Identification of a novel neuropathogenic Theiler's murine encephalomyelitis virus

Theiler's murine encephalitis viruses (TMEV) are divided into two subgroups based on their neurovirulence. Persistent strains resemble Theiler's original viruses (referred to as the TO subgroup), which largely induce a subclinical polioencephalomyelitis during the acute phase of the disease and can persist in the spinal cord of susceptible animals, inducing a chronic demyelinating disease. In contrast, members of the neurovirulent subgroup cause an acute encephalitis characterized by the rapid onset of paralysis and death within days following intracranial inoculation. We report herein the characterization of a novel neurovirulent strain of TMEV, identified using pyrosequencing technology and referred to as NIHE. Complete coverage of the NIHE viral genome was obtained, and it shares <90% nucleotide sequence identity to known TMEV strains irrespective of subgroup, with the greatest sequence variability being observed in genes encoding the leader and capsid proteins. The histopathological analysis of infected brain and spinal cord demonstrate inflammatory lesions and neuronal necrosis during acute infection with no evidence of viral persistence or chronic disease. Intriguingly, genetic analysis indicates the putative expression of the L protein, considered a hallmark of strains within the persistent subgroup. Thus, the identification and characterization of a novel neurovirulent TMEV strain sharing features previously associated with both subgroups will lead to a deeper understanding of the evolution of TMEV strains and new insights into the determinants of neurovirulence.     

Topography and Land Cover of Watersheds Predicts the Distribution of the Environmental Pathogen Mycobacterium ulcerans in Aquatic Insects

Background

An understanding of the factors driving the distribution of pathogens is useful in preventing disease. Often we achieve this understanding at a local microhabitat scale; however the larger scale processes are often neglected. This can result in misleading inferences about the distribution of the pathogen, inhibiting our ability to manage the disease. One such disease is Buruli ulcer, an emerging neglected tropical disease afflicting many thousands in Africa, caused by the environmental pathogen Mycobacterium ulcerans. Herein, we aim to describe the larger scale landscape process describing the distribution of M. ulcerans.

Methodology

Following extensive sampling of the community of aquatic macroinvertebrates in Cameroon, we select the 5 dominant insect Orders, and conduct an ecological niche model to describe how the distribution of M. ulcerans positive insects changes according to land cover and topography. We then explore the generalizability of the results by testing them against an independent dataset collected in a second endemic region, French Guiana.

Principal Findings

We find that the distribution of the bacterium in Cameroon is accurately described by the land cover and topography of the watershed, that there are notable seasonal differences in distribution, and that the Cameroon model does not predict the distribution of M. ulcerans in French Guiana.

Conclusions/Significance

Future studies of M. ulcerans would benefit from consideration of local structure of the local stream network in future sampling, and further work is needed on the reasons for notable differences in the distribution of this species from one region to another. This work represents a first step in the identification of large-scale environmental drivers of this species, for the purposes of disease risk mapping.     

Activation of Xenopus Eggs by the Kinase Inhibitor 6-DMAP Suggests a Differential Regulation of Cyclin B and p39 Proteolysis

In Xenopus eggs, metaphase II arrest is due to the cytostatic factor that maintains a high level of MPF activity. Kinases are important in this phenomenon since p39^mos and MAPK play a part in the cytostatic activity whereas p34^cdc2 is the catalytic subunit of MPF. Fertilization induces a rise in intracellular calcium leading to egg activation that can be mimicked by calcium-increasing agents such as calcium ionophore. We have performed on Xenopus eggs a biochemical comparison of the effects of the kinase inhibitor 6-DMAP and the calcium ionophore. Both drugs were able to induce pronucleus formation but the underlying molecular events were different. The inactivation of MAPK occurred earlier in eggs exposed to 6-DMAP. Cyclins B1 and B2 were stable and p39^mos was proteolysed in 6-DMAP-treated eggs while the three proteins underwent degradation in A23187-treated ones. These results suggest a differential regulation of ubiquitin-dependent proteolysis of cyclin B and p39^mos.     

The “Buruli Score”: Development of a Multivariable Prediction Model for Diagnosis of Mycobacterium ulcerans Infection in Individuals with Ulcerative Skin Lesions,Akonolinga, Cameroon

Background

Access to laboratory diagnosis can be a challenge for individuals suspected of Buruli Ulcer (BU). Our objective was to develop a clinical score to assist clinicians working in resource-limited settings for BU diagnosis.

Methododology/Principal Findings

Between 2011 and 2013, individuals presenting at Akonolinga District Hospital, Cameroon, were enrolled consecutively. Clinical data were collected prospectively. Based on a latent class model using laboratory test results (ZN, PCR, culture), patients were categorized into high, or low BU likelihood. Variables associated with a high BU likelihood in a multivariate logistic model were included in the Buruli score. Score cut-offs were chosen based on calculated predictive values. Of 325 patients with an ulcerative lesion, 51 (15.7%) had a high BU likelihood. The variables identified for the Buruli score were: characteristic smell (+3 points), yellow color (+2), female gender (+2), undermining (+1), green color (+1), lesion hyposensitivity (+1), pain at rest (-1), size >5cm (-1), locoregional adenopathy (-2), age above 20 up to 40 years (-3), or above 40 (-5). This score had AUC of 0.86 (95%CI 0.82–0.89), indicating good discrimination between infected and non-infected individuals. The cut-off to reasonably exclude BU was set at scores <0 (NPV 96.5%; 95%CI 93.0–98.6). The treatment threshold was set at a cut-off ≥4 (PPV 69.0%; 95%CI 49.2–84.7). Patients with intermediate BU probability needed to be tested by PCR.

