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Mutation to Wider Virulence in Puccinia graminis f. sp. tritici: Evidence for the Existence of Loci Which Allow the Fungus To Overcome Several Host Stem Rust Resistance Genes Simultaneously.

Mutants of Puccinia graminis f. sp. tritici were obtained which were able to overcome simultaneously several host stem rust resistance (Sr) genes effective against the wild-type culture. These results suggest that, in addition to those Psr loci which relate specifically to host Sr genes in a "gene for gene" manner, one or more general loci may be present in this pathogen. The product(s) of these general genes may be necessary for the expression of various host Sr genes. The evolution of a super race capable of overcoming many Sr genes for resistance seems likely, as such a pathogen would not have to give up the many proteins predicted by the gene-for-gene relationship. Moreover, it appears that specificity in the wheat rust system is more complicated than suggested by the gene-for-gene concept.

Journal Article↗

Isolation of virus from a SARS patient and genome-wide analysis of genetic mutations related to pathogenesis and epidemiology from 47 SARS-CoV isolates.

Severe acute respiratory syndrome (SARS) caused by SARS-associated coronavirus (SARS-CoV) is a fatal disease. Prevention of future outbreaks is essential and requires understanding pathogenesis and evolution of the virus. We have isolated a SARS-CoV in China and analyzed 47 SARS-CoV genomes with the aims to reveal the evolution trends of the virus and provide insights into understanding pathogenesis and SARS epidemic. Specimen from a SARS patient was inoculated into cell culture. The presence of SARS-CoV was determined by RT-PCR and confirmed by electron microscopy. Virus was isolated followed by the determination of its genome sequences, which were then analyzed by comparing with other 46 SARS-CoV genomes. Genetic mutations with potential implications to pathogenesis and the epidemic were characterized. This viral genome consists of 29,728 nucleotides with overall organization in agreement with that of published isolates. A total of 348 positions were mutated on 47 viral genomes. Among them 22 had mutations in more than three genomes. Hot spots of nucleotide variations and unique trends of mutations were identified on the viral genomes. Mutation rates were different from gene to gene and were correlated well with periodical or geographic characteristics of the epidemic.

Adolescent↗

Defending against parasites: fungus-growing ants combine specialized behaviours and microbial symbionts to protect their fungus gardens.

Parasites influence host biology and population structure, and thus shape the evolution of their hosts. Parasites often accelerate the evolution of host defences, including direct defences such as evasion and sanitation and indirect defences such as the management of beneficial microbes that aid in the suppression or removal of pathogens. Fungus-growing ants are doubly burdened by parasites, needing to protect their crops as well as themselves from infection. We show that parasite removal from fungus gardens is more complex than previously realized. In response to infection of their fungal gardens by a specialized virulent parasite, ants gather and compress parasitic spores and hyphae in their infrabuccal pockets, then deposit the resulting pellet in piles near their gardens. We reveal that the ants' infrabuccal pocket functions as a specialized sterilization device, killing spores of the garden parasite Escovopsis. This is apparently achieved through a symbiotic association with actinomycetous bacteria in the infrabuccal pocket that produce antibiotics which inhibit Escovopsis. The use of the infrabuccal pocket as a receptacle to sequester Escovopsis, and as a location for antibiotic administration by the ants' bacterial mutualist, illustrates how the combination of behaviour and microbial symbionts can be a successful defence strategy for hosts.

Actinobacteria↗

Functional and genetic plasticities of the poliovirus genome: quasi-infectious RNAs modified in the 5'-untranslated region yield a variety of pseudorevertants.

Poliovirus RNA species with nucleotides 564 to 571 deleted or with a secondary structure domain (positions 564 to 629) replaced by a shorter irregular oligonucleotide have been engineered previously; these RNAs have been considered quasi-infectious (yielding a single late revertant plaque) and dead, respectively (E. Pilipenko, A. Gmyl, Y. Svitkin, S. Maslova, A. Sinyakov, and V. Agol, Cell 68:119-131, 1992). By using large amounts of these RNAs for transfections, revertant clones with a great variety of genetic changes (point mutations, insertions of foreign sequences, short or extended deletions) were isolated. The pattern of these changes supported the notion that an appropriately spaced oligopyrimidine-AUG tandem is important for efficient poliovirus RNA translation. Structural features within and around this tandem modulated the initiation efficiency. The functional and genetic plasticities of the poliovirus genome are briefly discussed.

Animals↗

The structure, biosynthesis and functions of glycosylphosphatidylinositol anchors, and the contributions of trypanosome research.

