Horizontal Gene Transfers in Mycoplasmas (Mollicutes).

The class Mollicutes (trivial name "mycoplasma") is composed of wall-less bacteria with reduced genomes whose evolution was long thought to be only driven by gene losses. Recent evidences of massive horizontal gene transfer (HGT) within and across species provided a new frame to understand the successful adaptation of these minimal bacteria to a broad range of hosts. Mobile genetic elements are being identified in a growing number of mycoplasma species, but integrative and conjugative elements (ICEs) are emerging as pivotal in HGT. While sharing common traits with other bacterial ICEs, such as their chromosomal integration and the use of a type IV secretion system to mediate horizontal dissemination, mycoplasma ICEs (MICEs) revealed unique features: their chromosomal integration is totally random and driven by a DDE recombinase related to the Mutator-like superfamily. Mycoplasma conjugation is not restricted to ICE transmission, but also involves the transfer of large chromosomal fragments that generates progenies with mosaic genomes, nearly every position of chromosome being mobile. Mycoplasmas have thus developed efficient ways to gain access to a considerable reservoir of genetic resources distributed among a vast number of species expanding the concept of minimal cell to the broader context of flowing information.

Mycoplasma agalactiae Secretion of ß-(1→6)-Glucan, a Rare Polysaccharide in Prokaryotes, Is Governed by High-Frequency Phase Variation.

UNLABELLED: Mycoplasmas are minimal, wall-less bacteria but have retained the ability to secrete complex carbohydrate polymers that constitute a glycocalyx. In members of the Mycoplasma mycoides cluster, which are important ruminant pathogens, the glycocalyx includes both cell-attached and cell-free polysaccharides. This report explores the potential secretion of polysaccharides by M. agalactiae, another ruminant pathogen that belongs to a distant phylogenetic group. Comparative genomic analyses showed that M. agalactiae possesses all the genes required for polysaccharide secretion. Notably, a putative synthase gene (gsmA) was identified, by in silico reconstruction of the biosynthetic pathway, that could be involved in both polymerization and export of the carbohydrate polymers. M. agalactiae polysaccharides were then purified in vitro and found to be mainly cell attached, with a linear ß-(1→6)-glucopyranose structure [ß-(1→6)-glucan]. Secretion of ß-(1→6)-glucan was further shown to rely on the presence of a functional gsmA gene, whose expression is subjected to high-frequency phase variation. This event is governed by the spontaneous intraclonal variation in length of a poly(G) tract located in the gsmA coding sequence and was shown to occur in most of the M. agalactiae clinical isolates tested in this study. M. agalactiae susceptibility to serum-killing activity appeared to be dictated by ON/OFF switching of ß-(1→6)-glucan secretion, suggesting a role of this phenomenon in survival of the pathogen when it invades the host bloodstream. Finally, ß-(1→6)-glucan secretion was not restricted to M. agalactiae but was detected also in M. mycoides subsp. capri PG3(T), another pathogen of small ruminants. IMPORTANCE: Many if not all bacteria are able to secrete polysaccharides, either attached to the cell surface or exported unbound into the extracellular environment. Both types of polysaccharides can play a role in bacterium-host interactions. Mycoplasmas are no exception despite their poor overall metabolic capacity. We showed here that M. agalactiae secretes a capsular ß-(1→6)-glucopyranose thanks to a specific glycosyltransferase with synthase activity. This secretion is governed by high-frequency ON/OFF phase variation that might be crucial in mycoplasma host dissemination, as cell-attached ß-(1→6)-glucopyranose increases serum-killing susceptibility. Our results provide functional genetic data about mycoplasmal glycosyltransferases with dual functions, i.e., assembly and export of the sugar polymers across the cell membrane. Furthermore, we demonstrated that nonprotein epitopes can be subjected to surface antigenic variation in mycoplasmas. Finally, the present report contributes to unravel the role of secreted polysaccharides in the virulence and pathogenicity of these peculiar bacteria.

Volume and accreditation, but not specialty, affect quality standards in colonoscopy.

