Constitutive proteins of lumpy skin disease virion assessed by next-generation proteomics.

IMPORTANCE: Lumpy skin disease virus (LSDV) is the causative agent of an economically important cattle disease which is notifiable to the World Organisation for Animal Health. Over the past decades, the disease has spread at an alarming rate throughout the African continent, the Middle East, Eastern Europe, the Russian Federation, and many Asian countries. While multiple LDSV whole genomes have made further genetic comparative analyses possible, knowledge on the protein composition of the LSDV particle remains lacking. This study provides for the first time a comprehensive proteomic analysis of an infectious LSDV particle, prompting new efforts toward further proteomic LSDV strain characterization. Furthermore, this first incursion within the capripoxvirus proteome represents one of very few proteomic studies beyond the sole Orthopoxvirus genus, for which most of the proteomics studies have been performed. Providing new information about other chordopoxviruses may contribute to shedding new light on protein composition within the Poxviridae family.

An Update of Evidence for Pathogen Transmission by Ticks of the Genus Hyalomma.

Current and likely future changes in the geographic distribution of ticks belonging to the genus Hyalomma are of concern, as these ticks are believed to be vectors of many pathogens responsible for human and animal diseases. However, we have observed that for many pathogens there are no vector competence experiments, and that the level of evidence provided by the scientific literature is often not sufficient to validate the transmission of a specific pathogen by a specific Hyalomma species. We therefore carried out a bibliographical study to collate the validation evidence for the transmission of parasitic, viral, or bacterial pathogens by Hyalomma spp. ticks. Our results show that there are very few validated cases of pathogen transmission by Hyalomma tick species.

Risk of Monkeypox virus (MPXV) transmission through the handling and consumption of food.

Monkeypox (MPX) is a zoonotic infectious disease caused by Monkeypox virus (MPXV), an enveloped DNA virus belonging to the Poxviridae family and the Orthopoxvirus genus. Since early May 2022, a growing number of human cases of Monkeypox have been reported in non-endemic countries, with no history of contact with animals imported from endemic and enzootic areas, or travel to an area where the virus usually circulated before May 2022. This qualitative risk assessment aimed to investigate the probability that MPXV transmission occurs through food during its handling and consumption. The risk assessment used "top-down" (based on epidemiological data) and "bottom-up" (following the agent through the food chain to assess the risk of foodborne transmission to human) approaches, which were combined. The "top-down" approach first concluded that bushmeat was the only food suspected as a source of contamination in recorded cases of MPXV, by contact or ingestion. The "bottom-up" approach then evaluated the chain of events required for a human to become ill after handling or consuming food. This approach involves several conditions: (i) the food must be contaminated with MPXV (naturally, by an infected handler or after contact with a contaminated surface); (ii) the food must contain viable virus when it reaches the handler or consumer; (iii) the person must be exposed to the virus and; (iv) the person must be infected after exposure. Throughout the risk assessment, some data gaps were identified and highlighted. The conclusions of the top-down and bottom-up approaches are consistent and suggest that the risk of transmission of MPXV through food is hypothetical and that such an occurrence was never reported. In case of contamination, cooking (e.g., 12 min at 70°C) could be considered effective in inactivating Poxviridae in foods. Recommendations for risk management are proposed. To our knowledge, this is the first risk assessment performed on foodborne transmission of MPXV.

Development and Optimization of Indirect ELISAs for the Detection of Anti-Capripoxvirus Antibodies in Cattle, Sheep, and Goat Sera.

Sheeppox (SPP), goatpox (GTP), and lumpy skin disease (LSD) are economically significant pox diseases of ruminants, caused by sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV), respectively. SPPV and GTPV can infect both sheep and goats, while LSDV mainly affects cattle. The recent emergence of LSD in Asia and Europe and the repeated incursions of SPP in Greece, Bulgaria, and Russia highlight how these diseases can spread outside their endemic regions, stressing the urgent need to develop high-throughput serological surveillance tools. We expressed and tested two recombinant truncated proteins, the capripoxvirus homologs of the vaccinia virus C-type lectin-like protein A34 and the EEV glycoprotein A36, as antigens for an indirect ELISA (iELISA) to detect anti-capripoxvirus antibodies. Since A34 outperformed A36 by showing no cross-reactivity to anti-parapoxvirus antibodies, we optimized an A34 iELISA using two different working conditions, one for LSD in cattle and one for SPP/GTP in sheep and goats. Both displayed sound sensitivities and specificities: 98.81% and 98.72%, respectively, for the LSD iELISA, and 97.68% and 95.35%, respectively, for the SPP/GTP iELISA, and did not cross-react with anti-parapoxvirus antibodies of cattle, sheep, and goats. These assays could facilitate the implementation of capripox control programs through serosurveillance and the screening of animals for trade.

