Harnessing Attenuation-Related Mutations of Viral Genomes: Development of a Serological Assay to Differentiate between Capripoxvirus-Infected and -Vaccinated Animals.

Sheeppox, goatpox, and lumpy skin disease caused by the sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV), respectively, are diseases that affect millions of ruminants and many low-income households in endemic countries, leading to great economic losses for the ruminant industry. The three viruses are members of the Capripoxvirus genus of the Poxviridae family. Live attenuated vaccines remain the only efficient means for controlling capripox diseases. However, serological tools have not been available to differentiate infected from vaccinated animals (DIVA), though crucial for proper disease surveillance, control, and eradication efforts. We analysed the sequences of variola virus B22R homologue gene for SPPV, GTPV, and LSDV and observed significant differences between field and vaccine strains in all three capripoxvirus species, resulting in the truncation and absence of the B22R protein in major vaccines within each of the viral species. We selected and expressed a protein fragment present in wildtype viruses but absent in selected vaccine strains of all three species, taking advantage of these alterations in the B22R gene. An indirect ELISA (iELISA) developed using this protein fragment was evaluated on well-characterized sera from vaccinated, naturally and experimentally infected, and negative cattle and sheep. The developed wildtype-specific capripox DIVA iELISA showed >99% sensitivity and specificity for serum collected from animals infected with the wildtype virus. To the best of our knowledge, this is the first wildtype-specific, DIVA-capable iELISA for poxvirus diseases exploiting changes in nucleotide sequence alterations in vaccine strains.

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.

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.

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.

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.

Protective efficacy of a single immunization with capripoxvirus-vectored recombinant peste des petits ruminants vaccines in presence of pre-existing immunity.

Sheeppox, goatpox and peste des petits ruminants (PPR) are highly contagious ruminant diseases widely distributed in Africa, the Middle East and Asia. Capripoxvirus (CPV)-vectored recombinant PPR vaccines (rCPV-PPR vaccines), which have been developed and shown to protect against both Capripox (CP) and PPR, would be critical tools in the control of these important diseases. In most parts of the world, these disease distributions overlap each other leaving concerns about the potential impact that pre-existing immunity against either disease may have on the protective efficacy of these bivalent rCPV-PPR vaccines. Currently, this question has not been indisputably addressed. Therefore, we undertook this study, under experimental conditions designed for the context of mass vaccination campaigns of small ruminants, using the two CPV recombinants (Kenya sheep-1 (KS-1) strain-based constructs) developed previously in our laboratory. Pre-existing immunity was first induced by immunization either with an attenuated CPV vaccine strain (KS-1) or the attenuated PPRV vaccine strain (Nigeria 75/1) and animals were thereafter inoculated once subcutaneously with a mixture of CPV recombinants expressing either the hemagglutinin (H) or the fusion (F) protein gene of PPRV (10(3) TCID50/animal of each). Finally, these animals were challenged with a virulent CPV strain followed by a virulent PPRV strain 3 weeks later. Our study demonstrated full protection against CP for vaccinated animals with prior exposure to PPRV and a partial protection against PPR for vaccinated animals with prior exposure to CPV. The latter animals exhibited a mild clinical form of PPR and did not show any post-challenge anamnestic neutralizing antibody response against PPRV. The implications of these results are discussed herein and suggestions made for future research regarding the development of CPV-vectored vaccines.

Use of the Capripoxvirus homologue of Vaccinia virus 30 kDa RNA polymerase subunit (RPO30) gene as a novel diagnostic and genotyping target: development of a classical PCR method to differentiate Goat poxvirus from Sheep poxvirus.

Sheep poxvirus (SPPV), Goat poxvirus (GTPV) and Lumpy skin disease virus (LSDV) are Capripoxviruses (CaPVs) responsible for causing severe poxvirus disease in sheep, goats and cattle, respectively. Serological differentiation of CaPVs is not possible and strain identification has relied on the implicitly accepted hypothesis that the viruses show well defined host specificity. However, it is now known that cross infections can occur and authentication of identity based on the host animal species from which the strain was first isolated, is not valid and should be replaced with molecular techniques to allow unequivocal strain differentiation. To identify a diagnostic target for strain genotyping, the CaPV homologue of the Vaccinia virus E4L gene which encodes the 30 kDa DNA-dependent RNA polymerase subunit, RPO30 was analyzed. Forty-six isolates from different hosts and geographical origins were included. Most CaPVs fit into one of the three different groups according to their host origins: the SPPV, the GTPV and the LSDV group. A unique 21-nucleotide deletion was found in all SPPV isolates which was exploited to develop a RPO30-based classical PCR test to differentiate SPPV from GTPV that will allow rapid differential diagnosis of disease during CaPV outbreaks in small ruminants.

