Japanese encephalitis virus replication is negatively regulated by autophagy and occurs on LC3-I- and EDEM1-containing membranes

Autophagy is a lysosomal degradative pathway that has diverse physiological functions and plays crucial roles in several viral infections. Here we examine the role of autophagy in the life cycle of JEV, a neurotropic flavivirus. JEV infection leads to induction of autophagy in several cell types. JEV replication was significantly enhanced in neuronal cells where autophagy was rendered dysfunctional by ATG7 depletion, and in Atg5-deficient mouse embryonic fibroblasts (MEFs), resulting in higher viral titers. Autophagy was functional during early stages of infection however it becomes dysfunctional as infection progressed resulting in accumulation of misfolded proteins. Autophagy-deficient cells were highly susceptible to virus-induced cell death. We also observed JEV replication complexes that are marked by nonstructural protein 1 (NS1) and dsRNA colocalized with endogenous LC3 but not with GFP-LC3. Colocalization of NS1 and LC3 was also observed in Atg5 deficient MEFs, which contain only the nonlipidated form of LC3. Viral replication complexes furthermore show association with a marker of the ER-associated degradation (ERAD) pathway, EDEM1 (ER degradation enhancer, mannosidase α-like 1). Our data suggest that virus replication occurs on ERAD-derived EDEM1 and LC3-I-positive structures referred to as EDEMosomes. While silencing of ERAD regulators EDEM1 and SEL1L suppressed JEV replication, LC3 depletion exerted a profound inhibition with significantly reduced RNA levels and virus titers. Our study suggests that while autophagy is primarily antiviral for JEV and might have implications for disease progression and pathogenesis of JEV, nonlipidated LC3 plays an important autophagy independent function in the virus life cycle.     

Universality splitting in distribution of number of miRNA co-targets

In a recent work (Basu et al., in EPL 105:28007, 2014) it was pointed out that the link-weight distribution of microRNA co-target network of a wide class of species are universal up to scaling. The number cell types, widely accepted as a measure of complexity, turns out to be proportional to these scale-factor. In this article we discuss additional universal features of these networks and show that, this universality splits if one considers distribution of number of common targets of three or more number of microRNAs. These distributions for different species can be collapsed onto two distinct set of universal functions, revealing the fact that the species which appeared in early evolution have different complexity measure compared to those appeared late.     

The Genome Sequence of the Fungal Pathogen Fusarium virguliforme That Causes Sudden Death Syndrome in Soybean

Fusarium virguliforme causes sudden death syndrome (SDS) of soybean, a disease of serious concern throughout most of the soybean producing regions of the world. Despite the global importance, little is known about the pathogenesis mechanisms of F. virguliforme. Thus, we applied Next-Generation DNA Sequencing to reveal the draft F. virguliforme genome sequence and identified putative pathogenicity genes to facilitate discovering the mechanisms used by the pathogen to cause this disease.

Methodology/Principal Findings

We have generated the draft genome sequence of F. virguliforme by conducting whole-genome shotgun sequencing on a 454 GS-FLX Titanium sequencer. Initially, single-end reads of a 400-bp shotgun library were assembled using the PCAP program. Paired end sequences from 3 and 20 Kb DNA fragments and approximately 100 Kb inserts of 1,400 BAC clones were used to generate the assembled genome. The assembled genome sequence was 51 Mb. The N50 scaffold number was 11 with an N50 Scaffold length of 1,263 Kb. The AUGUSTUS gene prediction program predicted 14,845 putative genes, which were annotated with Pfam and GO databases. Gene distributions were uniform in all but one of the major scaffolds. Phylogenic analyses revealed that F. virguliforme was closely related to the pea pathogen, Nectria haematococca. Of the 14,845 F. virguliforme genes, 11,043 were conserved among five Fusarium species: F. virguliforme, F. graminearum, F. verticillioides, F. oxysporum and N. haematococca; and 1,332 F. virguliforme-specific genes, which may include pathogenicity genes. Additionally, searches for candidate F. virguliforme pathogenicity genes using gene sequences of the pathogen-host interaction database identified 358 genes.

Conclusions

The F. virguliforme genome sequence and putative pathogenicity genes presented here will facilitate identification of pathogenicity mechanisms involved in SDS development. Together, these resources will expedite our efforts towards discovering pathogenicity mechanisms in F. virguliforme. This will ultimately lead to improvement of SDS resistance in soybean.     

Surgical Site Infections and Other Postoperative Complications following Prophylactic Anticoagulation in Total Joint Arthroplasty

Background

Anticoagulants reduce the risk of venous thromboembolism (VTE) after total joint replacement. However, concern remains that pharmacologic VTE prophylaxis can lead to bleeding, which may impact on postoperative complications such as infections and reoperations.

