Low back pain is a highly prevalent clinical problem and intervertebral disc (IVD) degeneration is now accepted as the major pathophysiological mechanism responsible for this condition. Accumulating evidence suggests that inflammation plays a crucial role in the progression of human IVD degeneration, with macrophages being pointed as the key immune cell players in this process since their infiltration in degenerated IVD samples has been extensively demonstrated. Since they are highly plastic, macrophages can play different roles depending on the microenvironmental cues. The study of inflammation associated with IVD degeneration has been somehow neglected and one of the reasons is related with lack of adequate models. To overcome this, we established and characterized a new model of IVD organ culture under pro-inflammatory conditions to further dissect the role of macrophages in IVD associated immune response. For that, human monocyte-derived macrophages were co-cultured either with bovine caudal IVD punches in the presence of the pro-inflammatory cytokine IL-1β, or IVD-conditioned medium (CM), to investigate how IVD-produced factors influence macrophage phenotype. After 72 h, metabolic activity, gene expression and cytokine profile of macrophages and IVD cells were measured. Our results show that macrophages and IVDs remain metabolically active in the presence of IL-1β, significantly upregulate CCR7 gene expression and increase production of IL-6 on macrophages. When treating macrophages with IL-1β-IVD-CM, CCR7 upregulation follows the same trend, while for IL-6 an opposite effect was observed. On the other hand, macrophages interfere with IVD ECM remodeling, decreasing MMP3 expression and downregulating aggrecan and collagen II gene expression in the presence of IL-1β. Overall, the co-culture model established in this study can be considered a suitable approach to address the cellular and molecular pathways that regulate macrophage-IVD crosstalk, suggesting that degenerated IVD tissue tends to polarize human macrophages toward a more pro-inflammatory profile, which seems to aggravate IVD degeneration. This model could be used to improve the knowledge of the mechanisms that link IVD degeneration and the immune response.

Guanylate binding proteins (GBPs) are paramount in the host immunity by providing defense against invading pathogens. Multigene families related to the immune system usually show that the duplicated genes can either undergo deletion, gain new functions, or become non-functional. Here, we show that in muroids, the Gbp genes followed an unusual pattern of gain and loss of genes. Muroids present a high diversity and plasticity regarding Gbp synteny, with most species presenting two Gbp gene clusters. The phylogenetic analyses revealed seven different Gbps groups. Three of them clustered with GBP2, GBP5 and GBP6 of primates. Four new Gbp genes that appear to be exclusive to muroids were identified as Gbpa, b, c and d. A duplication event occurred in the Gbpa group in the common ancestor of Muridae and Cricetidae (~20 Mya), but both copies were deleted from the genome of Mus musculus, M. caroli and Cricetulus griseus. The Gbpb gene emerged in the ancestor of Muridae and Cricetidae and evolved independently originating Gbpb1 in Muridae, Gbpb2 and Gbpb3 in Cricetidae. Since Gbpc appears only in three species, we hypothesize that it was present in the common ancestor and deleted from most muroid genomes. The second Gbp gene cluster, Gbp6, is widespread across all muroids, indicating that this cluster emerged before the Muridae and Cricetidae radiation. An expansion of Gbp6 occurred in M. musculus and M. caroli probably to compensate the loss of Gbpa and b. Gbpd is divided in three groups and is present in most muroids suggesting that a duplication event occurred in the common ancestor of Muridae and Cricetidae. However, in Grammomys surdaster and Mus caroli, Gbpd2 is absent, and in Arvicanthis niloticus, Gbpd1 appears to have been deleted. Our results further demonstrated that primate GBP1, GBP3 and GBP7 are absent from the genome of muroids and showed that the Gbp gene annotations in muroids were incorrect. We propose a new classification based on the phylogenetic analyses and the divergence between the groups. Extrapolations to humans based on functional studies of muroid Gbps should be re-evaluated. The evolutionary analyses of muroid Gbp genes provided new insights about the evolution and function of these genes.

No abstract

Guanylate binding proteins (GBPs) represent an evolutionary ancient protein family widely distributed among eukaryotes. They are interferon (IFN)-inducible guanosine triphosphatases that belong to the dynamin superfamily. GBPs are known to have a major role in the cell-autonomous innate immune response against bacterial, parasitic and viral infections and are also involved in inflammasome activation. Evolutionary studies depicted that GBPs present a pattern of gain and loss of genes in each family with several genes pseudogenized and some genes more divergent, indicative for the birth-and-death evolution process. Most species harbor large GBP gene clusters encoding multiple paralogs. Previous functional studies mainly focused on mouse and human GBPs, but more data are becoming available, broadening the understanding of this multifunctional protein family. In this review, we will provide new insights and give a broad overview about GBP evolution, conservation and their roles in all studied species, including plants, invertebrates and vertebrates, revealing how far the described features of GBPs can be transferred to other species.