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.

A novel and sensitive real-time PCR system for universal detection of poxviruses.

Success in smallpox eradication was enabled by the absence of non-human reservoir for smallpox virus. However, other poxviruses with a wider host spectrum can infect humans and represent a potential health threat to humans, highlighted by a progressively increasing number of infections by (re)emerging poxviruses, requiring new improved diagnostic and epidemiological tools. We describe here a real-time PCR assay targeting a highly conserved region of the poxvirus genome, thus allowing a pan-Poxvirus detection (Chordopoxvirinae and Entomopoxvirinae). This system is specific (99.8% for vertebrate samples and 99.7% for arthropods samples), sensitive (100% for vertebrate samples and 86.3% for arthropods samples) and presents low limit of detection (< 1000 DNA copies/reaction). In addition, this system could be also valuable for virus discovery and epidemiological projects.

Phylodynamic study of the conserved RNA structure encompassing the hemagglutinin cleavage site encoding region of H5 and H7 low pathogenic avian influenza viruses.

Highly pathogenic avian influenza viruses (HPAIVs) evolve from low pathogenic avian influenza viruses (LPAIVs) of the H5 and H7 subtypes. This evolution is characterized by the acquisition of a multi-basic cleavage site (MBCS) motif in the hemagglutinin (HA) that leads to an extended viral tropism and severe disease in poultry. One key unanswered question is whether the risk of transition to HPAIVs is similar for all LPAIVs H5 or H7 strains, or whether specific determinants in the HA sequence of some H5 or H7 LPAIV strains correlate with a higher risk of transition to HPAIVs. Here, we determined if specific features of the conserved RNA stem-loop located at the HA cleavage site-encoding region could be detected along the LPAIV to HPAIV evolutionary pathway. Analysis of the thermodynamic stability of the predicted RNA structures showed no specific patterns common to HA sequences leading to HPAIVs and distinct from those remaining LPAIVs. However, RNA structure clustering analysis revealed that most of the American lineage ancestors leading to H7 emergences via recombination shared the same viral RNA (vRNA) structure topology at the HA1/HA2 boundary region. Our study thus identified predicted secondary RNA structures present in the HA of H7 viruses, which could promote genetic recombination and acquisition of a multibasic cleavage site motif (MBCS).

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.

Evaluation of the Antiviral Activity of Sephin1 Treatment and Its Consequences on eIF2α Phosphorylation in Response to Viral Infections.

The guanabenz derivative Sephin1 has recently been proposed to increase the levels of translation initiation factor 2 (eIF2α) phosphorylation by inhibiting dephosphorylation by the protein phosphatase 1-GADD34 (PPP1R15A) complex. As phosphorylation of eIF2α by protein kinase R (PKR) is a prominent cellular antiviral pathway, we evaluated the consequences of Sephin1 treatment on virus replication. Our results provide evidence that Sephin1 downregulates replication of human respiratory syncytial virus, measles virus, human adenovirus 5 virus, human enterovirus D68, human cytomegalovirus, and rabbit myxoma virus. However, Sephin1 proved to be inactive against influenza virus, as well as against Japanese encephalitis virus. Sephin1 increased the levels of phosphorylated eIF2α in cells exposed to a PKR agonist. By contrast, in virus-infected cells, the levels of phosphorylated eIF2α did not always correlate with the inhibition of virus replication by Sephin1. This work identifies Sephin1 as an antiviral molecule in cell culture against RNA, as well as DNA viruses belonging to phylogenetically distant families.

A simple method to estimate the number of doses to include in a bank of vaccines. The case of Lumpy Skin Disease in France.

A simple method to estimate the size of the vaccine bank needed to control an epidemic of an exotic infectious disease in case of introduction into a country is presented. The method was applied to the case of a Lumpy Skin disease (LSD) epidemic in France. The size of the stock of vaccines needed was calculated based on a series of simple equations that use some trigonometric functions and take into account the spread of the disease, the time required to obtain good vaccination coverage and the cattle density in the affected region. Assuming a 7-weeks period to vaccinate all the animals and a spread of the disease of 7.3 km/week, the vaccination of 740 716 cattle would be enough to control an epidemic of LSD in France in 90% of the simulations (608 196 cattle would cover 75% of the simulations). The results of this simple method were then validated using a dynamic simulation model, which served as reference for the calculation of the vaccine stock required. The differences between both models in different scenarios, related with the time needed to vaccinate the animals, ranged from 7% to 10.5% more vaccines using the simple method to cover 90% of the simulations, and from 9.0% to 13.8% for 75% of the simulations. The model is easy to use and may be adapted for the control of different diseases in different countries, just by using some simple formulas and few input data.