Conclusions/Significance

We developed a decisional algorithm based on a clinical score assessing BU probability. The Buruli score still requires further validation before it can be recommended for wide use.     

Biochemical and Immunological Properties of the Mouse Brain Enolases Purified by a Simple Method

Abstract: A simple and rapid purification method is presented for the two mouse cerebral isozymes of enolase (EC 4.2.1.11), E₁ and E₃. The purity of the preparations was ascertained by electrophoresis under two different conditions. The biochemical and immunological properties of E₁ and E₃ were compared. The molecular weight of the cerebral enolases was analysed by column chromatography on Sephadex G 150 and by electrophoresis in the presence of SDS. Both E₁ and E₃ are homodimers with a subunit of molecular weight of 50,000. The procedure also yields a semi-purified fraction of E₂. Conditions of in vitro formation of E₂ from pure or semi-purified fractions of E₁ and E₃ show that it is likely to be a real hybrid, rather than an aggregate and that it is probably not an artefact formed during the purification. The K_m values (K_m= 3–4·10^?5 M) for the substrate are not significantly different amongst the three forms. However, E₁ and E₂ but not E₃ are inhibited by excess substrate. Antisera against E₁ and E₃ have been obtained from rabbit and goat, respectively. Antibodies against each protein do not show any cross-reactivity with each other. There is, however, a broad species cross-reactivity, showing conservation of each enolase form during evolution. Both anti-E₁ and anti-E₃ sera react with the E₂ enolase fraction, in agreement with its hybrid structure. Anti-E₃ serum does not react with extracts of other tested organs. Brain enolase 1 resembles liver enolase in its biochemical and immunological properties. A slight cross-reactivity of anti-E₁ serum with muscle extracts is observed. Heterogeneity of brain enolase 1 is observed by both biochemical and immunological methods; the nature of this heterogeneity is discussed.     

A new disease-related mutation for mitochondrial encephalopathy lactic acidosis and strokelike episodes (MELAS) syndrome affects the ND4 subunit of the respiratory complex I.

The molecular lesions in two patients exhibiting classical clinical manifestations of MELAS (mitochondrial encephalopathy, lactic acidosis, and strokelike episodes) syndrome have been investigated. A recently reported disease-related A----G base substitution at nt 3243 of the mtDNA, in the DHU loop of tRNA(Leu), was detected by restriction-enzyme analysis of the relevant PCR-amplified segment of the mtDNA of one patient but was not observed, by either restriction-enzyme analysis or nucleotide sequencing, in the other. To define the molecular lesion in the patient who does not have the A----G base substitution at nt 3243, the total mitochondrial genome of the patient has been sequenced. An A----G base substitution at nt 11084, leading to a Thr-to-Ala amino acid replacement in the ND4 subunit of the respiratory complex I, is suggested to be a disease-related mutation.     

Developmental changes of blood group A-active glycosphingolipids with type 1 and type 2 chains in rat small intestine

Blood group A-active glycosphingolipids of the small intestine, A-6 and A-12, which have been characterized previously in the adult rat [Breimer ME, Hansson GC, Karlsson K-A, Leffler H (1982) J Biol Chem 257:906–12], were found to appear during postnatal development, using immunostaining on thin layer chromatograms with two monoclonal anti-A antibodies, A005 and A581. In this system, A005 was found to be specific for the A determinant based on the type 2 chain, while A581 reacted mainly with the A determinant based on the type 1 chain and only weakly with the A determinant based on the type 2 chain. A-6 Type 1 was detected first at 18 days after birth. Its concentration increased markedly during the fourth week. A-6 Type 2 was detected, at a very low level, in neonates. Its concentration increased between days 15 and 20 and then decreased almost to the neonate level by 28 days. Dodecaglycosylceramide A-12 followed the same pattern of reactivity as A-6 type 1 with A581, and remained strongly reactive with A005 after 20 days. Linear A-6 and branched A-12 appeared simultaneously. Antibodies directed against blood group H determinants based on the type 1 or type 2 chains did not detect any H structure which might have appeared as a precursor of either A-6 or A-12 at the early stages of postnatal development.Abbreviations A-6, A-12, H-5, H-10 etc the glycolipids are abbreviated by giving blood group activity, and number of sugars (see also Fig. 1) - G_M3 G_M3-ganglioside, H³NeuAc-LcCer - PBS phosphate-buffered saline     

Inhibition of protein tyrosine phosphatases blocks calcium-induced activation of metaphase II-arrested oocytes of Xenopus laevis.

We have studied the effect of a protein tyrosine phosphatases (PTP) inhibitor on calcium-induced activation of Xenopus laevis oocytes arrested at metaphase II. Ammonium molybdate microinjection blocked pronucleus formation following A23187 treatment while cortical granules still underwent exocytosis. Pronuclei still occurred in ammonium molybdate-injected oocytes following 6-DMAP addition. Changes that usually occurred following A23187 exposure were inhibited in the presence of ammonium molybdate in the oocyte: MAPK dephosphorylation, p34(cdc2) rephosphorylation and cyclin B2 and p39(mos) proteolysis. These results suggest that a PTP is involved in the activation of the ubiquitin-dependent degradation machinery.