The discovery of glycosylphosphatidylinositol (GPI) membrane anchors has had a significant impact on several areas of eukaryote cell biology. Studies of the African trypanosome, which expresses a dense surface coat of GPI-anchored variant surface glycoprotein, have played important roles in establishing the general structure of GPI membrane anchors and in delineating the pathway of GPI biosynthesis. The major cell-surface molecules of related parasites are also rich in GPI-anchored glycoproteins and/or GPI-related glycophospholipids, and differences in substrate specificity between enzymes of trypanosomal and mammalian GPI biosynthesis may have potential for the development of anti-parasite therapies. Apart from providing stable membrane anchorage, GPI anchors have been implicated in the sequestration of GPI-anchored proteins into specialised membrane microdomains, known as lipid rafts, and in signal transduction events.

Animals↗

Detection of a unique genotype of monkey B virus (Cercopithecine herpesvirus 1) indigenous to native Japanese macaques (Macaca fuscata).

The Japanese macaque or snow monkey (Macaca fuscata) is an autochthonous monkey in Japan. It has long been assumed that the monkey population was not infected with Cercopithecine herpesvirus 1 (monkey B virus [BV]) since cases of human BV infection have never been reported in Japan. Although serologic testing of captive snow monkeys in Japan revealed antibodies to BV, it was thought that native Japanese macaques had either been infected with herpes simplex virus from humans or with BV from other imported macaque species. To clarify this issue, we performed polymerase chain reaction (PCR) analysis to amplify BV sequences from trigeminal ganglia of 30 Japanese macaque monkeys that were seropositive for BV. Sequences from two BV genes, UL27 (360 bp) and UL19 (1.0 Kbp), from 3 of 30 monkeys were amplified. Results of restriction fragment length polymorphism analysis and DNA sequencing of the fragments provided evidence that native Japanese macaques are infected with BV. Phylogenetic analysis indicated that these monkeys harbor their own genotype of BV that is different from other known BV genotypes, and provided additional evidence supporting the co-evolution of BV and macaques.

Animals↗

[Recombinant analysis of classical swine fever virus].

To study the possible recombinant relationship among differently derived classical swine fever virus, the coding regions of 21 isolates were analyzed to detect recombination and breakpoints through gene trees comparison and quartet analyses. The results show nucleotide area corresponding to E0, E1 and E2 as a possible recombinant tract between ALD ( D49532) and GPE-(D49533) while NS5A-NS5B of the isolate 39 (AF407339) appears to be derived from a virulent Shimen strain (AF092448) sequence. This suggests that intertypic exchanges of genetic materials during mixed infections under natural or laboratorial conditions can lead classical swine fever virus to adapt to the changes of host environment.

Classical Swine Fever Virus↗

CadC is the preferential target of a convergent evolution driving enteroinvasive Escherichia coli toward a lysine decarboxylase-defective phenotype.

Enteroinvasive E. coli (EIEC), like Shigella, is the etiological agent of bacillary dysentery, a particularly severe syndrome in children in developing countries. All EIEC strains share with Shigella the inability to synthesize lysine decarboxylase (the LDC phenotype). The lack of this function is considered a pathoadaptive mutation whose emergence was necessary to obtain the full expression of invasiveness. Cadaverine, the product of lysine decarboxylation, is a small polyamine which interferes mainly with the inflammatory process induced by dysenteric bacteria. Genes coding for lysine decarboxylase and its transporter constitute a single operon (cadBA) and are expressed at low pH under the positive control of CadC. This regulator is an inner membrane protein that is able to sense pH variation and to respond by transcriptionally activating the cadBA genes. In this study we show that, unlike in Shigella, mutations affecting the cad locus in the EIEC strains we have analyzed are not followed by a novel gene arrangement and that the LCD(-) phenotype is dependent mainly on inactivation of the cadC gene. Introduction of a functional CadC restores cadaverine expression in all EIEC strains harboring either an IS2 element or a defective cadC promoter. Comparative analysis between the cad regions of S. flexneri and EIEC suggests that the LDC(-) phenotype has been attained by different strategies within the E. coli species.

Base Sequence↗

Description of a novel intimin variant (type zeta) in the bovine O84:NM verotoxin-producing Escherichia coli strain 537/89 and the diagnostic value of intimin typing.