BACKGROUND: The Global Rating Scale, defined by the Joint Advisory Group for Gastrointestinal Endoscopy, requires monitoring of endoscopic performance indicators. There are known variations in colonoscopic performance, and investigation of factors causing this is needed. This study aimed to analyse the impact of endoscopist specialty and procedural volume on the quality of colonoscopy. METHODS: Data collected prospectively from a UK hospital endoscopy service between June 2007 and January 2010 were analysed. The main endpoint was the adenoma detection rate (ADR). Secondary endpoints were polyp detection rate (PDR), reported caecal intubation rate (CIR) and reported complications. Multivariable binary regression models were built to adjust for confounding patient-level and endoscopist-level variation. RESULTS: A total of 10,026 colonoscopies were included, with an overall ADR of 19.2 per cent, a CIR of 90.2 per cent and a perforation rate of 0.06 per cent. In univariable analyses, surgeons had a higher ADR and higher PDR, but lower CIR, compared with physicians. Surgeons had a significantly different case mix in terms of age, sex and indication for colonoscopy. After adjusting for this case mix in multivariable analysis, specialty was no longer a significant predictor of ADR; however, surgeons retained their higher PDR and physicians their higher CIR. Endoscopists accredited for screening and those performing more than 100 colonoscopies per year had a higher ADR. CONCLUSION: Adjusting for case mix, physicians and surgeons performed equally well in terms of ADR. Accreditation and a higher annual number of colonoscopies were more important factors in achieving quality standards.

Evolution of cell recognition by viruses.

Evolution of receptor specificity by viruses has several implications for viral pathogenesis, host range, virus-mediated gene targeting, and viral adaptation after organ transplantation and xenotransplantation, as well as for the emergence of viral diseases. Recent evidence suggests that minimal changes in viral genomes may trigger a shift in receptor usage for virus entry, even into the same cell type. A capacity to exploit alternative entry pathways may reflect the ancient evolutionary origins of viruses and a possible role as agents of horizontal gene transfers among cells.

Animal vaccination and the evolution of viral pathogens.

Despite reducing disease, vaccination rarely protects against infection and many pathogens persist within vaccinated animal populations. Circulation of viral pathogens within vaccinated populations may favour the development of vaccine resistance with implications for the evolution of virus pathogenicity and the emergence of variant viruses. The high rate of mutations during replication of ribonucleic acid (RNA) viruses is conducive to the development of escape mutants. In vaccinated cattle, unusual mutations have been found in the major antigenic site of foot and mouth disease virus, which is also involved in receptor recognition. Likewise, atypical changes have been detected in the immunodominant region of bovine respiratory syncytial virus. Large deoxyribonucleic acid (DNA) viruses are able to recombine, generating new genotypes, as shown by the potential of glycoprotein E-negative vaccine strains of bovine herpesvirus-1 to recombine with wild-type strains. Marek's disease virus is often quoted as an example of vaccine-induced change in pathogenicity. The reasons for this increase in virulence have not been elucidated and possible explanations are discussed.

Biochemical and structural studies with neutralizing antibodies raised against foot-and-mouth disease virus.

The function of a loop exposed on the aphthovirus capsid (the G-H loop of protein VP1) has been explored by combining genetic and structural studies with viral mutants. The loop displays a dual function of receptor recognition and interaction with neutralizing antibodies. Remarkably, some amino acid residues play a critical role in both such disparate functions. Therefore residues subjected to antibody pressure for variation may nevertheless maintain a role in receptor recognition for which invariance is a requirement. Evolution of FMDV in cell culture may relax the requirements at this site and allow further increase of antigenic diversification. Essential residues at one stage of virus evolution may become dispensable at another not very distant point in the evolutionary landscape. Implications for FMDV evolution and vaccine design are discussed.

A monoclonal ELISA for bovine viral diarrhoea pestivirus antigen detection in persistently infected cattle.

Detection of cattle persistently infected with bovine viral diarrhoea virus (BVDV) is crucial to controlling mucosal disease. A sandwich enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies raised against the 48-kDa glycoprotein and the 120/80-kDa protein was developed for detecting antigens in leucocytes of 3 persistently BVDV-infected calves. The test is simple, sensitive and rapid. Moreover the same ELISA was able to recognise Belgian field isolates of BVDV. These results show that the test can be applied in the field.

A specific PCR to differentiate between gE negative vaccine and wildtype bovine herpesvirus type 1 strains.

In the context of infectious bovine rhinotracheitis (IBR) control programmes using glycoprotein E (gE) deleted marker vaccines, a PCR assay was developed to allow the genotypic differentiation between wildtype bovine herpesvirus type 1 (BoHV-1) and gE negative strains. This assay is based on the PCR amplification of a 281 bp DNA fragment within the gE gene. The specificity of the amplification was confirmed by restriction endonuclease analysis and nucleotide sequencing of the PCR product. Its ability to determine the gE genotype of BoHV-1 strains was demonstrated on isolates coming from 20 experimental calves infected with four different BoHV-1 strains. This PCR assay may be a useful tool for monitoring the spread of live marker vaccine and the gE genotype of viral field isolates.

