Neuroinvasion and anosmia are independent phenomena upon infection with SARS-CoV-2 and its variants.

Anosmia was identified as a hallmark of COVID-19 early in the pandemic, however, with the emergence of variants of concern, the clinical profile induced by SARS-CoV-2 infection has changed, with anosmia being less frequent. Here, we assessed the clinical, olfactory and neuroinflammatory conditions of golden hamsters infected with the original Wuhan SARS-CoV-2 strain, its isogenic ORF7-deletion mutant and three variants: Gamma, Delta, and Omicron/BA.1. We show that infected animals develop a variant-dependent clinical disease including anosmia, and that the ORF7 of SARS-CoV-2 contributes to the induction of olfactory dysfunction. Conversely, all SARS-CoV-2 variants are neuroinvasive, regardless of the clinical presentation they induce. Taken together, this confirms that neuroinvasion and anosmia are independent phenomena upon SARS-CoV-2 infection. Using newly generated nanoluciferase-expressing SARS-CoV-2, we validate the olfactory pathway as a major entry point into the brain in vivo and demonstrate in vitro that SARS-CoV-2 travels retrogradely and anterogradely along axons in microfluidic neuron-epithelial networks.

Prevalence of blood and skin trypanosomes in domestic and wild fauna from two sleeping sickness foci in Southern Cameroon.

Although studies on African Trypanosomiases revealed a variety of trypanosome species in the blood of various animal taxa, animal reservoirs of Trypanosoma brucei gambiense and anatomical niches such as skin have been overlooked in most epidemiological settings. This study aims to update epidemiological data on trypanosome infections in animals from human African trypanosomiasis (HAT) foci of Cameroon. Blood and skin snips were collected from 291 domestic and wild animals. DNA was extracted from blood and skin snips and molecular approaches were used to identify different trypanosomes species. Immunohistochemical analyses were used to confirm trypanosome infections in skin snips. PCR revealed 137 animals (47.1%) with at least one trypanosome species in the blood and/or in the skin. Of these 137 animals, 90 (65.7%) and 32 (23.4%) had trypanosome infections respectively in the blood and skin. Fifteen (10.9%) animals had trypanosome infections in both blood and skin snip. Animals from the Campo HAT focus (55.0%) were significantly (X2 = 17.6; P< 0.0001) more infected than those (29.7%) from Bipindi. Trypanosomes of the subgenus Trypanozoon were present in 27.8% of animals while T. vivax, T. congolense forest type and savannah type were detected in 16.5%, 10.3% and 1.4% of animals respectively. Trypanosoma b. gambiense infections were detected in the blood of 7.6% (22/291) of animals. No T. b. gambiense infection was detected in skin. This study highlights the presence of several trypanosome species in the blood and skin of various wild and domestic animals. Skin appeared as an anatomical reservoir for trypanosomes in animals. Despite methodological limitations, pigs, sheep, goats and wild animals were confirmed as potential reservoirs of T. b. gambiense. These animal reservoirs must be considered for the designing of control strategies that will lead to sustainable elimination of HAT.

Splenic clearance of rigid erythrocytes as an inherited mechanism for splenomegaly and natural resistance to malaria.