Infections with verotoxin-producing Escherichia coli (VTEC) has resulted in increasing numbers of human illnesses annually. These illnesses usually result from the ability of VTEC to cause the attaching and effacing lesions (AE lesion). The AE phenotype is encoded by the locus of enterocyte effacement (LEE) pathogenicity island. A key adhesion factor involved is the outer membrane protein intimin, encoded by the eae gene within the LEE. Intimin types alpha, beta, gamma, delta, and epsilon have been described previously. Each intimin represents distinct phylogenetic lineages of LEE-positive strains. A new intimin type zeta was identified in a VTEC strain of the serotype O84:NM (nonmotile) that was isolated from a calf with diarrhea. zeta intimin showed the highest similarity (88%) of its amino acid sequence to the alpha intimin. For diagnostic purposes, we established a polymerase chain reaction (PCR) method for diagnosis of the key virulence traits of VTEC (i.e., verotoxins and intimins). This method also distinguishes between the toxins (VT1 and VT2) and the six intimin types. By applying the PCR method, intimin zeta in strains of other VTEC serotypes O84:H2, O92:NM, O119:H25, and O150:NM was identified. Because the intimin types represent distinctive phylogenetic E. coli lineages, application of the intimin subtyping PCR offers significant benefits. These include improving diagnosis of VTEC infection and increasing the understanding of evolution of attaching and effacing VTEC and other LEE-positive bacteria.

Adhesins, Bacterial↗

[Comparison of a bivalent anti-foot-and-mouth disease vaccine with oil adjuvant to a vaccine with DEAE-dextran adjuvant in swine].

The report compares the compatibility, efficacity and serology response of two bivalent anti-foot-and-mouth disease vaccines (OC). One is oil-adjuvanted and the other based on diethyl-amino-ethyl-dextran (DEAE-Dextran). After vaccination no general clinical or local reactions are normally observed. Tissular reactions at the inoculation site are more severe with the oil-adjuvanted vaccine than with the DEAE-Dextran vaccine. Meat inspection, about three months after injection of the vaccine, showed the lesions to be in regression and negligible. The control of activity has been performed by the virulence test. For the oil-adjuvanted vaccine, protection against O1-Lausanne, 5, 21 and 90 days after vaccination is 90%, 80-100% and 55% respectively. For the DEAE-Dextran vaccine, these levels of protection are 80% after 5 days and 20-70% after 21 days. The percentage of swine protected against C-Noville 35 days after vaccination is 100% for the oil-adjuvanted vaccine and 70% for the DEAE-Dextran vaccine. The evolution of the amount of serum antibodies was followed for three months by the method of seroneutralization on cell cultures.

Adjuvants, Immunologic↗

Amplification of 'variola virus-specific' sequences in German cowpox virus isolates.

In 1995 a polymerase chain reaction (PCR) protocol describing the specific detection of variola virus, the causative agent of smallpox, was published by Knight and others. Virulent variola major strains could be differentiated from less virulent variola minor strains because of the distinct amplicon sizes. Here, we applied this PCR protocol to DNA from various orthopoxvirus isolates. There was no amplification with the orthopoxvirus species vaccinia, monkeypox, mousepox, or camelpox viruses. However, amplification was observed in six out of 15 cowpox virus strains investigated. The size of the amplicons corresponded exactly with the size described for variola minor strains and the nucleotide sequence identity accounted for 97%. Findings are discussed with respect to the evolution of orthopoxvirus species assuming that variola virus most probably stems from a rodent-transmitted cowpox virus-like progenitor.

Animals↗

Genetic and molecular characterization of a dental pathogen using genome-wide approaches.

Actinobacillus actinomycetemcomitans causes periodontitis, a costly chronic infection that affects a large number of patients. The pathogenesis of this dental infection is a multifactorial process that results in a serious degenerative disease of the periodontium. Although significant progress has been achieved after the identification of this Gram-negative bacterium as the etiological agent of this infection, much remains to be done to understand in detail the bacterial factors and host-pathogen interactions involved in the pathogenesis of this disease. Classic research approaches have resulted in the identification of important virulence factors and cellular processes, although they have provided a rather narrow picture of some of the steps of this complex process. In contrast, a much wider picture could be obtained with the application of tools such as bioinformatics and genomics. These tools will provide global information regarding the differential expression of genes encoding factors and processes that lead to the pathogenesis of this disease. Furthermore, comparative genomics has the potential of helping us to understand the emergence and evolution of this human pathogen. This genome-wide approach should provide a more complete picture of the pathogenesis process of this disease, and will facilitate the development of efficient diagnostic, preventive, and therapeutic measures for this disease.

Actinobacillus Infections↗

Asymmetry in host and parasitoid diffuse coevolution: when the red queen has to keep a finger in more than one pie.