Risk of introduction of Lumpy Skin Disease into France through imports of cattle.

The lumpy skin disease (LSD) virus belongs to the genus Capripoxvirus and causes a disease in cattle with economic impacts. In November 2014, the disease was first reported in Europe (in Cyprus); it was then reported in Greece (in August 2015) and has spread through different Balkan countries since 2016. Although vector transmission is predominant in at-risk areas, long-distance transmission usually occurs through movements of infected cattle. In order to estimate the threat for France, a quantitative import risk analysis (QIRA) model was developed to assess the risk of LSD being introduced into France by imports of cattle. Based on available information and using a stochastic model, the probability of a first outbreak of LSD in France following the import of batches of infected live cattle for breeding or fattening was estimated to be 5.4 × 10-4 (95% probability interval [PI]: 0.4 × 10-4 ; 28.7 × 10-4 ) in summer months (during high vector activity) and 1.8 × 10-4 (95% PI: 0.14 × 10-4 ; 15 × 10-4 ) in winter months. The development of a stochastic QIRA made it possible to quantify the risk of LSD being introduced into France through imports of live cattle. This tool is of prime importance because the LSD situation in the Balkans is continuously changing. Indeed, this model can be updated to process new information on the changing health situation in addition to new data from the TRAde Control and Expert System (TRACES, EU database). This model is easy to adapt to different countries and to other diseases.

Oncolytic properties of non-vaccinia poxviruses.

Vaccinia virus, a member of the Poxviridae family, has been extensively used as an oncolytic agent and has entered late stage clinical development. In this study, we evaluated the potential oncolytic properties of other members of the Poxviridae family. Numerous tumor cell lines were infected with ten non-vaccinia poxviruses to identify which virus displayed the most potential as an oncolytic agent. Cell viability indicated that tumor cell lines were differentially susceptible to each virus. Raccoonpox virus was the most potent of the tested poxviruses and was highly effective in controlling cell growth in all tumor cell lines. To investigate further the oncolytic capacity of the Raccoonpox virus, we have generated a thymidine kinase (TK)-deleted recombinant Raccoonpox virus expressing the suicide gene FCU1. This TK-deleted Raccoonpox virus was notably attenuated in normal primary cells but replicated efficiently in numerous tumor cell lines. In human colon cancer xenograft model, a single intratumoral inoculation of the recombinant Raccoonpox virus, in combination with 5-fluorocytosine administration, produced relevant tumor growth control. The results demonstrated significant antitumoral activity of this new modified Raccoonpox virus armed with FCU1 and this virus could be considered to be included into the growing armamentarium of oncolytic virotherapy for cancer.

Risk of introduction of lumpy skin disease in France by the import of vectors in animal trucks.

BACKGROUND: The lumpy skin disease virus (LSDV) is a dsDNA virus belonging to the Poxviridae family and the Capripoxvirus genus. Lumpy skin diseases (LSD) is a highly contagious transboundary disease in cattle producing major economic losses. In 2014, the disease was first reported in the European Union (in Cyprus); it was then reported in 2015 (in Greece) and has spread through different Balkan countries in 2016. Indirect vector transmission is predominant at small distances, but transmission between distant herds and between countries usually occurs through movements of infected cattle or through vectors found mainly in animal trucks. METHODS AND PRINCIPAL FINDINGS: In order to estimate the threat for France due to the introduction of vectors found in animal trucks (cattle or horses) from at-risk countries (Balkans and neighbours), a quantitative import risk analysis (QIRA) model was developed according to the international standard. Using stochastic QIRA modelling and combining experimental/field data and expert opinion, the yearly risk of LSDV being introduced by stable flies (Stomoxys calcitrans), that travel in trucks transporting animals was between 6 x 10-5 and 5.93 x 10-3 with a median value of 89.9 x 10-5; it was mainly due to the risk related to insects entering farms in France from vehicles transporting cattle from the at-risk area. The risk related to the transport of cattle going to slaughterhouses or the transport of horses was much lower (between 2 x 10-7 and 3.73 x 10-5 and between 5 x 10-10 and 3.95 x 10-8 for cattle and horses, respectively). The disinsectisation of trucks transporting live animals was important to reduce this risk. CONCLUSION AND SIGNIFICANCE: The development of a stochastic QIRA made it possible to quantify the risk of LSD being introduced in France through the import of vectors that travel in trucks transporting animals. This tool is of prime importance because the LSD situation in the Balkans is continuously changing. Indeed, this model can be updated to process new information on vectors and the changing health situation, in addition to new data from the TRAde Control and Expert System (TRACES, EU database). This model is easy to adapt to different countries and to other vectors and diseases.

