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To determine the population structure of the cryptococcosis agents in China, we analyzed the genotype of 120 Cryptococcus neoformans and 9 Cryptococcus gattii strains isolated from 1980 through 2006 from cryptococcosis patients residing in 16 provinces of mainland China. A total of 71% (91/129) of the clinical strains isolated from 1985 through 2006 were from patients without any apparent risk factors. Only 8.5% (11/129) were from AIDS patients; the remaining 20.5% (27/129) were from patients with underlying diseases other than HIV infection. One hundred twenty of the 129 isolates were C. neoformans serotype A, mating type MATα strains that exhibited an identical M13-based VNI subtype, which was distinguishable from the reference VNI molecular type. The 9 remaining isolates were serotype B, MATα strains of C. gattii and portrayed a typical VGI molecular type. Data analyzed from multilocus sequences showed no variation and that these Chinese C. neoformans isolates belong to a cluster that has phylogenetically diverged from the VNI reference strain. Our finding that most cryptococcosis patients in China had no apparent risk factor is in stark contrast with reports from other countries.

Importance: Studies mapping genetic heterogeneity of clinical isolates of M. tuberculosis for determining their strain lineage and drug resistance by whole-genome sequencing are limited in high tuberculosis burden settings. We carried out whole-genome sequencing of 242 M. tuberculosis isolates from drug-sensitive and drug-resistant tuberculosis patients, identified and collected as part of the TB Portals Program, to have a comprehensive insight into the genetic diversity of M. tuberculosis in Southern India. We report several genetic variations in M. tuberculosis that may confer resistance to antitubercular drugs. Further wide-scale efforts are required to fully characterize M. tuberculosis genetic diversity at a population level in high tuberculosis burden settings for providing precise tuberculosis treatment., Competing Interests: The authors declare no conflict of interest.

Astroviruses (AstVs) can cause of severe infection of the central nervous system (CNS) in immunocompromised individuals. Here, we identified a human AstV of the VA1 genotype, HAstV-NIH, as the cause of fatal encephalitis in an immunocompromised adult. We investigated the cells targeted by AstV, neurophysiological changes, and host responses by analyzing gene expression, protein expression, and cellular morphology in brain tissue from three cases of AstV neurologic disease (AstV-ND). We demonstrate that neurons are the principal cells targeted by AstV in the brain and that the cerebellum and brainstem have the highest burden of infection. Detection of VA1 AstV in interconnected brain structures such as thalamus, deep cerebellar nuclei, Purkinje cells, and pontine nuclei indicates that AstV may spread between connected neurons transsynaptically. We found transcriptional dysregulation of neural functions and disruption of both excitatory and inhibitory synaptic innervation of infected neurons. Importantly, transcriptional dysregulation of neural functions occurred in fatal cases, but not in a patient that survived AstV-ND. We show that the innate, but not adaptive immune response was transcriptionally driving host defense in the brain of immunocompromised patients with AstV-ND. Both transcriptome and molecular pathology studies showed that most of the cellular changes were associated with CNS-intrinsic cells involved in phagocytosis and injury repair (microglia, perivascular/parenchymal border macrophages, and astrocytes), but not CNS-extrinsic cells (T and B cells), suggesting an imbalance of innate and adaptive immune responses to AstV infection in the brain as a result of the underlying immunodeficiencies. These results show that VA1 AstV infection of the brain in immunocompromised humans is associated with imbalanced host defense responses, disruption of neuronal somatodendritic compartments and synapses and increased phagocytic cellular activity. Improved understanding of the response to viral infections of the human CNS may provide clues for how to manipulate these processes to improve outcomes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