Methods and Findings

From the Global Orthopedic Registry (GLORY), we reviewed 3,755 patients in US who elected for primary total hip or knee arthroplasty, received either warfarin or low molecular weight heparin (LMWH) as VTE prophylactics, and had up-to-90-day follow-up after discharge. We compared incidence rates of VTE, infections and other complications between LMWH and warfarin groups, and used multivariate analyses with propensity score weighting to generate the odds ratio (OR). Patients receiving LMWH tended to be older and higher in the American Society of Anesthesiologists grade scores. In contrast, warfarin was used more frequently for hip arthroplasty with longer duration among patients with more pre-existing comorbidity (all P<0.02). A weight variable was created with propensity score to account for differences in covariate distributions. Propensity score-weighted analyses showed no differences in VTE complications. However, compared to warfarin, LMWH was associated with significantly higher rates of bleeding (6.2% vs. 2.1%; OR = 3.82, 95% confidence interval [CI], 2.64 to 5.52), blood transfusion (29.4% vs. 22.0%; OR = 1.75, 95% CI, 1.51 to 2.04), reoperations (2.4% vs. 1.3%; OR = 1.77, 95% CI, 1.07 to 2.93) and infections (1.6% vs. 0.6%; OR = 2.79, 95% CI, 1.42 to 5.45). Similar results were obtained from compliant uses of warfarin (26%) and LMWH (62%) according to clinical guidelines. While surgical site infections were mostly superficial, current study was underpowered to compare incidence rates of deep infections (<1.0%).

Conclusions

Surgical site infections and reoperations in 3 months following primary total joint arthroplasty may be associated with anticoagulant use that exhibited higher bleeding risk. Long-term complications and deep wound infections remain to be studied.     

Molecular Evidence of Increased Resistance to Anti-Folate Drugs in Plasmodium falciparum in North-East India: A Signal for Potential Failure of Artemisinin Plus Sulphadoxine-Pyrimethamine Combination Therapy

North-east India, being a corridor to South-east Asia, is believed to play an important role in transmitting drug resistant Plasmodium falciparum malaria to India and South Asia. North-east India was the first place in India to record the emergence of drug resistance to chloroquine as well as sulphadoxine/pyrimethamine. Presently chloroquine resistance is widespread all over the North-east India and resistance to other anti-malarials is increasing. In this study both in vivo therapeutic efficacy and molecular assays were used to screen the spectrum of drug resistance to chloroquine and sulphadoxine/pyrimethamine in the circulating P. falciparum strains. A total of 220 P. falciparum positives subjects were enrolled in the study for therapeutic assessment of chloroquine and sulphadoxine/pyrimethamine and assessment of point mutations conferring resistances to these drugs were carried out by genotyping the isolates following standard methods. Overall clinical failures in sulphadoxine/pyrimethamine and chloroquine were found 12.6 and 69.5% respectively, while overall treatment failures recorded were 13.7 and 81.5% in the two arms. Nearly all (99.0%) the isolates had mutant pfcrt genotype (76T), while 68% had mutant pfmdr-1 genotype (86Y). Mutation in dhps 437 codon was the most prevalent one while dhfr codon 108 showed 100% mutation. A total of 23 unique haplotypes at the dhps locus and 7 at dhfr locus were found while dhps-dhfr combined loci revealed 49 unique haplotypes. Prevalence of double, triple and quadruple mutations were common while 1 haplotype was found with all five mutated codons (F/AGEGS/T) at dhps locus. Detection of quadruple mutants (51I/59R/108N/164L) in the present study, earlier recorded from Car Nicobar Island, India only, indicates the presence of high levels of resistance to sulphadoxine/pyrimethamine in north-east India. Associations between resistant haplotypes and the clinical outcomes and emerging resistance in sulphadoxine/pyrimethamine in relation to the efficacy of the currently used artemisinin combination therapy are discussed.     

Metabotropic glutamate receptor 1 recycles to the cell surface in protein phosphatase 2A‐dependent manner in non‐neuronal and neuronal cell lines

Trafficking of G protein‐coupled receptors plays a crucial role in controlling the precise signalling of the receptor as well as its proper regulation. Metabotropic glutamate receptor 1 (mGluR1), a G protein‐coupled receptor, is a member of the group I mGluR family. mGluR1 plays a critical role in neuronal circuit formation and also in multiple types of synaptic plasticity. This receptor has also been reported to be involved in various neuropsychiatric diseases. Other than the central nervous system, mGluR1 plays crucial roles in various non‐neuronal cells like hepatocytes, skin cells, etc. Although it has been reported that mGluR1 gets endocytosed on ligand application, the events after the internalization of the receptor has not been studied. We show here that mGluR1 internalizes on ligand application. Subsequent to endocytosis, majority of the receptors localize at the recycling compartment and no significant presence of the receptor was noticed in the lysosome. Furthermore, mGluR1 returned to the cell membrane subsequent to ligand‐mediated internalization. We also show here that the recycling of mGluR1 is dependent on the activity of protein phosphatase 2A. Thus, our data suggest that the ligand‐mediated internalized receptors recycle back to the cell surface in protein phosphatase 2A‐dependent manner.