Benefit-Cost Analysis of Foot-and-Mouth Disease Vaccination at the Farm-Level in South Vietnam.

This study aimed to analyze the financial impact of foot-and-mouth disease (FMD) outbreaks in cattle at the farm-level and the benefit-cost ratio (BCR) of biannual vaccination strategy to prevent and eradicate FMD for cattle in South Vietnam. Production data were collected from 49 small-scale dairy farms, 15 large-scale dairy farms, and 249 beef farms of Long An and Tay Ninh province using a questionaire. Financial data of FMD impacts were collected using participatory tools in 37 villages of Long An province. The net present value, i.e., the difference between the benefits (additional revenue and saved costs) and costs (additional costs and revenue foregone), of FMD vaccination in large-scale dairy farms was 2.8 times higher than in small-scale dairy farms and 20 times higher than in beef farms. The BCR of FMD vaccination over 1 year in large-scale dairy farms, small-scale dairy farms, and beef farms were 11.6 [95% confidence interval (95% CI) 6.42-16.45], 9.93 (95% CI 3.45-16.47), and 3.02 (95% CI 0.76-7.19), respectively. The sensitivity analysis showed that varying the vaccination cost had more effect on the BCR of cattle vaccination than varying the market price. This benefit-cost analysis of biannual vaccination strategy showed that investment in FMD prevention can be financially profitable, and therefore sustainable, for dairy farmers. For beef cattle, it is less certain that vaccination is profitable. Additional benefit-cost analysis study of vaccination strategies at the national-level would be required to evaluate and adapt the national strategy to achieve eradication of this disease in Vietnam.

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.

Cowpox Virus: A New and Armed Oncolytic Poxvirus.

Oncolytic virus therapy has recently been recognized as a promising new therapeutic approach for cancer treatment. In this study, we are proposing for the first time to evaluate the in vitro and in vivo oncolytic capacities of the Cowpox virus (CPXV). To improve the tumor selectivity and oncolytic activity, we developed a thymidine kinase (TK)-deleted CPXV expressing the suicide gene FCU1, which converts the non-toxic prodrug 5-fluorocytosine (5-FC) into cytotoxic 5-fluorouracil (5-FU) and 5-fluorouridine-5'-monophosphate (5-FUMP). This TK-deleted virus replicated efficiently in human tumor cell lines; however, it was notably attenuated in normal primary cells, thus displaying a good therapeutic index. Furthermore, this new recombinant poxvirus rendered cells sensitive to 5-FC. In vivo, after systemic injection in mice, the TK-deleted variant caused significantly less mortality than the wild-type strain. A biodistribution study demonstrated high tumor selectivity and low accumulation in normal tissues. In human xenograft models of solid tumors, the recombinant CPXV also displayed high replication, inducing relevant tumor growth inhibition. This anti-tumor effect was improved by 5-FC co-administration. These results demonstrated that CPXV is a promising oncolytic vector capable of expressing functional therapeutic transgenes.

A Q Method Approach to Evaluating Farmers' Perceptions of Foot-and-Mouth Disease Vaccination in Vietnam.

This study aims to explore the farmers' perceptions of foot-and-mouth disease (FMD) vaccination using a reflexive research method called Q methodology. A structured sample was composed of 46 farmers selected according to gender, farming experience, level of education, and production type. Statements relevant to the farmers' perceptions of and attitudes toward FMD vaccination, related to confidence, logistics, costs, and impacts of vaccination were developed. Results were analyzed by principal component analysis and factor analysis. The influence of demographics and characterized variables on the respondent's contribution to each factor was also tested. Regarding the different beliefs and behavior toward FMD vaccination, the common perceptions held by Vietnamese cattle and pig farmers were divided into three discourses named Confidence (24 subjects), Belief (12 subjects), and Challenge (6 subjects). The identified discourses represented 57.3% of the variances. Consensus points were found, such as the feeling of being more secure after FMD vaccination campaigns; the fact that farmers take vaccination decisions themselves without being influenced by other stakeholders; the opinion that FMD vaccination is cheaper than the costs of treating a sick animal; and that vaccines provided by governmental authorities are of high quality. Part of the studied population did not consider vaccination to be the first choice strategy in prevention. This raises the question of how to improve the active participation of farmers in the FMD vaccine strategy. Taking into consideration farmers' perceptions can help to implement feasible vaccination strategies at the local level.