The classical laboratory mouse strains are genetic mosaics of three Mus musculus subspecies that occupy distinct regions of Eurasia. These strains and subspecies carry infectious and endogenous mouse leukemia viruses (MLVs) that can be pathogenic and mutagenic. MLVs evolved in concert with restrictive host factors with some under positive selection, including the XPR1 receptor for xenotropic/polytropic MLVs (X/P-MLVs) and the post-entry restriction factor Fv1 . Since positive selection marks host-pathogen genetic conflicts, we examined MLVs for counter-adaptations at sites that interact with XPR1, Fv1 , and the CAT1 receptor for ecotropic MLVs (E-MLVs). Results describe different co-adaptive evolutionary paths within the ranges occupied by these virus-infected subspecies. The interface of CAT1, and the otherwise variable E-MLV envelopes, is highly conserved; antiviral protection is afforded by the Fv4 restriction factor. XPR1 and X/P-MLVs variants show coordinate geographic distributions, with receptor critical sites in envelope, under positive selection but with little variation in envelope and XPR1 in mice carrying P-ERVs. The major Fv1 target in the viral capsid is under positive selection, and the distribution of Fv1 alleles is subspecies-correlated. These data document adaptive, spatial and temporal, co-evolutionary trajectories at the critical interfaces of MLVs and the host factors that restrict their replication.

Malaria parasites use the RhopH complex for erythrocyte invasion and channel-mediated nutrient uptake. As the member proteins are unique to Plasmodium spp., how they interact and traffic through subcellular sites to serve these essential functions is unknown. We show that RhopH is synthesized as a soluble complex of CLAG3, RhopH2, and RhopH3 with 1:1:1 stoichiometry. After transfer to a new host cell, the complex crosses a vacuolar membrane surrounding the intracellular parasite and becomes integral to the erythrocyte membrane through a PTEX translocon-dependent process. We present a 2.9 Å single-particle cryo-electron microscopy structure of the trafficking complex, revealing that CLAG3 interacts with the other subunits over large surface areas. This soluble complex is tightly assembled with extensive disulfide bonding and predicted transmembrane helices shielded. We propose a large protein complex stabilized for trafficking but poised for host membrane insertion through large-scale rearrangements, paralleling smaller two-state pore-forming proteins in other organisms., Competing Interests: MS, JD, AM, JS, GD, PS, PO, MR, BC, KW, SD No competing interests declared, SS Reviewing editor, eLife

Mycobacterium tuberculosis (M.tb) is the leading cause of death from an infectious disease. Drug resistant tuberculosis (DR-TB) threatens to exacerbate challenges in diagnostics and treatment. It is important to monitor strains circulating in countries with heavy burden of DR-TB, to make informed decisions about treatment, and because in these countries there is an elevated probability that DR-TB may advance to the totally drug resistant form. The TB Portals Program (TBPP, https://TBPortals.niaid.nih.gov) formed a global network of participating institutions and hospitals collecting and analyzing de-identified clinical, imaging and socioeconomic data, augmenting these with genomic sequencing results. TB Portals database includes complete M.tb genomes, with the information about spoligotypes, strains, and genomic variants related to drug resistance. Within the framework of TB Portals, we created Data Exploration Portal (DEPOT), to facilitate visualization and statistical analysis of user-defined cohorts from the entire TB Portals database. A continuing TB Portals research objective is to actively monitor and examine genomic variability that may account for observed differences in DR-TB incident rates and/or difficulties with diagnosis and treatment. Our analysis identified that several genomic variants implicated in drug resistance or improved fitness of the pathogen, were significantly more frequent in M.tb strains circulating in Belarus in comparison with other countries. Further studies are necessary to reveal whether the corresponding genomic variants may explain unusually high burden of drug-resistant M.tb in Belarus and suggest improvements for diagnostic and drug therapies., (Published by Elsevier B.V.)

