complement regulatory protein decay-accelerating factor (DAF/CD55) is an attachment receptor for enterovirus 70 (EV70), a member of the Picornaviridae, commonly associated with an eye infection in humans known as acute hemorrhagic conjunctivitis. In early work, the EV70 receptor on erythrocytes, responsible for its hemagglutinating activity, was shown to be sensitive to neuraminidase, implying an essential role for sialic acid in virus attachment. Here, we extend these results to show that cell surface sialic acid is required for EV70 binding to nucleated cells susceptible to virus infection and that sialic acid binding is important in productive infection. Through the use of site-directed mutagenesis to eliminate the single N-linked glycosylation site of DAF and of a chimeric receptor protein in which the O-glycosylated domain of DAF was replaced by a region of the HLA-B44 molecule, a role in EV70 binding for the sialic acid residues of DAF was excluded, suggesting the existence of at least one additional, sialylated EV70-binding factor at the cell surface. Treatment of cells with metabolic inhibitors of glycosylation excluded a role for the N-linked oligosaccharides of glycoproteins but suggested that O-linked glycosylation is important for EV70 binding.

Animals↗

A multifunctional domain in human CRM1 (exportin 1) mediates RanBP3 binding and multimerization of human T-cell leukemia virus type 1 Rex protein.

Human CRM1 (hCRM1) functions in the Rex-mediated mRNA export of human T-cell leukemia virus type 1 (HTLV-1) as an export receptor and as an inducing factor for Rex multimerization on its cognate RNA. Although there are only 24 amino acid differences between hCRM1 and rat CRM1 (rCRM1), rCRM1 can hardly support Rex activity, suggesting a role for rCRM1 as a determinant restricting the host range of HTLV-1. Here, we used a series of mutants, which were generated by interchanging residues of these CRM1s, to examine the relationship of hCRM1 functions. The functions for Rex multimerization and binding to nuclear export signals are mapped to different amino acid residues, and these are separable, suggesting that CRM1 not only functions as an export receptor but also participates in the formation of the RNA export complex through higher-ordered interaction with Rex. The region for the interaction with RanBP3, comprising four residues (amino acids [aa] 411, 414, 474, and 481), and the region for Rex multimerization, including two residues (aa 411 and 414), form an overlapped domain. Our results provide the molecular basis underlying the species-specific ability of HTLV-1 to propagate in human cells.

Amino Acid Sequence↗

Molecular determinants of the virulence and infectivity of California serogroup bunyaviruses.

California bunyaviruses cause encephalitis in mammalian hosts after peripheral infection. The virulence of these viruses is determined by their ability to replicate sequentially in striated muscle, cause viremia, and invade and replicate in the central nervous system. These viruses are also able to infect vector mosquitoes following ingestion of a blood meal containing virus. Bunyaviruses are negative stranded RNA viruses with a trisegmented genome, and the large, medium, and small RNA segments encode the polymerase, the glycoproteins, and the nucleoprotein, respectively. Reassortants between virulent and avirulent virus clones have been used to map virulence determinants in mice as well as determinants of infectivity in mosquitoes. Attenuation in mice and infectivity in mosquitoes of some virus clones maps to the medium RNA segment, implying that the virus glycoproteins, which are involved in virus entry, play a role in virulence. Attenuation in mice and mosquito infectivity of other clones maps to the large RNA segment, suggesting that cell-specific differences in the function of the viral polymerase can also determine virulence and host range.

Animals↗

Phosphatidylserine treatment relieves the block to retrovirus infection of cells expressing glycosylated virus receptors.

BACKGROUND: A major determinant of retrovirus host range is the presence or absence of appropriate cell-surface receptors required for virus entry. Often orthologs of functional receptors are present in a wide range of species, but amino acid differences can render these receptors non-functional. In some cases amino acid differences result in additional N-linked glycosylation that blocks virus infection. The latter block to retrovirus infection can be overcome by treatment of cells with compounds such as tunicamycin, which prevent the addition of N-linked oligosaccharides. RESULTS: We have discovered that treatment of cells with liposomes composed of phosphatidylserine (PS) can also overcome the block to infection mediated by N-linked glycosylation. Importantly, this effect occurs without apparent change in the glycosylation state of the receptors for these viruses. This effect occurs with delayed kinetics compared to previous results showing enhancement of virus infection by PS treatment of cells expressing functional virus receptors. CONCLUSION: We have demonstrated that PS treatment can relieve the block to retrovirus infection of cells expressing retroviral receptors that have been rendered non-functional by glycosylation. These findings have important implications for the current model describing inhibition of virus entry by receptor glycosylation.

