Cas9+ conditionally-immortalized macrophages as a tool for bacterial pathogenesis and beyond.

Macrophages play critical roles in immunity, development, tissue repair, and cancer, but studies of their function have been hampered by poorly-differentiated tumor cell lines and genetically-intractable primary cells. Here we report a facile system for genome editing in non-transformed macrophages by differentiating ER-Hoxb8 myeloid progenitors from Cas9-expressing transgenic mice. These conditionally immortalized macrophages (CIMs) retain characteristics of primary macrophages derived from the bone marrow yet allow for easy genetic manipulation and a virtually unlimited supply of cells. We demonstrate the utility of this system for dissection of host genetics during intracellular bacterial infection using two important human pathogens: Listeria monocytogenes and Mycobacterium tuberculosis

First detection of kobuvirus in farm animals in Brazil and the Netherlands.

Animal kobuviruses have been described in pigs, cattle, sheep and bats in countries in Asia and Europe. The virus can be detected in fecal and serum samples of infected animals with or without diarrhea, but most of the clinical as well as epidemiological features of kobuvirus infection are still unknown. This study reports the first detection of kobuvirus in farm animals from Brazil and the Netherlands and the molecular analysis of the detected strains. In Brazil, 53% (61/115) of the pigs (suckling, weaned and sows) were shedding porcine kobuvirus in feces, while in the Netherlands 16.7% (3/18) of the tested weaned pigs were infected. Kobuviruses detected in fecal samples of pigs in Brazil showed association (p = 0.0002) with diarrhea. In pig serum, kobuvirus was detected at different ages (3, 21, 36, 60, 75, and 180 days), with an overall rate of 76.7% (23/30). The sequencing of amplicons detected in serum of pigs of different ages suggested reinfection and no persistent infection. Kobuvirus was also detected in sheep and cattle feces from Brazil and the Netherlands, respectively. Phylogenetic analyses of Brazilian and Dutch kobuviruses from pig, cattle and sheep revealed genetic variability, particularly in one strain detected in sheep feces, which was more closely related to human Aichi virus. The molecular and phylogenetic analyses performed with other published kobuvirus strains and the strains presented in this study, showed that, in most of the cases, kobuvirus seems to group according to host species, but not to geographical region of origin. The data presented in this study contribute to the comprehension of kobuvirus epidemiology and also to the molecular identification of kobuvirus strains circulating worldwide

Changes on the viral capsid surface during the evolution of porcine circovirus type 2 (PCV2) from 2009 till 2018 may lead to a better receptor binding

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases (PCVAD). Three major PCV2 genotypes (PCV2a, PCV2b, and PCV2d) have been identified globally. Despite their worldwide distribution, the prevalence and genetic evolution of PCV2 in Belgium has not previously been determined. In this study, 319 samples from animals suffering from diseases likely to be associated with PCV2 were collected from 2009 to 2018 and analysed by virus titration. The overall prevalence of PCV2 in PCVAD-suspected cases was 15.7 per cent (50/319). The phylogenetic analysis demonstrated that at least three genotypes (PCV2a, PCV2b, and PCV2d) circulated in Belgium from 2009 till 2018, and that PCV2 evolved from PCV2a to PCV2b and from PCV2d-1 to PCV2d-2. Sequence comparison among the forty-three PCV2 isolates showed that they had 89.7-100 per cent nucleotide-sequence and 88.5-100 per cent amino-acid-sequence identities. Three amino acid sites were under positive selection. Three-dimensional analysis of genotype-specific amino acids revealed that most of the mutations were on the outside of the cap protein with a few conserved mutations present on the inner side. Mutations toward more basic amino acids were found on the upper and tail parts of two connecting capsid proteins which form one big contact region, most probably involved in receptor binding. The lower part was relatively conserved. This polarity change together with the formation of an extruding part drive the virus to a more efficient GAG receptor binding. Taken together, these results showed a genotype shift from PCV2a to PCV2b and later on from PCV2d-1 to PCV2d-2, and a PCV2 evolution toward a better receptor binding capacity

Evaluation morphométrique des chevaux pur-sang Arabe en Algérie: mensurations corporelles et proposition d’équations barymétriques

Cette étude vise à la caractérisation morphobiométrique des chevaux de course pur-sang arabe et à l’estimation d’équations barymétriques adaptées à cette race. La caractérisation a concerné 98 chevaux, dont 44 femelles et 54 mâles, tous âgés de trois ans et plus, auprès de 77 propriétaires-éleveurs dans 3 hippodromes d’Algérie (Zemmouri, Tiaret et Caroubier). Dix-neuf mensurations étaient relevées ainsi que le poids vif (PV). Le poids moyen est de 456,2 +/- 43,0 kg, variant de 335 kg à 545 kg. La sélection des variables à inclure dans les équations barymétriques a été réalisée à l’aide de la procédure stepwise du SAS. Quatre mensurations parmi les 19 réalisées ont été retenues pour la proposition d’équations d’estimation du poids vif des chevaux : le périmètre thoracique (PT), la hauteur à la croupe (HC), la longueur de l’encolure (LE) et le tour de l’encolure (TE). Ainsi, les équations proposées pour les mâles et pour les femelles sont respectivement de : PV= 7,024*PT - 787,119 (R²=0,99); PV=6,207*PT + 0,633*HC + 0,668*TE - 0,878*LE - 746,370 (R²=0,96). Les résultats de cette étude devraient permettre aux propriétaires-éleveurs et entraineurs de suivre aisément le poids de leurs chevaux. Ce suivi est nécessaire pour adapter l’activité et l’alimentation des chevaux et favoriser leur performance en course

Persistence pays: how viruses promote host group survival.