Rabbit hemorrhagic disease (RHD) causes high mortality and morbidity in European rabbits (Oryctolagus cuniculus). In Africa, the presence of the causative agent, the rabbit hemorrhagic disease virus (RHDV), was first confirmed in 1992 (genotype Lagovirus europaeus/GI.1). In 2015, the new genotype Lagovirus europaeus/GI.2 (RHDV2/b) was detected in Tunisia. Currently, GI.2 strains are present in several North and Sub-Saharan African countries. Considerable economic losses have been observed in industrial and traditional African rabbitries due to RHDV. Like other RNA viruses, this virus presents high recombination rates, with the emergence of GI.2 being associated with a recombinant strain. Recombination events have been detected with both pathogenic (GI.1b and GII.1) and benign (GI.3 and GI.4) strains. We obtained complete genome sequences of Tunisian GI.2 strains collected between 2018 and 2020 and carried out phylogenetic analyses. The results revealed that Tunisian strains are GI.3P-GI.2 strains that were most likely introduced from Europe. In addition, the results support the occurrence of multiple introductions of GI.2 into Africa, stressing the need for characterizing complete genome sequences of the circulating lagoviruses to uncover their origin. Continued monitoring and control of rabbit trade will grant a better containment of the disease and reduce the disease-associated economic losses.

Viruses that affect lagomorphs have decades of reported history of spillover events.One of these viruses is the causative agent of the so-called rabbit or 'lagomorph' haemorrhagic disease (e.g. Lagovirus europaeus/GI.1 and L. europaeus/GI.2). In particular, L. europaeus/GI.2 has shown a great capacity to recombine with existing lagoviruses. In fact, it has replaced the former GI.1 genotype in the wild, and recently, an increase on spillover events has been detected among several lagomorph species including European and North American species of hares. In this study, we report for the first time the infection of a wild Iberian hare with GI.2 (RHDV2/b), potential shedding and associated histopathological alterations. We identify the recombinant GI.4P-GI.2 as causative of the infection and discuss plausible causes regarding the origin of the spillover event and its potential consequences for the Iberian hare wild populations, which is an endemic species of the Iberian Peninsula as well as an important game and prey species for many predators, including endangered species.

Adenoviruses (AdV) are pathogens capable of infecting animals and humans leading to a wide spectrum of diseases. They have a widespread geographical dissemination and infect several species, including red squirrels (Sciurus vulgaris). In the Iberian Peninsula, there are no reports of infection of red squirrels by AdV. In this study, we report for the first time the detection of squirrel AdV in a red squirrel trapped in central Portugal. The phylogenetic analysis showed that the sequences obtained for the hexon and the penton base genes were similar to the Korean squirrel AdV strain and strongly divergent from the strains present in red squirrels in Europe. These findings indicate the emergence of a new strain circulating in Europe and raise concerns regarding the conservation of the red squirrel.

The European rabbit (Oryctolagus cuniculus) populations of the Iberian Peninsula have been severely affected by the emergence of the rabbit haemorrhagic disease virus (RHDV) Lagovirus europaeus/GI.2 (RHDV2/b). Bushflies and blowflies (Muscidae and Calliphoridae families, respectively) are important RHDV vectors in Oceania, but their epidemiological role is unknown in the native range of the European rabbit. In this study, scavenging flies were collected between June 2018 and February 2019 in baited traps at one site in southern Portugal, alongside a longitudinal capture-mark-recapture study of a wild European rabbit population, aiming to provide evidence of mechanical transmission of GI.2 by flies. Fly abundance, particularly from Calliphoridae and Muscidae families, peaked in October 2018 and in February 2019. By employing molecular tools, we were able to detect the presence of GI.2 in flies belonging to the families Calliphoridae, Muscidae, Fanniidae and Drosophilidae. The positive samples were detected during an RHD outbreak and absent in samples collected when no evidence of viral circulation in the local rabbit population was found. We were able to sequence a short viral genomic fragment, confirming its identity as RHDV GI.2. The results suggest that scavenging flies may act as mechanical vectors of GI.2 in the native range of the southwestern Iberian subspecies O. cuniculus algirus. Future studies should better assess their potential in the epidemiology of RHD and as a tool for monitoring viral circulation in the field.