BACKGROUND: In malaria-endemic areas, subjects from specific groups like Fulani have a peculiar protection against malaria, with high levels of IgM but also frequent anaemia and splenomegaly. The mechanisms underlying this phenotype remain elusive. METHODS: In a cohort study set up in Benin, West Africa, after a careful evaluation of malaria-related phenotypes, we measured the deformability of circulating erythrocytes in genetically distinct groups (including Fulani) living in sympatry, using ektacytometry and microsphiltration, a mimic of how the spleen clears rigid erythrocytes. Heritability of erythrocytes deformability was calculated, followed by a genome-wide association study (GWAS) of the same phenotype. FINDINGS: Compared to non-Fulani, Fulani displayed a higher deformability of circulating erythrocytes, pointing to an enhanced clearance of rigid erythrocytes by the spleen. This phenotype was observed in individuals displaying markers of Plasmodium falciparum infection. The heritability of this new trait was high, with a strong multigenic component. Five of the top 10 genes selected by a population structure-adjusted GWAS, expressed in the spleen, are potentially involved in splenic clearance of erythrocytes (CHERP, MB, PALLD, SPARC, PDE10A), through control of vascular tone, collagen synthesis and macrophage activity. INTERPRETATION: In specific ethnic groups, genetically-controlled processes likely enhance the innate retention of infected and uninfected erythrocytes in the spleen, explaining splenomegaly, anaemia, cryptic intrasplenic parasite loads, hyper-IgM, and partial protection against malaria. Beyond malaria-related phenotypes, inherited splenic hyper-filtration of erythrocytes may impact the pathogenesis of other hematologic diseases. FUNDING: ANR, National Geographic Society, IMEA, IRD, and Région Ile-de-France.

A live measles-vectored COVID-19 vaccine induces strong immunity and protection from SARS-CoV-2 challenge in mice and hamsters.

Several COVID-19 vaccines have now been deployed to tackle the SARS-CoV-2 pandemic, most of them based on messenger RNA or adenovirus vectors.The duration of protection afforded by these vaccines is unknown, as well as their capacity to protect from emerging new variants. To provide sufficient coverage for the world population, additional strategies need to be tested. The live pediatric measles vaccine (MV) is an attractive approach, given its extensive safety and efficacy history, along with its established large-scale manufacturing capacity. We develop an MV-based SARS-CoV-2 vaccine expressing the prefusion-stabilized, membrane-anchored full-length S antigen, which proves to be efficient at eliciting strong Th1-dominant T-cell responses and high neutralizing antibody titers. In both mouse and golden Syrian hamster models, these responses protect the animals from intranasal infectious challenge. Additionally, the elicited antibodies efficiently neutralize in vitro the three currently circulating variants of SARS-CoV-2.

Generating genomic platforms to study Candida albicans pathogenesis.

The advent of the genomic era has made elucidating gene function on a large scale a pressing challenge. ORFeome collections, whereby almost all ORFs of a given species are cloned and can be subsequently leveraged in multiple functional genomic approaches, represent valuable resources toward this endeavor. Here we provide novel, genome-scale tools for the study of Candida albicans, a commensal yeast that is also responsible for frequent superficial and disseminated infections in humans. We have generated an ORFeome collection composed of 5099 ORFs cloned in a Gateway™ donor vector, representing 83% of the currently annotated coding sequences of C. albicans. Sequencing data of the cloned ORFs are available in the CandidaOrfDB database at http://candidaorfeome.eu. We also engineered 49 expression vectors with a choice of promoters, tags and selection markers and demonstrated their applicability to the study of target ORFs transferred from the C. albicans ORFeome. In addition, the use of the ORFeome in the detection of protein-protein interaction was demonstrated. Mating-compatible strains as well as Gateway™-compatible two-hybrid vectors were engineered, validated and used in a proof of concept experiment. These unique and valuable resources should greatly facilitate future functional studies in C. albicans and the elucidation of mechanisms that underlie its pathogenicity.

Plasmodium falciparum parasite population structure and gene flow associated to anti-malarial drugs resistance in Cambodia.