Capripoxvirus G-protein-coupled chemokine receptor: a host-range gene suitable for virus animal origin discrimination.

The genus Capripoxvirus within the family Poxviridae comprises three closely related viruses, namely goat pox, sheep pox and lumpy skin disease viruses. This nomenclature is based on the animal species from which the virus was first isolated, respectively, goat, sheep and cattle. Since capripoxviruses are serologically identical, their specific identification relies exclusively on the use of molecular tools. We describe here the suitability of the G-protein-coupled chemokine receptor (GPCR) gene for use in host-range grouping of capripoxviruses. The analysis of 58 capripoxviruses showed three tight genetic clusters consisting of goat pox, sheep pox and lumpy skin disease viruses. However, a few discrepancies exist with the classical virus-host origin nomenclature: a virus isolated from sheep is grouped in the goat poxvirus clade and vice versa. Intra-group diversity was further observed for the goat pox and lumpy skin disease virus isolates. Despite the presence of nine vaccine strains, no genetic determinants of virulence were identified on the GPCR gene. For sheep poxviruses, the addition or deletion of 21 nucleic acids (7 aa) was consistently observed in the 5' terminal part of the gene. Specific signatures for each cluster were also identified. Prediction of the capripoxvirus GPCR topology, and its comparison with other known mammalian GPCRs and viral homologues, revealed not only a classical GPCR profile in the last three-quarters of the protein but also unique features such as a longer N-terminal end with a proximal hydrophobic alpha-helix and a shorter serine-rich C-tail.

Surface expression of an immunodominant malaria protein B cell epitope by yellow fever virus.

The yellow fever 17D virus (YF17D) has several characteristics that are desirable for the development of new, live attenuated vaccines. We approached its development as a vector for heterologous antigens by studying the expression of a humoral epitope at the surface of the E protein based on the results of modelling its three-dimensional structure. This model indicated that the most promising insertion site is between beta-strands f and g, a site that is exposed at the external surface of the virus. The large deletion of six residues from the fg loop of the E protein from yellow fever virus, compared to tick-born encephalitis virus, leaves space at the dimer interface for a large insertion without creating steric hindrance. We have tested this hypothesis by inserting a model humoral epitope from the circumsporozoite protein of Plasmodium falciparum consisting of triple NANP repeats. Recombinant virus (17D/8) expressing this insertion flanked by two glycine residues at each end, is specifically neutralized by a monoclonal antibody to the model epitope. Furthermore, mouse antibodies raised to the recombinant virus recognize the parasite protein in an ELISA assay. Serial passage analysis confirmed the genetic stability of the insertion made in the viral genome and the resulting 17D/8 virus is significantly more attenuated in mouse neurovirulence tests than the 17DD vaccine. The fg loop belongs to the dimerization domain of the E protein and lies at the interface between monomers. This domain undergoes a low pH transition, which is related to the fusion of the viral envelope to the endosome membrane. It is conceivable that a slower rate of fusion, resulting from the insertion close to the dimer interface, may delay the onset of virus production and thereby lead to a milder infection of the host. This would account for the more attenuated phenotype of the recombinant virus in the mouse model and lower extent of replication in cultured cells. The vectorial capacity of the yellow fever virus is being further explored for the expression and presentation of other epitopes, including those mediating T-cell responses.

The yellow fever 17D vaccine virus as a vector for the expression of foreign proteins: development of new live flavivirus vaccines

The Flaviviridae is a family of about 70 mostly arthropod-borne viruses many of which are major public health problems with members being present in most continents. Among the most important are yellow fever (YF), dengue with its four serotypes and Japanese encephalitis virus. A live attenuated virus is used as a cost effective, safe and efficacious vaccine against YF but no other live flavivirus vaccines have been licensed. The rise of recombinant DNA technology and its application to study flavivirus genome structure and expression has opened new possibilities for flavivirus vaccine development. One new approach is the use of cDNAs encopassing the whole viral genome to generate infectious RNA after in vitro transcription. This methodology allows the genetic mapping of specific viral functions and the design of viral mutants with considerable potential as new live attenuated viruses. The use of infectious cDNA as a carrier for heterologous antigens is gaining importance as chimeric viruses are shown to be viable, immunogenic and less virulent as compared to the parental viruses. The use of DNA to overcome mutation rates intrinsic of RNA virus populations in conjunction with vaccine production in cell culture should improve the reliability and lower the cost for production of live attenuated vaccines. The YF virus despite a long period ignored by researchers probably due to the effectiveness of the vaccine has made a come back, both in nature as human populations grow and reach endemic areas as well as in the laboratory being a suitable model to understand the biology of flaviviruses in general and providing new alternatives for vaccine development through the use of the 17D vaccine strain