Background: Recurrence of drug-resistant tuberculosis (DR-TB) after treatment occurs through relapse of the initial infection or reinfection by a new drug-resistant strain. Outbreaks of DR-TB in high burden regions present unique challenges in determining recurrence status for effective disease management and treatment. In the Republic of Moldova the burden of DR-TB is exceptionally high, with many cases presenting as recurrent., Methods: We performed a retrospective analysis of Mycobacterium tuberculosis from Moldova to better understand the genomic basis of drug resistance and its effect on the determination of recurrence status in a high DR-burden environment. To do this we analyzed genomes from 278 isolates collected from 189 patients, including 87 patients with longitudinal samples. These pathogen genomes were sequenced using Illumina technology, and SNP panels were generated for each sample for use in phylogenetic and network analysis. Discordance between genomic resistance profiles and clinical drug-resistance test results was examined in detail to assess the possibility of mixed infection., Results: There were clusters of multiple patients with 10 or fewer differences among DR-TB samples, which is evidence of person-to-person transmission of DR-TB. Analysis of longitudinally collected isolates revealed that many infections exhibited little change over time, though 35 patients demonstrated reinfection by divergent (number of differences > 10) lineages. Additionally, several same-lineage sample pairs were found to be more divergent than expected for a relapsed infection. Network analysis of the H3/4.2.1 clade found very close relationships among 61 of these samples, making differentiation of reactivation and reinfection difficult. There was discordance between genomic profile and clinical drug sensitivity test results in twelve samples, and four of these had low level (but not statistically significant) variation at DR SNPs suggesting low-level mixed infections., Conclusions: Whole-genome sequencing provided a detailed view of the genealogical structure of the DR-TB epidemic in Moldova, showing that reinfection may be more prevalent than currently recognized. We also found increased evidence of mixed infection, which could be more robustly characterized with deeper levels of genomic sequencing.

The NIAID TB Portals Program (TBPP) established a unique and growing database repository of socioeconomic, geographic, clinical, laboratory, radiological, and genomic data from patient cases of drug-resistant tuberculosis (DR-TB). Currently, there are 2,428 total cases from nine country sites (Azerbaijan, Belarus, Moldova, Georgia, Romania, China, India, Kazakhstan, and South Africa), 1,611 (66%) of which are multidrug- or extensively-drug resistant and 1,185 (49%), 863 (36%), and 952 (39%) of which contain X-ray, computed tomography (CT) scan, and genomic data, respectively. We introduce the Data Exploration Portal (TB DEPOT, https://depot.tbportals.niaid.nih.gov) to visualize and analyze these multi-domain data. The TB DEPOT leverages the TBPP integration of clinical, socioeconomic, genomic, and imaging data into standardized formats and enables user-driven, repeatable, and reproducible analyses. It furthers the TBPP goals to provide a web-enabled analytics platform to countries with a high burden of multidrug-resistant TB (MDR-TB) but limited IT resources and inaccessible data, and enables the reusability of data, in conformity with the NIH's Findable, Accessible, Interoperable, and Reusable (FAIR) principles. TB DEPOT provides access to "analysis-ready" data and the ability to generate and test complex clinically-oriented hypotheses instantaneously with minimal statistical background and data processing skills. TB DEPOT is also promising for enhancing medical training and furnishing well annotated, hard to find, MDR-TB patient cases. TB DEPOT, as part of TBPP, further fosters collaborative research efforts to better understand drug-resistant tuberculosis and aid in the development of novel diagnostics and personalized treatment regimens., Competing Interests: The authors have declared that no competing interests exist.

Hepatitis B virus (HBV)-associated acute liver failure (ALF) is a dramatic clinical syndrome leading to death or liver transplantation in 80% of cases. Due to the extremely rapid clinical course, the difficulties in obtaining liver specimens, and the lack of an animal model, the pathogenesis of ALF remains largely unknown. Here, we performed a comprehensive genetic and functional characterization of the virus and the host in liver tissue from HBV-associated ALF and compared the results with those of classic acute hepatitis B in chimpanzees. In contrast with acute hepatitis B, HBV strains detected in ALF livers displayed highly mutated HBV core antigen (HBcAg), associated with increased HBcAg expression ex vivo, which was independent of viral replication levels. Combined gene and miRNA expression profiling revealed a dominant B cell disease signature, with extensive intrahepatic production of IgM and IgG in germline configuration exclusively targeting HBcAg with subnanomolar affinities, and complement deposition. Thus, HBV ALF appears to be an anomalous T cell-independent, HBV core-driven B cell disease, which results from the rare and unfortunate encounter between a host with an unusual B cell response and an infecting virus with a highly mutated core antigen., Competing Interests: The authors declare no conflict of interest.

