An equine iPSC-based phenotypic screening platform identifies pro- and anti-viral molecules against West Nile virus.

Outbreaks of West Nile virus (WNV) occur periodically, affecting both human and equine populations. There are no vaccines for humans, and those commercialised for horses do not have sufficient coverage. Specific antiviral treatments do not exist. Many drug discovery studies have been conducted, but since rodent or primate cell lines are normally used, results cannot always be transposed to horses. There is thus a need to develop relevant equine cellular models. Here, we used induced pluripotent stem cells to develop a new in vitro model of WNV-infected equine brain cells suitable for microplate assay, and assessed the cytotoxicity and antiviral activity of forty-one chemical compounds. We found that one nucleoside analog, 2'C-methylcytidine, blocked WNV infection in equine brain cells, whereas other compounds were either toxic or ineffective, despite some displaying anti-viral activity in human cell lines. We also revealed an unexpected proviral effect of statins in WNV-infected equine brain cells. Our results thus identify a potential lead for future drug development and underscore the importance of using a tissue- and species-relevant cellular model for assessing the activity of antiviral compounds.

Transcriptomic Studies Suggest a Coincident Role for Apoptosis and Pyroptosis but Not for Autophagic Neuronal Death in TBEV-Infected Human Neuronal/Glial Cells.

Tick-borne encephalitis virus (TBEV), a member of the Flaviviridae family, Flavivirus genus, is responsible for neurological symptoms that may cause permanent disability or death. With an incidence on the rise, it is the major arbovirus affecting humans in Central/Northern Europe and North-Eastern Asia. Neuronal death is a critical feature of TBEV infection, yet little is known about the type of death and the molecular mechanisms involved. In this study, we used a recently established pathological model of TBEV infection based on human neuronal/glial cells differentiated from fetal neural progenitors and transcriptomic approaches to tackle this question. We confirmed the occurrence of apoptotic death in these cultures and further showed that genes involved in pyroptotic death were up-regulated, suggesting that this type of death also occurs in TBEV-infected human brain cells. On the contrary, no up-regulation of major autophagic genes was found. Furthermore, we demonstrated an up-regulation of a cluster of genes belonging to the extrinsic apoptotic pathway and revealed the cellular types expressing them. Our results suggest that neuronal death occurs by multiple mechanisms in TBEV-infected human neuronal/glial cells, thus providing a first insight into the molecular pathways that may be involved in neuronal death when the human brain is infected by TBEV.

Model of persistent foot-and-mouth disease virus infection in multilayered cells derived from bovine dorsal soft palate.

Foot-and-mouth disease virus (FMDV) causes a highly contagious vesicular disease in livestock, with serious consequences for international trade. The virus persists in the nasopharynx of cattle and this slows down the process to obtain an FMDV-free status after an outbreak. To study biological mechanisms, or to identify molecules that can be targeted to diagnose or interfere with persistence, we developed a model of persistent FMDV infection in bovine dorsal soft palate (DSP). Primary DSP cells were isolated after commercial slaughter and were cultured in multilayers at the air-liquid interface. After 5 weeks of culture without further passage, the cells were infected with FMDV strain O/FRA/1/2001. Approximately, 20% of cells still had a polygonal morphology and displayed tight junctions as in stratified squamous epithelia. Subsets of cells expressed cytokeratin and most or all cells expressed vimentin. In contrast to monolayers in medium, multilayers in air demonstrated only a limited cytopathic effect. Integrin αV ß6 expression was observed in mono- but not in multilayers. FMDV antigen, FMDV RNA and live virus were detected from day 1 to 28, with peaks at day 1 and 2. The proportion of infected cells was highest at 24 hr (3% and 36% of cells at an MOI of 0.01 and 1, respectively). At day 28 after infection, at a time when animals that still harbour FMDV are considered carriers, FMDV antigen was detected in 0.2%-2.1% of cells, in all layers, and live virus was isolated from supernatants of 6/8 cultures. On the consensus level, the viral genome did not change within the first 24 hr after infection. Only a few minor single nucleotide variants were detected, giving no indication of the presence of a viral quasispecies. The air-liquid interface model of DSP brings new possibilities to investigate FMDV persistence in a controlled manner.

Seroprevalence and molecular characterization of foot-and-mouth disease virus in Chad.

This study aimed at determining the seroprevalence of foot-and-mouth disease (FMD) in domestic ruminants and at characterizing the virus strains circulating in four areas of Chad (East Batha, West Batha, Wadi Fira and West Ennedi). The study was carried out between October and November 2016. A total of 1,520 sera samples (928 cattle, 216 goats, 254 sheep and 122 dromedaries) were collected randomly for FMD serological analyses. Nine epithelial tissue samples were also collected from cattle showing clinical signs, for FMDV isolation and characterization. Serological results showed an overall NSP seroprevalence of 40% (375/928) in cattle in our sample (95% CrI [19-63]). However, seroprevalences of 84% (27/32), 78% (35/45) and 84% (21/25) were estimated in cattle over 5 years of age in East Batha, West Batha and Wadi Fira, respectively. In cattle under 1 year of age, 67% (18/27) seroprevalence was estimated in Wadi Fira, 64% (14/22) in East Batha and 59% (13/22) in West Batha. It was found that the high seroprevalences have been obtained in areas where pastures are shared by several different herds but also in farms where two to three species (bovine, caprine and ovine) are raised together. ELISA PrioCHECK® FMDV types O and A and in-house solid phase competition ELISA serotyping results showed that the four O, A, SAT1 and SAT2 serotypes have circulated in Chad in 2016. However, the type SAT2 dominated with an overall seroprevalence of 43% (29/67) and was present in the four areas investigated. The phylogenetic analyses of the VP1 coding sequence allowed determining the serotype SAT2 topotype VII, close to viral strains found in Cameroon in 2015 with a similarity of 98.60%.