Structural and functional analysis of bovine herpesvirus 1 minor glycoproteins.

This paper focuses on the structure and functions of bovine herpesvirus 1 minor glycoproteins gH, gE, gG and gp42. It reviews the progress which has been made in their identification and characterization, in the study of their temporal expression and processing in infected cells, and finally in the understanding of their biological activities. In addition, aspects discussed include a comparison with two other alphaherpesviruses, namely herpes simplex virus and pseudorabies virus.

Population dynamics in the evolution of RNA viruses.

RNA virus quasispecies are subjected to processes of positive Darwinian selection, to a very active and continuous negative selection and to random genetic drift. The course of RNA virus evolution is often unpredictable, and recent results suggest that even highly conserved motifs, once regarded as essential for infectivity, may be rendered dispensable by singular evolutionary events. An immediate consequence of the quasispecies genetic organization of RNA viruses is a surprising ability to gain fitness once a minimal replication ability is established in a biological environment. The unique features of RNA genetics should not be underestimated since they are at the basis of the emergence of new viral diseases and of the current difficulties to control many diseases associated variable viruses.

[Quasispecies and molecular evolution of viruses]. / Cuasiespecies y evolución molecular de virus.

Ribonucleic acid (RNA) viruses evolve as complex distributions of genetically different but closely related variants termed viral quasispecies. The precise genome of a quasispecies cannot be defined, since the consensus genome is an average of many variants. The dynamics of quasispecies has considerable implications for the understanding of the adaptability and pathogenic potential of viruses, and in addition, for the design of preventive and therapeutic measures for the diseases caused by these viruses. The authors summarise current knowledge on the structure of quasispecies, and the biological implications of this structure.

[Preventive pentamidine inhalation in patients with HIV infection (CD3 and CD4) in ambulatory practice]. / Pentamidin-Inhalationsprophylaxe bei Patienten mit HIV-Infektion (CD3 und CD4) in der ambulanten Praxis.

Over a time-period of twelve months in a one-center drug monitoring trial a) the efficacy of pentamidine-aerosol for the prophylaxis of pneumocystis carinii pneumonia and b) the patient compliance was investigated. The results of 73 patients who have been treated since the fourth quarter 1988, were evaluated. None of the patients developed pneumocystis carinii pneumonia. Two patients on suspicion of beginning pneumocystis carinii pneumonia improved their clinical and radiological state with higher dosages of pentamidine. 86% of the patients had inhalations for at least once a month, which can be accounted as good compliance. According to our experience pentamidine inhalation prophylaxis is appropriate to outpatient treatment.

[Variability and development of viral populations: assessment and implications]. / Variabilité et evolution des populations virales: bilan et implications.

RNA virus populations consist of complex distributions of closely related but not identical genomes known as viral quasi-species. The quasi-species concept describes the dynamics of these genomes subjected to a continuous process of variation, competition, and selection. Quasi-species dynamics has broad implications not only in the understanding of the molecular mechanisms underlying adaptation of RNA viruses but also in the design of strategies for control and prevention of viral disease. Viral load and genetic heterogeneity have a determinant influence on the adaptation of RNA virus to their environment. Vaccines designed to control diseases caused by highly variable viruses must contain several B and T epitopes to provide an ample and diversified immune response. Similarly, antiviral drugs should be used in combination therapy to minimize selection of resistant viruses. The theoretical model of quasi-species has opened the way for new antiviral therapies based on augmentation of the mutation rate during replication of viral RNA. Finally the quasi-species concept provides the basis for defining the selective factors that could influence the evolution of RNA virus and promote the emergence or reemergence of viral diseases.

An unusual presentation of acquired hypothyroidism: the Van Wyk-Grumbach syndrome.