The TB Portals program is an international consortium of physicians, radiologists, and microbiologists from countries with a heavy burden of drug-resistant tuberculosis working with data scientists and information technology professionals. Together, we have built the TB Portals, a repository of socioeconomic/geographic, clinical, laboratory, radiological, and genomic data from patient cases of drug-resistant tuberculosis backed by shareable, physical samples. Currently, there are 1,299 total cases from five country sites (Azerbaijan, Belarus, Moldova, Georgia, and Romania), 976 (75.1%) of which are multidrug or extensively drug resistant and 38.2%, 51.9%, and 36.3% of which contain X-ray, computed tomography (CT) scan, and genomic data, respectively. The top Mycobacterium tuberculosis lineages represented among collected samples are Beijing, T1, and H3, and single nucleotide polymorphisms (SNPs) that confer resistance to isoniazid, rifampin, ofloxacin, and moxifloxacin occur the most frequently. These data and samples have promoted drug discovery efforts and research into genomics and quantitative image analysis to improve diagnostics while also serving as a valuable resource for researchers and clinical providers. The TB Portals database and associated projects are continually growing, and we invite new partners and collaborations to our initiative. The TB Portals data and their associated analytical and statistical tools are freely available at https://tbportals.niaid.nih.gov/.

Rambaut et al show that the erratum to our report on Ebola virus Makona evolution not only corrected sample dates modified by others in GenBank but also corrected an additional transcriptional error in our original analysis. We agree with their observation that both factors contributed to our revised evolutionary rate estimate but continue to stand by our revised estimate and conclusions., (Copyright © 2016, American Association for the Advancement of Science.)

Analysis of hepatitis C virus (HCV) replication and quasispecies distribution within the tumor of patients with HCV-associated hepatocellular carcinoma (HCC) can provide insight into the role of HCV in hepatocarcinogenesis and, conversely, the effect of HCC on the HCV lifecycle. In a comprehensive study of serum and multiple liver specimens from patients with HCC who underwent liver transplantation, we found a sharp and significant decrease in HCV RNA in the tumor compared with surrounding nontumorous tissues, but found no differences in multiple areas of control non-HCC cirrhotic livers. Diminished HCV replication was not associated with changes in miR-122 expression. HCV genetic diversity was significantly higher in livers containing HCC compared with control non-HCC cirrhotic livers. Tracking of individual variants demonstrated changes in the viral population between tumorous and nontumorous areas, the extent of which correlated with the decline in HCV RNA, suggesting HCV compartmentalization within the tumor. In contrast, compartmentalization was not observed between nontumorous areas and serum, or in controls between different areas of the cirrhotic liver or between liver and serum. Our findings indicate that HCV replication within the tumor is restricted and compartmentalized, suggesting segregation of specific viral variants in malignant hepatocytes.

Rhesus cytomegalovirus (RhCMV) 68-1 is the prototypic strain of RhCMV that has been used for pathogenesis and vaccine development. We determined the complete sequence of the RhCMV 68-1 UL/b' region directly from the original urine from which RhCMV 68-1 was isolated in 1968, and compared it to other RhCMVs. The laboratory passaged RhCMV 68-1 has inversions, deletions, and stop codons in UL/b' that are absent in the original isolate and other low passage RhCMV isolates. Fourteen of the 17 open reading frames (ORFs) in 68-1 UL/b' in the original isolate share >95% amino acid identity with low passage RhCMV. The original isolate retains 6 ORFs that encode α-chemokine-like proteins, including RhUL146 and RhUL146b that share only 92% and 81% amino acid identity, respectively, with a contemporary low passage RhCMV isolate. Identification of the original RhCMV 68-1 UL/b' sequence is important for using RhCMV 68-1 in pathogenesis and vaccine studies., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Xenotropic mouse leukemia viruses (X-MLVs) are broadly infectious for mammals except most of the classical strains of laboratory mice. These gammaretroviruses rely on the XPR1 receptor for entry, and the unique resistance of laboratory mice is due to two mutations in different putative XPR1 extracellular loops. Cells from avian species differ in susceptibility to X-MLVs, and 2 replacement mutations in the virus-resistant chicken XPR1 (K496Q and Q579E) distinguish it from the more permissive duck and quail receptors. These substitutions align with the two mutations that disable the laboratory mouse XPR1. Mutagenesis of the chicken and duck genes confirms that residues at both sites are critical for virus entry. Among 32 avian species, the 2 disabling XPR1 mutations are found together only in the chicken, an omnivorous, ground-dwelling fowl that was domesticated in India and/or Southeast Asia, which is also where X-MLV-infected house mice evolved. The receptor-disabling mutations are also present separately in 5 additional fowl and raptor species, all of which are native to areas of Asia populated by the virus-infected subspecies Mus musculus castaneus. Phylogenetic analysis showed that the avian XPR1 gene is under positive selection at sites implicated in receptor function, suggesting a defensive role for XPR1 in the avian lineage. Contact between bird species and virus-infected mice may thus have favored selection of mouse virus-resistant receptor orthologs in the birds, and our data suggest that similar receptor-disabling mutations were fixed in mammalian and avian species exposed to similar virus challenges.