BACKGROUND: Coevolution between pairs of antagonistic species is generally considered an endless "arms race" between attack and defense traits to counteract the adaptive responses of the other species. PRESENTATION OF THE HYPOTHESIS: When more than two species are involved, diffuse coevolution of hosts and parasitoids could be asymmetric because consumers can choose their prey whereas preys do not choose their predator. This asymmetry may lead to differences in the rate of evolution of the antagonistic species in response to selection. The more long-standing the coevolution of a given pair of antagonistic populations, the higher should be the fitness advantage for the consumer. Therefore, the main prediction of the hypothesis is that the consumer trophic level is more likely to win the coevolution race. TESTING THE HYPOTHESIS: We propose testing the asymmetry hypothesis by focusing on the tritrophic system plant/aphid/aphid parasitoid. The analysis of the genetic variability in the virulence of several parasitoid populations and in the defenses of several aphid species or several clones of the same aphid species could be compared. Moreover, the analysis of the neutral population genetic structure of the parasitoid as a function of the aphid host, the plant host and geographic isolation may complement the detection of differences between host and parasitoid trophic specialization. IMPLICATIONS OF THE HYPOTHESIS: Genetic structures induced by the arms race between antagonistic species may be disturbed by asymmetry in coevolution, producing neither rare genotype advantages nor coevolutionary hotspots. Thus this hypothesis profoundly changes our understanding of coevolution and may have important implications in terms of pest management.

Journal Article↗

Toxin genes on pathogenicity islands: impact for microbial evolution.

Toxin-specific genes are often located on mobile genetic elements such as phages, plasmids and pathogenicity islands (PAIs). The uropathogenic E. coli strain 536 carries two alpha-hemolysin gene clusters, which are part of the pathogenicity islands I536 and II536, respectively. Using different genetic techniques, two additional PAIs were identified in the genome of the E. coli strain 536, and it is likely that further PAIs are located on the genome of this strain. Pathogenicity islands are often associated with tRNA genes. In the case of the E. coli strain 536, the PAI-associated tRNA gene leuX, which encodes a minor leucyl-tRNA, affects the expression of various virulence traits including alpha-hemolysin production. The exact mode of action of the tRNA5Leu-dependent gene expression has to be identified in the future.

Bacterial Toxins↗

Sabin type 2 polioviruses with intertypic vaccine/vaccine recombinant genomes.

Attenuated strains of the Sabin oral poliovirus vaccine replicate in the human gut and, in rare cases, cause vaccine-associated paralytic poliomyelitis. In the present study, 15 vaccine-derived strains isolated from patients with vaccine-associated paralytic poliomyelitis and from healthy vaccinees were examined. Four distant sequences of the poliovirus genome (5' NCR, VP3/VP1, VP1/2A, and 3DPol/3' NCR) were targeted, and the reverse-transcribed segments were amplified by polymerase chain reaction followed by restriction fragment length polymorphism analysis with four restriction enzymes. Among the 15 isolates (11 Sabin type 2, 3 Sabin type 1, and 1 Sabin type 3), four Sabin type 2 isolates (36%) were found to be intertypic vaccine/vaccine recombinant in the 3DPol/3' NCR region of the viral genome. The recombinant genotypes identified were S2/S2/S1 for two isolates and S2/S2/S2/S3 and S2/S2/S1/S2 for each of the other two isolates, respectively. Recombinant viruses with unmodified segments in the 5' NCR and the VP3/VP1 regions of the viral genome, a modified segment in the VPI/2A region only for one strain, and an often recombinant segment in the 3DPol/3' NCR parts of the genome were so identified. These findings provide strong evidence that recombination is a frequent phenomenon in type 2 poliovirus vaccine strains and suggest that recombination may be an important mechanism of the natural evolution of polioviruses of Sabin type 2 origin, perhaps even one of the mechanisms of reversion of attenuated vaccine strains toward neurovirulence.

Base Sequence↗

The avian influenza virus nucleoprotein gene and a specific constellation of avian and human virus polymerase genes each specify attenuation of avian-human influenza A/Pintail/79 reassortant viruses for monkeys.