Establishment of persistent foot-and-mouth disease virus (FMDV) infection in MDBK cells.

In addition to acute infection and disease, foot-and-mouth disease virus (FMDV) can cause persistent infection in ruminants. Such "carrier" animals represent a potential risk for FMDV transmission to susceptible animals. However, the mechanisms and the factors that determine FMDV persistence remain unknown. We describe here the establishment of FMDV type O persistent infection in a bovine epithelial cell line (Madin-Darby bovine kidney; MDBK). Preliminary experiments to assess the permissivity of MDBK cells to FMDV O infection revealed an unusual pattern of infection: after the initial phase of acute cell lysis, new monolayers formed within 48-72 h post-infection. We found that some cells survived cytolytic infection and subsequently regrew, thereby demonstrating that this bovine cell line can be persistently infected with FMDV type O. Further evidence that MDBK cells were persistently infected with FMDV includes: (i) detection of viral RNA in cells as well as in cell culture supernatants, (ii) detection of viral antigens in the cells by immunofluorescence analysis, and (iii) production of infectious viral particles for up to 36 cell passages. Furthermore, preliminary sequence analysis of persistent virus revealed a single nucleotide substitution within the VP1 coding region, resulting in the V50A amino acid substitution. This bovine model of FMDV persistence holds promise for the investigation of the viral and cellular molecular determinants that promote FMDV persistence.

First isolation and molecular characterization of foot-and-mouth disease virus in Benin.

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals. It is one of the most economically devastating diseases affecting livestock animals. In West Africa, where constant circulation of FMD virus (FMDV) is assumed, very few studies on the characterization of circulating strains have been published. This study describes the first isolation and characterization of FMDV in Benin. FMDV was isolated from 42 samples. Antigen Capture Elisa (Ag-ELISA) and VP1 coding sequence analysis revealed 33 strains of serotype O and 9 strains of serotype A. Phylogenetic analysis of the VP1 sequence revealed two different groups of type O isolates and one group of A isolates. VP1 sequence comparison with the sequences available in the GenBank database revealed a close relationship of the Benin isolates with topotype O of West Africa and with African topotype A of genotype VI. Knowledge of the recent strains circulating in Benin should contribute to better selection of vaccine strains and enable the updating of molecular epidemiology data available for West Africa in general.

Gag-specific immune enhancement of lentiviral infection after vaccination with an adenoviral vector in an animal model of AIDS.

The evaluation of vaccine strategies in animal models is essential for the development of a vaccine against HIV. In efficacy trials conducted in non-human primate models of AIDS, vaccines based on adenoviruses compared favourably with other vaccine vectors. To determine whether this strategy could be transposed to another animal model, and by extension, to humans, we have evaluated the efficacy of adenoviral vectors in a natural model of AIDS, infection of the cat by the feline immunodeficiency virus (FIV). Recombinant canine adenoviruses expressing the envelope glycoproteins or the Gag protein of a primary strain of FIV were constructed. Three groups of six cats were immunised twice with vectors expressing FIV antigens or with a vector expressing an irrelevant antigen, green fluorescent protein, by intramuscular and subcutaneous routes. Humoral responses were elicited against the transgene product in 6/6, 3/6 and 0/6 cats after immunisation against green fluorescent protein, Gag or the envelope glycoproteins, respectively. Six weeks after the second administration, cats were challenged by the intraperitoneal route with the homologous strain, and viral burden in plasma was followed by quantitative RT-PCR. Immunisation with FIV antigens did not afford protection. Rather, viral RNA was detected at earlier time points in cats immunised against Gag than in cats immunised with a vector expressing an irrelevant antigen. Such immune-mediated enhancement did not appear to have a long-range impact on viral set point or inversion of the CD4(+)/CD8(+) ratio. Thus, in the feline AIDS model pre-existing immunity against a viral antigen exacerbated acute phase infection.

Aspergillus fumigatus conidia inhibit tumour necrosis factor- or staurosporine-induced apoptosis in epithelial cells.

A major innate immune response to inhaled conidia of the opportunistic pathogen Aspergillus fumigatus (Af) is the synthesis of pro-inflammatory cytokines, which include tumour necrosis factor (TNF)-alpha, a known inducer of apoptosis. Modulation of host cell apoptosis has been reported to be one of the mechanisms whereby pathogens overcome host cell defences. Our study was designed to investigate whether or not Af conidia could modulate apoptosis induced by TNF-alpha or staurosporine (STS). Exposure of epithelial cells treated by these inducers and exposed to Af conidia decreased the number of apoptotic cells detected by Annexin V staining, analysis of nuclear morphology, terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end-labelling reaction and immunoblotting. Inhibition of apoptosis by Af conidia was seen in cells of the A549 pneumocyte II line, human tracheal epithelial 16HBE and primary human respiratory cells. Inhibition of apoptosis by Af conidia was also observed when apoptosis was induced by co-cultivating A549 cells with activated human alveolar macrophages. Unlike Af conidia, conidia of Cladosporium cladosporioides as well as latex beads or killed Af conidia have no inhibitory effect on TNF-alpha or STS-induced apoptosis. For TNF-induced apoptosis, the observed anti-apoptotic effect of Af conidia was found to be associated with a significant reduction of caspase-3.