Acquired antimalarial drug resistance produces treatment failures and has led to periods of global disease resurgence. In Plasmodium falciparum, resistance is known to arise through genome-level changes such as mutations and gene duplications. We now report an epigenetic resistance mechanism involving genes responsible for the plasmodial surface anion channel, a nutrient channel that also transports ions and antimalarial compounds at the host erythrocyte membrane. Two blasticidin S-resistant lines exhibited markedly reduced expression of clag genes linked to channel activity, but had no genome-level changes. Silencing aborted production of the channel protein and was directly responsible for reduced uptake. Silencing affected clag paralogs on two chromosomes and was mediated by specific histone modifications, allowing a rapidly reversible drug resistance phenotype advantageous to the parasite. These findings implicate a novel epigenetic resistance mechanism that involves reduced host cell uptake and is a worrisome liability for water-soluble antimalarial drugs.

Chronic hepatitis C may follow a mild and stable disease course or progress rapidly to cirrhosis and liver-related death. The mechanisms underlying the different rates of disease progression are unknown. Using serial, prospectively collected samples from cases of transfusion-associated hepatitis C, we identified outcome-specific features that predict long-term disease severity. Slowly progressing disease correlated with an early alanine aminotransferase peak and antibody seroconversion, transient control of viremia, and significant induction of IFN-γ and MIP-1β, all indicative of an effective, albeit insufficient, adaptive immune response. By contrast, rapidly progressive disease correlated with persistent and significant elevations of alanine aminotransferase and the profibrogenic chemokine MCP-1 (CCL-2), greater viral diversity and divergence, and a higher rate of synonymous substitution. This study suggests that the long-term course of chronic hepatitis C is determined early in infection and that disease severity is predicted by the evolutionary dynamics of hepatitis C virus and the level of MCP-1, a chemokine that appears critical to the induction of progressive fibrogenesis and, ultimately, the ominous complications of cirrhosis.

Background: Neisseria meningitidis serogroup B has been predominant in Brazil, but no broadly effective vaccine is available to prevent endemic meningococcal disease. To understand genetic diversity among serogroup B strains in Brazil, we selected a nationally representative sample of clinical disease isolates from 2004, and a temporally representative sample for the state of São Paulo (1988-2006) for study (n = 372)., Methods: We performed multi-locus sequence typing (MLST) and sequence analysis of five outer membrane protein (OMP) genes, including novel vaccine targets fHbp and nadA., Results: In 2004, strain B:4:P1.15,19 clonal complex ST-32/ET-5 (cc32) predominated throughout Brazil; regional variation in MLST sequence type (ST), fetA, and porB was significant but diversity was limited for nadA and fHbp. Between 1988 and 1996, the São Paulo isolates shifted from clonal complex ST-41/44/Lineage 3 (cc41/44) to cc32. OMP variation was associated with but not predicted by cc or ST. Overall, fHbp variant 1/subfamily B was present in 80% of isolates and showed little diversity. The majority of nadA were similar to reference allele 1., Conclusions: A predominant serogroup B lineage has circulated in Brazil for over a decade with significant regional and temporal diversity in ST, fetA, and porB, but not in nadA and fHbp.

