ANCHOR-tagged equine herpesvirus 1: A new tool for monitoring viral infection and discovering new antiviral compounds.

Equine herpesvirus 1 (EHV-1) is a causative agent of respiratory disorders, abortion and myeloencephalopathy in horses and has an important impact on equine health and economy. Several bacterial artificial chromosomes have already been developed and enabled identification and functional characterization of EHV-1 genes. Unfortunately, little is known about its replication. Here, the ANCHOR system was inserted by targeted homologous recombination into the equine herpesvirus genome. This insertion led to the conversion of EHV-1 DNA to auto-fluorescent spots easily detectable by fluorescence microscopy, and enabled production of an auto-fluorescent EHV-1 ANCHORGFP with tropism and replication kinetic like the parental strain. High resolution imaging allowed first visualization of EHV-1 replication from apparition of first viral genome to large replicative centers, in single cells or inside syncytia. Combined with high content microscopy, EHV-1 ANCHORGFP leads to identification of auranofin and azacytidine-5 as new potential antivirals to treat EHV-1 infection.

Antipoxvirus Activity Evaluation of Optimized Corroles Based on Development of Autofluorescent ANCHOR Myxoma Virus.

A series of 43 antiviral corrole-based molecules have been tested on myxoma virus (Lausanne-like T1MYXV strain). An autofluorescent MYXV, with an ANCHOR cassette, has been used for the studies. A2B-fluorocorroles display various toxicities, from 40 being very toxic (CC50 = 1.7 µM) to nontoxic 38 (CC50 > 50 µM), whereas A3-fluorocorroles, with one to three fluorine atoms, are not toxic (with the exception of corroles 9, 10, and 22). In vitro, these compounds show a good selectivity index when used alone. Corrole 35 seems to be the most promising compound, which displays a high selectivity index with the lowest IC50. Interestingly, this "Hit" corrole is easy to synthesize in a two-step reaction. Upscaling production up to 25 g has been carried out for in vivo tests. In vivo studies on New Zealand white rabbits infected with myxoma virus show that symptoms are delayed and animal weight is increased upon treatment, while no acute toxicity of the corrole molecule was detected.

A Novel Imaging Approach for Single-Cell Real-Time Analysis of Oncolytic Virus Replication and Efficacy in Cancer Cells.

Oncolytic viruses (OVs) are novel cancer gene therapies that are moving toward the forefront of modern medicines. However, their full therapeutic potential is hindered by the lack of convenient and reliable strategies to visualize and quantify OV growth kinetics and therapeutic efficacy in live cells. In this study, we present an innovative imaging approach for single-cell real-time analysis of OV replication and efficacy in cancer cells. We selected SG33 as a prototypic new OV that derives from wild-type Myxoma virus (MYXV). Lausanne Toulouse 1 (T1) was used as control. We equipped SG33 and T1 genomes with the ANCHOR system and infected a panel of cell lines. The ANCHOR system is composed of a fusion protein (OR-GFP) that specifically binds to a short nonrepetitive DNA target sequence (ANCH) and spreads onto neighboring sequences by protein oligomerization. Its accumulation on the tagged viral DNA results in the creation of fluorescent foci. We found that (1) SG33 and T1-ANCHOR DNA can be readily detected and quantified by live imaging, (2) both OVs generate perinuclear replication foci after infection clustering into horse-shoe shape replication centers, and (3) SG33 replicates to higher levels as compared with T1. Lastly, as a translational proof of concept, we benchmarked SG33 replication and oncolytic efficacy in primary cancer cells derived from pancreatic adenocarcinoma (PDAC) both at the population and at the single-cell levels. In vivo, SG33 significantly replicates in experimental tumors to inhibit tumor growth. Collectively, we provide herein for the first time a novel strategy to quantify each step of OV infection in live cells and in real time by tracking viral DNA and provide first evidence of theranostic strategies for PDAC patients. Thus, this approach has the potential to rationalize the use of OVs for the benefit of patients with incurable diseases.

Fluorescent Tagged Vaccinia Virus Genome Allows Rapid and Efficient Measurement of Oncolytic Potential and Discovery of Oncolytic Modulators.

As a live biologic agent, oncolytic vaccinia virus has the ability to target and selectively amplify at tumor sites. We have previously reported that deletion of thymidine kinase and ribonucleotide reductase genes in vaccinia virus can increase the safety and efficacy of the virus. Here, to allow direct visualization of the viral genome in living cells, we incorporated the ANCH target sequence and the OR3-Santaka gene in the double-deleted vaccinia virus. Infection of human tumor cells with ANCHOR3-tagged vaccinia virus enables visualization and quantification of viral genome dynamics in living cells. The results show that the ANCHOR technology permits the measurement of the oncolytic potential of the double deleted vaccinia virus. Quantitative analysis of infection kinetics and of viral DNA replication allow rapid and efficient identification of inhibitors and activators of oncolytic activity. Our results highlight the potential application of the ANCHOR technology to track vaccinia virus and virtually any kind of poxvirus in living cells.

Large-scale lagovirus disease outbreaks in European brown hares (Lepus europaeus) in France caused by RHDV2 strains spatially shared with rabbits (Oryctolagus cuniculus).