Recently, we have realized that viruses numerically dominate all life. Although viruses are known to affect host survival in populations, this has not been previously evaluated in the context of host group selection. Group selection per se is not a currently accepted idea and its apparent occurrence is explained by statistical gene frequency models of kin selection. Viruses were not considered in such models. Prevalent views associate viruses and disease. Yet many viruses establish species-specific persistent, inapparent infections that are stable on an evolutionary time scale. Such persistent infections can have large effects on relative reproductive fitness of competing host populations. In this essay, I present arguments on how persistent infections can promote population survival. Mouse hepatitis virus is used as well studied examplar to re-evaluate the theoretical basis of the mouse haystack model of M Smith. This virus-centric re-examination concludes that viruses can indeed affect and promote relative group selection

Absence of Neu5Gc and Presence of Anti-Neu5Gc Antibodies in Humans-An Evolutionary Perspective.

The glycocalyx of human cells differs from that of many other mammals by the lack of the sialic acid N-glycolylneuraminic acid (Neu5Gc) and increased abundance of its precursor N-acetylneuraminic acid (Neu5Ac). Most humans also have circulating antibodies specifically targeting the non-human sialic acid Neu5Gc. Recently, several additional mammalian species have been found to also lack Neu5Gc. In all cases, loss-of-function mutations in the gene encoding the sialic acid-modifying enzyme CMAH are responsible for the drastic change in these species. Unlike other glycan antigens, Neu5Gc apparently cannot be produced by microbes, raising the question about the origin of these antibodies in humans. Dietary exposure and presentation on bacteria coating themselves with Neu5Gc from the diet are distinct possibilities. However, the majority of the non-human species that lack Neu5Gc do not consume diets rich in Neu5Gc, making it unlikely that they will have been immunized against this sialic acid. A notable exception are mustelids (ferrets, martens and their relatives) known for preying on various small mammal species rich in Neu5Gc. No studies exist on levels of anti-Neu5Gc antibodies in non-human species. Evolutionary scenarios for the repeated, independent fixation of CMAH loss-of-function mutations at various time points in the past include strong selection by parasites, especially enveloped viruses, stochastic effects of genetic drift, and directional selection via female immunity to paternal Neu5Gc. Convergent evolution of losses of the vertebrate-specific self-glycan Neu5Gc are puzzling and may represent a prominent way in which glycans become agents of evolutionary change in their own right. Such change may include the reconfiguration of innate immune lectins that use self-sialic acids as recognition patterns

Expression of Ifnlr1 on intestinal epithelial cells is critical to the antiviral effects of IFN-lambda against norovirus and reovirus

Lambda interferon (IFN-λ) has potent antiviral effects against multiple enteric viral pathogens, including norovirus and rotavirus, in both preventing and curing infection. Because the intestine includes a diverse array of cell types, however, the cell(s) upon which IFN-λ acts to exert its antiviral effects is unclear. Here, we sought to identify IFN-λ-responsive cells by generation of mice with lineage-specific deletion of the receptor for IFN-λ, Ifnlr1. We found that expression of IFNLR1 on intestinal epithelial cells (IECs) in the small intestine and colon is required for enteric IFN-λ antiviral activity. IEC Ifnlr1 expression also determines the efficacy of IFN-λ in resolving persistent murine norovirus (MNoV) infection and regulates fecal shedding and viral titers in tissue. Thus, the expression of Ifnlr1 by IECs is necessary for the response to both endogenous and exogenous IFN-λ. We further demonstrate that IEC Ifnlr1 expression is required for the sterilizing innate immune effects of IFN-λ by extending these findings in Rag1-deficient mice. Finally, we assessed whether our findings pertained to multiple viral pathogens by infecting mice specifically lacking IEC Ifnlr1 expression with reovirus. These mice phenocopied Ifnlr1-null animals, exhibiting increased intestinal tissue titers and enhanced reovirus fecal shedding. Thus, IECs are the critical cell type responding to IFN-λ to control multiple enteric viruses. This is the first genetic evidence that supports an essential role for IECs in IFN-λ-mediated control of enteric viral infection, and these findings provide insight into the mechanism of IFN-λ-mediated antiviral activity. IMPORTANCE Human noroviruses (HNoVs) are the leading cause of epidemic gastroenteritis worldwide. Type III interferons (IFN-λ) control enteric viral infections in the gut and have been shown to cure mouse norovirus, a small-animal model for HNoVs. Using a genetic approach with conditional knockout mice, we identified IECs as the dominant IFN-λ-responsive cells in control of enteric virus infection in vivo. Upon murine norovirus or reovirus infection, Ifnlr1 depletion in IECs largely recapitulated the phenotype seen in Ifnlr1(−/−) mice of higher intestinal tissue viral titers and increased viral shedding in the stool. Moreover, IFN-λ-mediated sterilizing immunity against murine norovirus requires the capacity of IECs to respond to IFN-λ. These findings clarify the mechanism of action of this cytokine and emphasize the therapeutic potential of IFN-λ for treating mucosal viral infections

Outlook Magazine, Winter 2015

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