Animals↗

[Molecular mechanism of replication and transcription of the influenza virus genome and host factors].

The genome of influenza A virus is a set of eight segmented- and single-stranded RNAs. A basic transcription and replication unit is the genome complexed with viral RNA-dependent RNA polymerases and nucleoprotein (NP). For the efficient transcription and replication of the genome, not only viral factors but also host cell-derived factors are required. Although receptor and protease molecules play important roles in infection and pathogenicity, it is also possible that host factors involved in the virus genome function determine these. PB2, for instance, is reported to be a possible candidate for determination of the host range of avian influenza viruses. Here we summarize recent progresses in the molecular mechanism of the influenza virus genome transcription and replication and discuss the involvement of host factors in these processes.

Animals↗

Development of Anguillicola crassus (Dracunculoidea, Anguillicolidae) in experimentally infected Balearic congers Ariosoma balearicum (Anguilloidea, Congridae).

The development of Anguillicola crassus in experimentally infected Ariosoma balearicum (Anguilloidea, Congridae) kept in seawater was studied in the laboratory. In parallel trials the effect of water salinity on the development of larval A. crassus in European eels Anguilla anguilla was also investigated using eels kept in seawater of a salinity of 34 per thousand. Both eel species were orally inoculated with L3 larvae of A. crassus and then maintained for up to 3 mo at 18 degrees C in seawater. 110 d post infection, no adult but larval (L3 and L4) stages of A. crassus were detected in the swimbladder wall of Balearic congers, although this period of time was sufficient for the parasites to develop to the adult stage in European eel kept in seawater. The results presented suggest that the definitive host specificity of A. crassus comprises species of the family Anguillidae (i.e. the genus Anguilla), but not members of the Congridae. Theoretically however, A. balearicum might serve as a metaparatenic host. Factors determining the definitive host range of A. crassus remain to be elucidated. Water salinity does not seem to act as a factor affecting definitive host specificity once the parasite has become ingested by the eel.

Air Sacs↗

Murine sarcoma virus pseudotypes used as immunogens against viral and chemical oncogenesis.

The purpose of this study was to develop an animal system of protective immunity against oncornaviruses and to test whether such immunization had an inhibitory effect upon chemical sarcomagenesis. Several murine sarcoma virus (MSV) pseudotypes were used as immunogens and tested against themselves, against other pseudotypes, against leukemogenesis by their helper viruses, and against sarcomagenesis by 3-methylcholanthrene. Five MSV pseudotypes were obtained by rescuing complete MSV from MSV-genome carrier, nonproducer hamster tumor cells, using five different leukemia viruses as helpers. The immunogenic properties of these pseudotypes could be specified on the basis of the following observations. 1) They all induced sarcomas in newborn mice and regressing sarcoma nodules in young adult mice. After regression, most mice remained free of neoplastic disease, but some developed sarcoma or leukemia relapses. 2) They had an individual host range pattern, usually determined by the helper virus, as tested by inoculation of a constant virus dose in BALB/c, C57BL/Ka, and Swiss mice. 3) They were all immunogenic, in the sense that the first virus inoculation prevented sarcoma induction by a second challenge, either viral or cellular. 4) They were cross-reactive in vivo, one pseudotype immunizing against another, in the combinations tested. 5) They were able to immunize against leukemogenesis induced by their helper viruses. This was shown by prevention of leukemic deaths by Rauscher and Friend viruses, by a slight prolongation of survival after challenge with the Precerutti-Law leukemia virus, and by inhibition of splenomegaly by Moloney leukemia virus. In a second stage of the study, we investigated whether immunization with any of the MSV psuedotypes had an inhibitory effect upon sarcomagenesis induced by near-threshold doses of 3-methylcholanthrene. The incidence of these sarcomas was essentially the same in virus-immunized and control mice. It was concluded that immunizing procedures able to prevent sarcomagenesis when the inducer is a virus did not have any consistent preventive effect when the inducer was a chemical.

Animals↗