BACKGROUND: Western Cambodia is recognized as the epicentre of emergence of Plasmodium falciparum multi-drug resistance. The emergence of artemisinin resistance has been observed in this area since 2008-2009 and molecular signatures associated to artemisinin resistance have been characterized in k13 gene. At present, one of the major threats faced, is the possible spread of Asian artemisinin resistant parasites over the world threatening millions of people and jeopardizing malaria elimination programme efforts. To anticipate the diffusion of artemisinin resistance, the identification of the P. falciparum population structure and the gene flow among the parasite population in Cambodia are essential. METHODS: To this end, a mid-throughput PCR-LDR-FMA approach based on LUMINEX technology was developed to screen for genetic barcode in 533 blood samples collected in 2010-2011 from 16 health centres in malaria endemics areas in Cambodia. RESULTS: Based on successful typing of 282 samples, subpopulations were characterized along the borders of the country. Each 11-loci barcode provides evidence supporting allele distribution gradient related to subpopulations and gene flow. The 11-loci barcode successfully identifies recently emerging parasite subpopulations in western Cambodia that are associated with the C580Y dominant allele for artemisinin resistance in k13 gene. A subpopulation was identified in northern Cambodia that was associated to artemisinin (R539T resistant allele of k13 gene) and mefloquine resistance. CONCLUSIONS: The gene flow between these subpopulations might have driven the spread of artemisinin resistance over Cambodia.

Draft Genome Sequences of Acinetobacter parvus CM11, Acinetobacter radioresistens CM38, and Stenotrophomonas maltophilia BR12, Isolated from Murine Proximal Colonic Tissue.

Here, we report three genome sequences of bacteria isolated from murine proximal colonic tissue and identified as Acinetobacter parvus CM11, Acinetobacter radioresistens CM38, and Stenotrophomonas maltophilia BR12.

Effects of mefloquine use on Plasmodium vivax multidrug resistance.

Numerous studies have indicated a strong association between amplification of the multidrug resistance-1 gene and in vivo and in vitro mefloquine resistance of Plasmodium falciparum. Although falciparum infection usually is not treated with mefloquine, incorrect diagnosis, high frequency of undetected mixed infections, or relapses of P. vivax infection triggered by P. falciparum infections expose non-P. falciparum parasites to mefloquine. To assess the consequences of such unintentional treatments on P. vivax, we studied variations in number of Pvmdr-1 (PlasmoDB accession no. PVX_080100, NCBI reference sequence NC_009915.1) copies worldwide in 607 samples collected in areas with different histories of mefloquine use from residents and from travelers returning to France. Number of Pvmdr-1 copies correlated with drug use history. Treatment against P. falciparum exerts substantial collateral pressure against sympatric P. vivax, jeopardizing future use of mefloquine against P. vivax. A drug policy is needed that takes into consideration all co-endemic species of malaria parasites.

Rapid 96-well plates DNA extraction and sequencing procedures to identify genome-wide transposon insertion sites in a difficult to lyse bacterium: Lactobacillus casei.

Random transposon mutagenesis followed by adequate screening methods is an unavoidable procedure to characterize genetics of bacterial adaptation to environmental changes. We have recently constructed a mutant library of Lactobacillus casei and we aimed to fully annotate it. However, we have observed that, for L. casei which is a difficult to lyse bacterium, methods used to identify the transposon insertion site in a few mutants (transposon rescue by restriction and recircularization or PCR-based methods) were not transposable for a larger number because they are too time-consuming and sometimes not reliable. Here, we describe a method for large-scale and reliable identification of transposon insertion sites in a L. casei mutant library of 9250 mutants. DNA extraction procedure based on silica membranes in 96-column format was optimized to obtain genomic DNA from a large number of mutants. Then reliable direct genomic sequencing was improved to fit the obtained genomic DNA extracts. Using this procedure, readable and identifiable sequences were obtained for 87% of the L. casei mutants. This method extends the applications of a library of this type, reduces the number of insertions needed to be screened, and allows selection of specific mutants from an arrayed and stored mutant library. This method is applicable to any already existing mutant library (obtained by transposon or insertional mutagenesis) and could be useful for other bacterial species, especially for highly lysis-resistant bacteria species such as lactic acid bacteria.

Streptococcus agalactiae clones infecting humans were selected and fixed through the extensive use of tetracycline.