Reassortant viruses which possessed the hemagglutinin and neuraminidase genes of wild-type human influenza A viruses and the remaining six RNA segments (internal genes) of the avian A/Pintail/Alberta/119/79 (H4N6) virus were previously found to be attenuated in humans. To study the genetic basis of this attenuation, we isolated influenza A/Pintail/79 X A/Washington/897/80 reassortant viruses which contained human influenza virus H3N2 surface glycoprotein genes and various combinations of avian or human influenza virus internal genes. Twenty-four reassortant viruses were isolated and first evaluated for infectivity in avian (primary chick kidney [PCK]) and mammalian (Madin-Darby canine kidney [MDCK]) tissue culture lines. Reassortant viruses with two specific constellations of viral polymerase genes exhibited a significant host range restriction of replication in mammalian (MDCK) tissue culture compared with that in avian (PCK) tissue culture. The viral polymerase genotype PB2-avian (A) virus, PB1-A virus, and PA-human (H) virus was associated with a 900-fold restriction, while the viral polymerase genotype PB2-H, PB1-A, and PA-H was associated with an 80,000-fold restriction of replication in MDCK compared with that in PCK. Fifteen reassortant viruses were subsequently evaluated for their level of replication in the respiratory tract of squirrel monkeys, and two genetic determinants of attenuation were identified. First, reassortant viruses which possessed the avian influenza virus nucleoprotein gene were as restricted in replication as a virus which possessed all six internal genes of the avian influenza A virus parent, indicating that the nucleoprotein gene is the major determinant of attenuation of avian-human A/Pintail/79 reassortant viruses for monkeys. Second, reassortant viruses which possessed the viral polymerase gene constellation of PB2-H, PB1-A, and PA-H, which was associated with the greater degree of host range restriction in vitro, were highly restricted in replication in monkeys. Since the avian-human influenza reassortant viruses which expressed either mode of attenuation in monkeys replicated to high titer in eggs and in PCK tissue culture, their failure to replicate efficiently in the respiratory epithelium of primates must be due to the failure of viral factors to interact with primate host cell factors. The implications of these findings for the development of live-virus vaccines and for the evolution of influenza A viruses in nature are discussed.

Animals↗

Interactions between octopine and nopaline plasmids in Agrobacterium tumefaciens.

Transfer of octopine Ti plasmids to strains already carrying an octopine Ti plasmid was found to occur at the same (high) frequency as transfer to Ti plasmid lacking recipients, showing that resident Ti plasmids do not exhibit entry exclusion towards incoming Ti plasmids. The resident octopine Ti plasmid was lost by the recipient after the entrance of the incoming Ti plasmid, which is indicative of the incompatibility between the Ti plasmids. Octopine Ti plasmids were found to become established only infrequently in recipients with a nopaline Ti plasmid and, vice versa, nopaline Ti plasmids were only rarely established in recipients with an octopine Ti plasmid. Rare clones in which the incoming octopine (nopaline) Ti plasmid had been established despite the presence of a nopaline (octopine) Ti plasmid appeared to harbor cointegrates consisting of the entire incoming Ti plasmid and the entire resident Ti plasmid. The integration event invariably had occurred in a region of the plasmids that is highly conserved in evolution and that is essential for oncogenicity. These results show that octopine and nopaline Ti plasmids cannot be maintained as separate replicons by one and the same cell. Therefore, be definition, these plasmids belong to the same incompatibility group, which has been names inc Rh-1. Agrobacterial non-Ti octopine and nopaline plasmids were found to belong to another incompatibility group. The tumorigenic properties of strains harboring two different Ti plasmids, in a cointegrate structure, were indicative of the virulence genes of both of them being expressed. The agrobacterial non-Ti octopine and nopaline plasmids did not influence the virulence properties encoded by the Ti plasmid.

Arginine↗

Selective targeting of indel-inferred differences in spatial structures of highly homologous proteins.

Recent findings have shown that the protein elongation factor-1alpha (EF-1alpha) from the eukaryotic pathogen Leishmania donovani possesses virulence properties. This was unexpected, since it has greater than 80% sequence identity with its human homologue. Given that EF-1alpha is essential for cell survival, in principle, it can be considered an attractive drug target. However, the challenge is to be able to selectively target the protein so as not to affect function of the human homologue. While a limited number of discrete differences were scattered throughout the sequence, most of the difference between these 2 homologues could be attributed to a 12-amino acid insert present in human EF-1alpha and absent from the leishmania sequence. In the present study, we modeled the spatial differences in structures of human and L. donovani EF-1alpha's inferred by this insertion-deletion (or "indel"). The protein models were used to develop antibodies directed specifically toward the deletion region of the pathogen protein. The strategy described allowed successful selective targeting of this putative leishmania virulence factor while avoiding recognition of the highly similar human EF-1alpha homologue. These findings may establish a new strategy for the development of antagonists directed against certain pathogenic targets having close human homologues.

Amino Acid Sequence↗