     

Development of Peptide-Based Lineage-Specific Serology for Chronic Chagas Disease: Geographical and Clinical Distribution of Epitope Recognition

Background

Chagas disease, caused by infection with the protozoan Trypanosoma cruzi, remains a serious public health issue in Latin America. Genetically diverse, the species is sub-divided into six lineages, known as TcI–TcVI, which have disparate geographical and ecological distributions. TcII, TcV, and TcVI are associated with severe human disease in the Southern Cone countries, whereas TcI is associated with cardiomyopathy north of the Amazon. T. cruzi persists as a chronic infection, with cardiac and/or gastrointestinal symptoms developing years or decades after initial infection. Identifying an individual''s history of T. cruzi lineage infection directly by genotyping of the parasite is complicated by the low parasitaemia and sequestration in the host tissues.

Methodology/Principal Findings

We have applied here serology against lineage-specific epitopes of the T. cruzi surface antigen TSSA, as an indirect approach to allow identification of infecting lineage. Chagasic sera from chronic patients from a range of endemic countries were tested by ELISA against synthetic peptides representing lineage-specific TSSA epitopes bound to avidin-coated ELISA plates via a biotin labelled polyethylene glycol-glycine spacer to increase rotation and ensure each amino acid side chain could freely interact with their antibodies. 79/113 (70%) of samples from Brazil, Bolivia, and Argentina recognised the TSSA epitope common to lineages TcII/TcV/TcVI. Comparison with clinical information showed that a higher proportion of Brazilian TSSApep-II/V/VI responders had ECG abnormalities than non-responders (38% vs 17%; p<0.0001). Among northern chagasic sera 4/20 (20%) from Ecuador reacted with this peptide; 1/12 Venezuelan and 1/34 Colombian samples reacted with TSSApep-IV. In addition, a proposed TcI-specific epitope, described elsewhere, was demonstrated here to be highly conserved across lineages and therefore not applicable to lineage-specific serology.

Conclusions/Significance

These results demonstrate the considerable potential for synthetic peptide serology to investigate the infection history of individuals, geographical and clinical associations of T. cruzi lineages.     

Emergent canine visceral leishmaniasis in Argentina: Comparative diagnostics and relevance to proliferation of human disease

BackgroundVisceral leishmaniasis (VL) is a zoonotic protozoal vector-borne disease that is a major public health challenge. In Argentina, canine (CVL) and human visceral leishmaniasis (HVL) have recently emerged. There is a lack of standardised diagnostic tests for CVL, which hinders control of CVL and HVL.Methodology/Principal findingsSampling was carried out in Puerto Iguazú, Argentina, comprising 190 asymptomatic, oligosymptomatic and polysymptomatic dogs. The following diagnostics were applied: microscopy of lymph node aspirate (LNA); three immunochromatographic rapid diagnostic tests (RDTs), prototype rK28-ICT, rK39-ICT (both Coris BioConcept), commercial rK39 (InBios); ELISA for IgG, IgG1 and IgG2, against rK28, rK39 or crude lysate antigen. DNA detection and analysis, with 30 dogs, was of the ITS1 region using skin samples, and loop-mediated isothermal amplification (LAMP; Eiken Loopamp) of buffy coat, skin scrape or LNA. 15.4% of dogs were positive by LNA microscopy. The rK28 RDT had higher seropositivity rate (61%) than either a prototype rK39 RDT (31.4%) or commercial rK39 RDT (18.8%), without cross-reactivity with six other pathogens. IgG anti-rK39 ELISA antibody titres, but not IgG2, were positively correlated with number of clinical signs. LAMP with LNA had a higher positivity rate than PCR; buffy coat sampling was more sensitive than skin scrape. ITS1 confirmed Leishmania (Leishmania) infantum as the agent of CVL. Leishmania (Viannia) spp. was detected in skin samples from two dogs, compatible with Leishmania (Viannia) braziliensis.Conclusions/SignificanceSeroprevalence confirmed rapid increase in CVL in Puerto Iguazú. The rK28 RDT test potentially has great value for improved point-of-care diagnosis. Given cost reduction and accessibility, commercial LAMP may be applicable to buffy coat. RDT biomarkers of CVL clinical status are required to combat spread of CVL and HVL. The presence of Viannia, perhaps as an agent of human mucocutaneous leishmaniasis (MCL), highlights the need for vigilance and surveillance.