Streptococcus agalactiae (Group B Streptococcus, GBS) is a commensal of the digestive and genitourinary tracts of humans that emerged as the leading cause of bacterial neonatal infections in Europe and North America during the 1960s. Due to the lack of epidemiological and genomic data, the reasons for this emergence are unknown. Here we show by comparative genome analysis and phylogenetic reconstruction of 229 isolates that the rise of human GBS infections corresponds to the selection and worldwide dissemination of only a few clones. The parallel expansion of the clones is preceded by the insertion of integrative and conjugative elements conferring tetracycline resistance (TcR). Thus, we propose that the use of tetracycline from 1948 onwards led in humans to the complete replacement of a diverse GBS population by only few TcR clones particularly well adapted to their host, causing the observed emergence of GBS diseases in neonates.

Dandruff is associated with disequilibrium in the proportion of the major bacterial and fungal populations colonizing the scalp.

The bacterial and fungal communities associated with dandruff were investigated using culture-independent methodologies in the French subjects. The major bacterial and fungal species inhabiting the scalp subject's were identified by cloning and sequencing of the conserved ribosomal unit regions (16S for bacterial and 28S-ITS for fungal) and were further quantified by quantitative PCR. The two main bacterial species found on the scalp surface were Propionibacterium acnes and Staphylococcus epidermidis, while Malassezia restricta was the main fungal inhabitant. Dandruff was correlated with a higher incidence of M. restricta and S. epidermidis and a lower incidence of P. acnes compared to the control population (p<0.05). These results suggested for the first time using molecular methods, that dandruff is linked to the balance between bacteria and fungi of the host scalp surface.

Plasmodium falciparum Na+/H+ exchanger (pfnhe-1) genetic polymorphism in Indian Ocean malaria-endemic areas.

To date, 11 studies conducted in different countries to test the association between Plasmodium falciparum Na(+)/H(+) exchanger gene (pfnhe-1; PF13_0019) polymorphisms and in vitro susceptibility to quinine have generated conflicting data. In this context and to extend our knowledge of the genetic polymorphism of Pfnhe gene, we have sequenced the ms4760 locus from 595 isolates collected in the Comoros (N = 250; an area with a high prevalence of chloroquine and sulfadoxine-pyrimethamine resistance) and Madagascar (N = 345; a low drug-resistance area). Among them, 29 different alleles were observed, including 8 (27%) alleles not previously described. Isolates from the Comoros showed more repeats in block II (DNNND), which some studies have found to be positively associated with in vitro resistance to quinine, compared with isolates from Madagascar. Additional studies are required to better define the mechanisms underlying quinine resistance, which involve multiple gene interactions.

Reduced impact of pyrimethamine drug pressure on Plasmodium malariae dihydrofolate reductase gene.

Molecular investigations performed following the emergence of sulfadoxine-pyrimethamine (SP) resistance in Plasmodium falciparum have allowed the identification of the dihydrofolate reductase (DHFR) enzyme as the target of pyrimethamine. Although clinical cases of Plasmodium malariae are not usually treated with antifolate therapy, incorrect diagnosis and the high frequency of undetected mixed infections has probably exposed non-P. falciparum parasites to antifolate therapy in many areas. In this context, we aimed to assess the worldwide genetic diversity of the P. malariae dhfr gene in 123 samples collected in Africa and Asia, areas with different histories of SP use. Among the 10 polymorphic sites found, we have observed 7 new mutations (K55E, S58R, S59A, F168S, N194S, D207G, and T221A), which led us to describe 6 new DHFR proteins. All isolates from African countries were classified as wild type, while new mutations and haplotypes were recognized as exclusive to Madagascar (except for the double mutations at nucleotides 341 and 342 [S114N] found in one Cambodian isolate). Among these nonsynonymous mutations, two were likely related to pyrimethamine resistance: S58R (corresponding to C59R in P. falciparum and S58R in Plasmodium vivax; observed in one Malagasy sample) and S114N (corresponding to S108N in P. falciparum and S117N in P. vivax; observed in three Cambodian samples).

Evolutionary genetic dissection of human interferons.