Eukaryotic cells utilize complex signaling systems to detect their environments, responding and adapting as new conditions arise during evolution. The basidiomycete fungus Cryptococcus neoformans is a leading cause of AIDS-related death worldwide and utilizes the calcineurin and protein kinase C-1 (Pkc1) signaling pathways for host adaptation and expression of virulence. In the present studies, a C-terminal zinc finger transcription factor, homologous both to the calcineurin-responsive zinc fingers (Crz1) of ascomycetes and to the Pkc1-dependent specificity protein-1 (Sp1) transcription factors of metazoans, was identified and named SP1 because of its greater similarity to the metazoan factors. Structurally, the Cryptococcus neoformans Sp1 (Cn Sp1) protein was found to have acquired an additional zinc finger motif from that of Crz1 and showed Pkc1-dependent phosphorylation, nuclear localization, and whole genome epistatic associations under starvation conditions. Transcriptional targets of Cn Sp1 shared functional similarities with Crz1 factors, such as cell wall synthesis, but gained the regulation of processes involved in carbohydrate metabolism, including trehalose metabolism, and lost others, such as the induction of autophagy. In addition, overexpression of Cn Sp1 in a pkc1Δ mutant showed restoration of altered phenotypes involved in virulence, including cell wall stability, nitrosative stress, and extracellular capsule production. Cn Sp1 was also found to be important for virulence of the fungus using a mouse model. In summary, these data suggest an evolutionary shift in C-terminal zinc finger proteins during fungal evolution, transforming them from calcineurin-dependent to PKC1-dependent transcription factors, helping to shape the role of fungal pathogenesis of C. neoformans.

Genetic conflicts between retroviruses and their receptors result in the evolution of novel host entry restrictions and novel virus envelopes, and such variants can influence trans-species transmission. We screened rodents and other mammals for sequence variation in the Xpr1 receptor for the mouse xenotropic or polytropic mouse leukemia viruses (X-MLVs or P-MLVs, respectively) of the gammaretrovirus family and for susceptibility to mouse-derived X/P-MLVs and to XMRV (xenotropic murine leukemia virus-related virus), an X-MLV-like virus isolated from humans with prostate cancer and chronic fatigue syndrome. We identified multiple distinct susceptibility phenotypes; these include the four known Xpr1 variants in Mus and a novel fifth Xpr1 gene found in Mus molossinus and Mus musculus. We describe the geographic and species distribution of the Mus Xpr1 variants but failed to find the X-MLV-restrictive laboratory mouse allele in any wild mouse. We used mutagenesis and phylogenetic analysis to evaluate the functional contributions made by constrained, variable, and deleted residues. Rodent Xpr1 is under positive selection, indicating a history of host-pathogen conflicts; several codons under selection have known roles in virus entry. All non-Mus mammals are susceptible to mouse X-MLVs, but some restrict other members of the X/P-MLV family, and the resistance of hamster and gerbil cells to XMRV indicates that XMRV has unique receptor requirements. We show that the hypervariable fourth extracellular XPR1 loop (ECL4) contains three evolutionarily constrained residues that do not contribute to receptor function, we identify two novel residues important for virus entry (I579 and T583), and we describe a unique pattern of ECL4 variation in the three virus-restrictive Xpr1 variants found in MLV-infected house mice; these mice carry different deletions in ECL4, suggesting either that these sites or loop size affects receptor function.

Mouse APOBEC3 (mA3) is a cytidine deaminase with antiviral activity. mA3 is linked to the Rfv3 virus resistance factor, a gene responsible for recovery from infection by Friend murine leukemia virus, and mA3 allelic variants differ in their ability to restrict mouse mammary tumor virus. We sequenced mA3 genes from 38 inbred strains and wild mouse species, and compared the mouse sequence and predicted structure with human APOBEC3G (hA3G). An inserted sequence was identified in the virus restrictive C57BL strain allele that disrupts a splice donor site. This insertion represents the long terminal repeat of the xenotropic mouse gammaretrovirus, and was acquired in Eurasian mice that harbor xenotropic retrovirus. This viral regulatory sequence does not alter splicing but is associated with elevated mA3 expression levels in spleens of laboratory and wild-derived mice. Analysis of Mus mA3 coding sequences produced evidence of positive selection and identified 10 codons with very high posterior probabilities of having evolved under positive selection. Six of these codons lie in two clusters in the N-terminal catalytically active cytidine deaminase domain (CDA), and 5 of those 6 codons are polymorphic in Rfv3 virus restrictive and nonrestrictive mice and align with hA3G CDA codons that are critical for deaminase activity. Homology models of mA3 indicate that the two selected codon clusters specify residues that are opposite each other along the predicted CDA active site groove, and that one cluster corresponds to an hAPOBEC substrate recognition loop. Substitutions at these clustered mA3 codons alter antiviral activity. This analysis suggests that mA3 has been under positive selection throughout Mus evolution, and identified an inserted retroviral regulatory sequence associated with enhanced expression in virus resistant mice and specific residues that modulate antiviral activity.