The association in young females of long-standing primary hypothyroidism, isosexual precocious pseudopuberty and multicystic enlarged ovaries was first described in 1960 by Van Wyk and Grumbach. Since then, sporadic case reports have contributed to clarifying the key features of this syndrome. The unique elements that lead to this diagnosis are FSH-dominated sexual precocity combined with a delayed bone age in the presence of hypothyroidism. It is important to recognise this syndrome because initiating simple thyroid hormone replacement completely resolves symptoms and hormone abnormalities, avoiding unnecessary investigations for malignancies or surgical intervention. We describe an 8-year-old girl with autoimmune thyroiditis and severe long-standing hypothyroidism presenting with the clinical features of Van Wyk-Grumbach syndrome, a secondary TSH-secreting adenoma and hyperprolactinaemia. In addition, this girl presented with microcytic anaemia, elevated erythrocyte sedimentation rate (ESR) and two unusual features - a newly developed streaky hyperpigmented skin lesion and parathyroid hormone suppression despite vitamin D deficiency. Thyroxine replacement normalised all hormone abnormalities and shrunk the pituitary adenoma within 9 months, but the new skin lesion persisted. We review the literature and explore the pathophysiology of known and new features that give rise to speculation indicating stimulation of the FSH G protein-coupled receptor by excessive TSH, but LH suppression by hyperprolactinaemia.

Cytomegalovirus retinitis under combination therapy with zidovudine and dideoxycytidine in advanced human immunodeficiency virus infection.

The incidence of cytomegalovirus (CMV) retinitis during combination therapy with zidovudine (azidothymidine AZT) and zalcitabine (dideoxycytidine ddC), was compared with that during monotherapy with AZT alone in patients with advanced human immunodeficiency virus (HIV) infection. A total of 85 patients with CD4 cell counts under 500/microliters were enrolled in a prospective, controlled study. Between August 1991 and June 1992, these patients were treated daily with 500 mg/kg AZT given alone (n = 42) or in combination with 0.02 mg/kg ddC (n = 43). The rate of occurrence of typical microvascular retinopathy with cotton-wool exudates was lower in patients receiving combination treatment than in those given monotherapy [10 patients (26%) receiving AZT/ddC vs 23 patients (56%) given AZT; P < or = 0.01, chi-square test]. CMV retinitis developed in 8 patients (19%) treated with AZT and in 6 patients (14%) treated with ddC and AZT (no significant difference). In contrast to recently published data, we found no decrease in the rate of occurrence of retinitis in the group under combined antiretroviral therapy but observed a significantly lower incidence of microvascular retinopathy.

Identification of 108K, 93K, and 42K glycoproteins of bovine herpesvirus-1 by monoclonal antibodies.

Three glycoproteins of bovine herpesvirus-1 (BHV-1) other than glycoproteins gI, gIII, and gIV were identified by monoclonal antibody (MAb) analyses. Monoclonal antibodies were obtained by immunization of mice with either BHV-1 envelope or virus infected cells, from which the glycoproteins gI, gIII, and gIV were removed by immunoaffinity. In the latter immunization procedure mice were tolerized either against normal cellular antigens with or without glycoproteins gI, gIII, gIV, and nucleocapsid. From 154 anti-BHV-1 hybridomas isolated, 39 MAbs precipitated a 108K glycoprotein. Two other glycoproteins of respectively 42K and 93K were precipitated each by one MAb. These three glycoproteins were detected in infected cell lysate. Nine anti-108K glycoprotein MAbs neutralized BHV-1 infectivity and three non-neutralizing MAbs were able to reduce plaque development when virus was grown in the presence of these MAbs. It is therefore suggested that this glycoprotein is involved in viral entry into the cell and in cell-to-cell spread of the virus.

Evidence for positive selection in the capsid protein-coding region of the foot-and-mouth disease virus (FMDV) subjected to experimental passage regimens.

We present sequence data from two genomic regions of foot-and-mouth disease virus (FMDV) subjected to several experimental passage regimens. Maximum-likelihood estimates of the nonsynonymous-to-synonymous rate ratio parameter (d(N)/d(S)) suggested the action of positive selection on some antigenic sites of the FMDV capsid during some experimental passages. These antigenic sites showed an accumulation of convergent amino acid replacements during massive serial cytolytic passages and also in persistent infections of FMDV in cell culture. This accumulation was most significant at the antigenic site A (the G-H loop of capsid VP1), which includes an Arg-Gly-Asp (RGD) cellular recognition motif. Our analyses also identified a subregion of VP3, part of the fivefold axis of FMDV particles, that also appeared to be subjected to positive selection of amino acid replacements. From these results, we can conclude that under the restrictive conditions imposed either by the presence of the monoclonal antibodies, by the persistent infections, or by the competition processes established between different variants of the viral population, amino acid replacement in some capsid-coding regions can be positively selected toward an increase of those mutants with a higher capability to infect the cell.