Interferons (IFNs) are cytokines that play a key role in innate and adaptive immune responses. Despite the large number of immunological studies of these molecules, the relative contributions of the numerous IFNs to human survival remain largely unknown. Here, we evaluated the extent to which natural selection has targeted the human IFNs and their receptors, to provide insight into the mechanisms that govern host defense in the natural setting. We found that some IFN-α subtypes, such as IFN-α6, IFN-α8, IFN-α13, and IFN-α14, as well as the type II IFN-γ, have evolved under strong purifying selection, attesting to their essential and nonredundant function in immunity to infection. Conversely, selective constraints have been relaxed for other type I IFNs, particularly for IFN-α10 and IFN-ε, which have accumulated missense or nonsense mutations at high frequencies within the population, suggesting redundancy in host defense. Finally, type III IFNs display geographically restricted signatures of positive selection in European and Asian populations, indicating that genetic variation at these genes has conferred a selective advantage to the host, most likely by increasing resistance to viral infection. Our population genetic analyses show that IFNs differ widely in their biological relevance, and highlight evolutionarily important determinants of host immune responsiveness.

Performance of the CareStart™ G6PD deficiency screening test, a point-of-care diagnostic for primaquine therapy screening.

Development of reliable, easy-to-use, rapid diagnostic tests (RDTs) to detect glucose-6-phosphate dehydrogenase (G6PD) deficiency at point of care is essential to deploying primaquine therapies as part of malaria elimination strategies. We assessed a kit under research and development called CareStart™ G6PD deficiency screening test (Access Bio, New Jersey, USA) by comparing its performance to quantitative G6PD enzyme activity using a standardized spectrophotometric method ('gold standard'). Blood samples (n = 903) were collected from Cambodian adults living in Pailin province, western Cambodia. G6PD enzyme activities ranged from 0 to 20.5 U/g Hb (median 12.0 U/g Hg). Based on a normal haemoglobin concentration and wild-type G6PD gene, the normal values of G6PD enzymatic activity for this population was 3.6 to 20.5 U/g Hg (95(th) percentiles from 5.5 to 17.2 U/g Hg). Ninety-seven subjects (10.7%) had <3.6 U/g Hg and were classified as G6PD deficient. Prevalence of deficiency was 15.0% (64/425) among men and 6.9% (33/478) among women. Genotype was analyzed in 66 G6PD-deficient subjects and 63 of these exhibited findings consistent with Viangchang genotype. The sensitivity and specificity of the CareStart™ G6PD deficiency screening test was 0.68 and 1.0, respectively. Its detection threshold was <2.7 U/g Hg, well within the range of moderate and severe enzyme deficiencies. Thirteen subjects (1.4%, 12 males and 1 female) with G6PD enzyme activities <2 U/g Hg were falsely classified as "normal" by RDT. This experimental RDT test here evaluated outside of the laboratory for the first time shows real promise, but safe application of it will require lower rates of falsely "normal" results.

Evolutionary genetics evidence of an essential, nonredundant role of the IFN-γ pathway in protective immunity.

Identifying how natural selection has affected immunity-related genes can provide insights into the mechanisms that have been crucial for our survival against infection. Rare disorders of either chain of the IFN-γ receptor, but not of IFN-γ itself, have been shown to confer predisposition to mycobacterial disease in patients otherwise normally resistant to most viruses. Here, we defined the levels of naturally occurring variation in the three specific genes controlling the IFN-γ pathway (IFNG, IFNGR1, IFNGR2) and assessed whether and how natural selection has acted on them. To this end, we resequenced the three genes in 186 individuals from sub-Saharan Africa, Europe, and East-Asia. Our results show that IFNG is subject to strong purifying selection against nonsynonymous variants. Conversely, IFNGR1 and IFNGR2 evolve under more relaxed selective constraints, although they are not completely free to accumulate amino acid variation having a major impact on protein function. In addition, we have identified signatures of population-specific positive selection, including at one intronic variant known to be associated with higher production of IFN-γ. The integration of our population genetic data into a clinical framework demonstrates that the IFN-γ pathway is essential and nonredundant in host defense, probably because of its role in protective immunity against mycobacteria.