Invasion of human red blood cells by the malaria parasite Plasmodium falciparum is a coordinated, multi-step process. Here, we describe three novel integral membrane proteins that colocalize on the inner membrane complex immediately beneath the merozoite plasma membrane. Each has six predicted transmembrane domains and is conserved in diverse apicomplexan parasites. Immunoprecipitation studies using specific antibodies reveal that these proteins assemble into a heteromeric complex. Each protein was also expressed on insect cells using the baculovirus vector system with a truncated SUMO tag that facilitates maximal expression and protein purification while permitting cleavage with SUMO protease to release unmodified parasite protein. The expressed proteins were successfully reconstituted into artificial liposomes, but were not recognized by human immune sera. Because all three genes are highly conserved in apicomplexan parasites, the complex formed by their encoded proteins likely serves an essential role for invasive merozoites.

The Fv1 virus resistance gene is a coopted endogenous retrovirus (ERV) sequence related to the gag gene of the MuERV-L ERV family. Three major Fv1 resistance alleles have been identified in laboratory mice, and they target virus capsid genes to produce characteristic patterns of resistance to mouse leukemia viruses (MLVs). We identified Fv1 in 3 of the 4 Mus subgenera; its absence from Coelomys and 1 of 3 species of Pyromys indicate Fv1 was acquired shortly after the origin of the Mus genus. We sequenced Fv1 genes from 21 mice representative of the major taxonomic groups of Mus. Two lines of evidence indicate that Fv1 has had antiviral function for 7 million years of evolution. First, 2 species of African pygmy mice (subgenus Nannomys) show an Fv1-like MLV resistance, and transduced cells expressing the Nannomys Fv1 gene reproduce this resistance pattern. Second, sequence comparisons suggest that Fv1 has been involved in genetic conflicts throughout Mus evolution. We found evidence for strong positive selection of Fv1 and identified 6 codons that show evidence of positive selection: 3 codons in the C-terminal region including 2 previously shown to contribute to Fv1 restriction in laboratory mice, and 3 codons in a 10-codon segment overlapping the major homology region of Fv1; this segment is known to be involved in capsid multimerization. This analysis suggests that Fv1 has had an antiviral role throughout Mus evolution predating exposure of mice to the MLVs restricted by laboratory mouse Fv1, and suggests a mechanism for Fv1 restriction.

Background: As pathogens that circumvent the host immune response are favoured by selection, so are host alleles that reduce parasite load. Such evolutionary processes leave their signature on the genes involved. Deciphering modes of selection operating on immune genes might reveal the nature of host-pathogen interactions and factors that govern susceptibility in host populations. Such understanding would have important public health implications., Methodology/findings: We analyzed polymorphisms in four mosquito immune genes (SP14D1, GNBP, defensin, and gambicin) to decipher selection effects, presumably mediated by pathogens. Using samples of Anopheles arabiensis, An. quadriannulatus and four An. gambiae populations, as well as published sequences from other Culicidae, we contrasted patterns of polymorphisms between different functional units of the same gene within and between populations. Our results revealed selection signatures operating on different time scales. At the most recent time scale, within-population diversity revealed purifying selection. Between populations and between species variation revealed reduced differentiation (GNBP and gambicin) at coding vs. noncoding- regions, consistent with balancing selection. McDonald-Kreitman tests between An. quadriannulatus and both sibling species revealed higher fixation rate of synonymous than nonsynonymous substitutions (GNBP) in accordance with frequency dependent balancing selection. At the longest time scale (>100 my), PAML analysis using distant Culicid taxa revealed positive selection at one codon in gambicin. Patterns of genetic variation were independent of exposure to human pathogens., Significance and Conclusions: Purifying selection is the most common form of selection operating on immune genes as it was detected on a contemporary time scale on all genes. Selection for "hypervariability" was not detected, but negative balancing selection, detected at a recent evolutionary time scale between sibling species may be rather common. Detection of positive selection at the deepest evolutionary time scale suggests that it occurs infrequently, possibly in association with speciation events. Our results provided no evidence to support the hypothesis that selection was mediated by pathogens that are transmitted to humans.