Rabbit haemorrhagic disease virus (RHDV) is a lagovirus that causes rabbit haemorrhagic disease (RHD) in European rabbits (Oryctolagus cuniculus). In 2010, a new genotype called RHDV2 emerged in France. It exhibits a larger host range than classical RHDV strains by sporadically infecting different hare species, including the European hare (Lepus europaeus). Phylogenetic analyses revealed that closely related RHDV2 strains circulate locally in both hares and rabbits, and therefore that RHDV2 strains infecting hares do not belong to a lineage that has evolved only in this species. We showed that RHDV2 is widely distributed in France and that it was responsible for more than a third of cases of lagovirus disease in European hare populations in 2015. The oldest RHDV2 positive hare was sampled in November 2013 and we reported two hares co-infected by EBHSV and RHDV2. All together, our results raise important epidemiological and evolutionary issues. In particular, along with the potential emergence of recombinant EBHSV/RHDV2 strains in hares, the enlargement of the host range changes the host population structure of RHDV2 and may alter the impact of the virus on rabbit and hare populations.

Wildlife Reservoir for Hepatitis E Virus, Southwestern France.

Pigs are a reservoir for hepatitis E virus (HEV). To determine the relative contribution of game to the risk for human HEV infection in southwestern France, we tested wildlife samples. HEV RNA was in 3.3% of wildlife livers, indicating that in this region, eating game meat is as risky as eating pork.

Expression of VP7, a Bluetongue virus group specific antigen by viral vectors: analysis of the induced immune responses and evaluation of protective potential in sheep.

Bluetongue virus (BTV) is an economically important Orbivirus transmitted by biting midges to domestic and wild ruminants. The need for new vaccines has been highlighted by the occurrence of repeated outbreaks caused by different BTV serotypes since 1998. The major group-reactive antigen of BTV, VP7, is conserved in the 26 serotypes described so far, and its role in the induction of protective immunity has been proposed. Viral-based vectors as antigen delivery systems display considerable promise as veterinary vaccine candidates. In this paper we have evaluated the capacity of the BTV-2 serotype VP7 core protein expressed by either a non-replicative canine adenovirus type 2 (Cav-VP7 R0) or a leporipoxvirus (SG33-VP7), to induce immune responses in sheep. Humoral responses were elicited against VP7 in almost all animals that received the recombinant vectors. Both Cav-VP7 R0 and SG33-VP7 stimulated an antigen-specific CD4+ response and Cav-VP7 R0 stimulated substantial proliferation of antigen-specific CD8+ lymphocytes. Encouraged by the results obtained with the Cav-VP7 R0 vaccine vector, immunized animals were challenged with either the homologous BTV-2 or the heterologous BTV-8 serotype and viral burden in plasma was followed by real-time RT-PCR. The immune responses triggered by Cav-VP7 R0 were insufficient to afford protective immunity against BTV infection, despite partial protection obtained against homologous challenge. This work underscores the need to further characterize the role of BTV proteins in cross-protective immunity.

Risk of zoonotic transmission of HEV from rabbits.

Hepatitis E virus strains from rabbits indicate that these mammals may be a reservoir for HEVs that cause infection in humans. Further issues remain to be clarified, including whether the genotype of rabbit HEV differs from human and swine HEV genotype 3 and whether rabbit HEV can infect human and other animals. HEV was found in farmed rabbits in several geographic areas of China, in USA and more recently in France. The prevalence of antibodies against HEV was 36%, 57% and 55% in rabbits from Virginia (USA), Gansu Province and Beijing (China), respectively. HEV RNA was detected in 16.5% of serum samples from farmed rabbits in Virginia, 7.5% in Gansu Province and 7.0% in Beijing. HEV RNA was detected in 7% of bile samples from farmed rabbits and in 23% of liver samples from wild rabbits in France. The full-length genomic sequences analysis indicates that all the rabbit strains belong to the same clade. Nucleotide sequences were 72.2-78.2% identical to HEV genotypes 1-4. Comparison with HEV sequences of human strains circulating in France and reference sequences identified a human strain closely related to rabbit HEV. A 93-nucleotide insertion in the X domain of the ORF1 of the human strain and in all the rabbit HEV strains was found. Moreover, the ability of rabbit HEV to cause cross-species infection in a pig model has recently been demonstrated. Rabbit HEV can replicate efficiently in human cell lines. Collectively, these data support the possibility of zoonotic transmission of HEV from rabbits.

Myxomavirus as a vector for the immunisation of sheep: protection study against challenge with bluetongue virus.

Recombinant poxviruses are well suited for the development of new vaccine vectors. Our previous data supported the idea that Myxomavirus (MYXV) is efficient at priming antibody responses in sheep. To provide definitive evidence on the potential of MYXV for vaccination against infectious diseases in ruminants, we investigated the immune protection provided by recombinant MYXV against bluetongue, a devastating disease in sheep. To test this concept, sheep were injected twice with an MYXV expressing the immunodominant VP2 protein (SG33-VP2). The SG33-VP2 vector promoted the production of neutralising antibodies and partially protected sheep against disease after challenge with a highly virulent strain of serotype-8 bluetongue virus (BTV-8). In contrast, an MYXV expressing both VP2 and VP5 proteins (SG33-VP2/5) elicited very little protection. The expression levels of the VP2 and VP5 proteins suggested that, greater than the co-expression of the VP5 protein which was previously thought to favour anti-VP2 antibody response, the high expression of VP2 may be critical in the MYXV context to stimulate a protective response in sheep. This highlights the requirement for a careful examination of antigen expression before any conclusion can be drawn on the respective role of the protective antigens. As a proof of principle, our study shows that an MYXV vaccine vector is possible in ruminants.