Celiac disease is an immune-mediated disorder triggered by ingestion of wheat gliadin and related proteins in genetically susceptible individuals. In addition to the characteristic enteropathy, celiac disease is associated with various extraintestinal manifestations, including neurologic complications such as neuropathy, ataxia, seizures, and neurobehavioral changes. The cause of the neurologic manifestations is unknown, but autoimmunity resulting from molecular mimicry between gliadin and nervous system proteins has been proposed to play a role. In this study, we sought to investigate the immune reactivity of the anti-gliadin Ab response toward neural proteins. We characterized the binding of affinity-purified anti-gliadin Abs from immunized animals to brain proteins by one- and two-dimensional gel electrophoresis, immunoblotting, and peptide mass mapping. The major immunoreactive protein was identified as synapsin I. Anti-gliadin Abs from patients with celiac disease also bound to the protein. Such cross-reactivity may provide clues into the pathogenic mechanism of the neurologic deficits that are associated with gluten sensitivity.

Purpose: To identify differentially expressed genes in healing mouse corneas by using cDNA microarrays., Methods: Transepithelial excimer laser ablations were performed on mouse corneas, and the wounds were allowed to heal partially in vivo for 18 to 22 hours. Total RNA was isolated from both normal and healing corneas and was used for synthesis of cDNA probes. 33P-labeled exponential cDNA probes were hybridized to mouse cDNA nylon arrays., Results: Of the 1176 genes on the nylon arrays, the expression of 37 was upregulated and that of 27 was downregulated more than fivefold in the healing corneas compared with the normal, uninjured corneas. Interleukin (IL)-1beta, laminin-5, and thrombospondin-1, which have been shown to be upregulated in healing corneas, were all found to be induced in the corneas in response to excimer laser treatment. Many genes were identified for the first time to be differentially regulated during corneal wound healing. Among the upregulated genes were intercellular adhesion molecule (ICAM)-1, macrophage inflammatory proteins, suppressors of cytokine signaling proteins (SOCS), IL-10 receptor, and galectin-7. Among the downregulated genes were connexin-31, a gap junction protein; ZO1 and occludin, tight junction proteins; and Smad2, a key component in the TGFbeta signaling pathway. Microarray data were validated on a limited number of genes by semiquantitative RT-PCR and Western blot analyses., Conclusions: Gene array technology was used to identify for the first time many genes that are differentially regulated during corneal wound healing. These differentially expressed genes have not previously been investigated in the context of wound healing and represent novel factors for further study of the mechanism of wound healing.

In sexual species, autosomal alleles are transmitted through multigeneration organismal pedigrees via pathways of descent involving both genders. Here, models assess the sampling properties of these gender-described transmission pathways. An isolation-by-distance model of mating was used to construct a series of computer population pedigrees by systematically varying neighborhood size and the timing of isolation events in sundered populations. For each known pedigree, a matrix of true coancestry coefficients between all individuals in the final generation was calculated and compared (using cophenetic correlations) to mean pairwise times to common ancestry as estimated by sampling varying numbers of gender-defined lineage routes available to individual alleles through that pedigree. When few lineage routes were sampled, agreement between the estimated and the true pedigree was poor and showed a large variance. Agreement improved as more lineage routes were incorporated and asymptotically approached plateau levels predictably relatable to the magnitude of population structure. Results underscore a distinction between the composite genealogical information in a population pedigree and the subsets of that information registered in allelic lineage pathways., (© 1998 The Society for the Study of Evolution.)