Evaluation of west nile virus diagnostic capacities in veterinary laboratories of the mediterranean and black sea regions

The increasing incidence of West Nile virus (WNV) in the Euro-Mediterranean area warrants the implementation of effective surveillance programs in animals. A crucial step in the fight against the disease is the evaluation of the capacity of the veterinary labs to accurately detect the infection in animal populations. In this context, the animal virology network of the MediLabSecure project organized an external quality assessment (EQA) to evaluate the WNV molecular and serological diagnostic capacities of beneficiary veterinary labs. Laboratories from 17 Mediterranean and Black Sea countries participated. The results of the triplex real time RT-PCR for simultaneous detection and differentiation of WNV lineage 1 (L1), lineage 2 (L2) and Usutu virus (USUV) were highly satisfactory, especially for L1 and L2, with detection rates of 97.9% and 100%, respectively. For USUV, 75% of the labs reported correct results. More limitations were observed for the generic detection of flaviviruses using conventional reverse-transcription polymerase chain reaction (RT-PCR), since only 46.1% reported correct results in the whole panel. As regards the serological panel, the results were excellent for the generic detection of WNV antibodies. More variability was observed for the specific detection of IgM antibodies with a higher percentage of incorrect results mainly in samples with low titers. This EQA provides a good overview of the WNV (and USUV) diagnostic performance of the involved veterinary labs and demonstrates that the implemented training program was successful in upgrading their diagnostic capacities

A gel-based PCR method to differentiate sheeppox virus field isolates from vaccine strains

Abstract Background Sheeppox (SPP) and goatpox (GTP) caused by sheeppox virus (SPPV) and goatpox virus (GTPV), respectively of the genus Capripoxvirus in the family Poxviridae, are severely afflicting small ruminants’ production systems in Africa and Asia. In endemic areas, SPP and GTP are controlled using vaccination with live attenuated vaccines derived from SPPV, GTPV or Lumpy skin disease virus (LSDV). Sometimes outbreaks occur following vaccination. In order to successfully control the spread of the virus, it is essential to identify whether the animals were infected by the field strain and the vaccine did not provide sufficient protection. Alternatively, in some cases the vaccine strain may cause adverse reactions in vaccinated animals or in rare occasions, re-gain virulence. Thus, diagnostic tools for differentiation of virulent strains from attenuated vaccine strains of the virus are needed. The aim of this study was to identify an appropriate diagnostic target region in the capripoxvirus genome by comparing the genomic sequences of SPPV field isolates with those of the most widely used SPP vaccine strains. Results A unique 84 base pair nucleotide deletion located between the DNA ligase gene and the VARV B22R homologue gene was found only in SPPV vaccines derived from the Romanian and Yugoslavian RM/65 strains and absent in SPPV field isolates originated from various geographical locations of Asia and Africa. In addition, we developed and evaluated a conventional PCR assay, exploiting the targeted intergenic region to differentiate SPPV vaccine virus from field isolates. The assay produced an amplicon size of 218 bp for the vaccine strains, while the SPPV field isolates resulted in a 302 bp PCR fragment. The assay showed good sensitivity and specificity, and the results were in full agreement with the sequencing data of the PCR amplicons. Conclusion The developed assay is an improvement of currently existing diagnostic tools and, when combined with a capripox virus species-specific assay, will enhance SPP and GTP diagnosis and surveillance and facilitate epidemiological investigations in countries using live attenuated SPP vaccines. In addition, for laboratories with limited resources, the assay provides a simple and cost-effective alternative for sequencing

An HRM assay to differentiate sheeppox virus vaccine strains from sheeppox virus field isolates and other capripoxvirus species

International audienceSheep poxvirus (SPPV), goat poxvirus (GTPV) and lumpy skin disease virus (LSDV) affect small ruminants and cattle causing sheeppox (SPP), goatpox (GTP) and lumpy skin disease (LSD) respectively. In endemic areas, vaccination with live attenuated vaccines derived from SPPV, GTPV or LSDV provides protection from SPP and GTP. As live poxviruses may cause adverse reactions in vaccinated animals, it is imperative to develop new diagnostic tools for the differentiation of SPPV field strains from attenuated vaccine strains. Within the capripoxvirus (CaPV) homolog of the variola virus B22R gene, we identified a unique region in SPPV vaccines with two deletions of 21 and 27 nucleotides and developed a High-Resolution Melting (HRM)-based assay. The HRM assay produces four distinct melting peaks, enabling the differentiation between SPPV vaccines, SPPV field isolates, GTPV and LSDV. This HRM assay is sensitive, specific, and provides a cost-effective means for the detection and classification of CaPVs and the differentiation of SPPV vaccines from SPPV field isolates

Additional file 3: of A gel-based PCR method to differentiate sheeppox virus field isolates from vaccine strains

Figure S2. Determination of the limits of detection of the PCR assay. Defined amount for the plasmid genotype standard (104, 103, 100, 80, 60, 40, 20, 10, 1 and 0) for SPPV vaccine (A) and SPPV field isolates (B) were tested in parallel reactions and run on agarose gel. (PDF 77 kb

Additional file 2: of A gel-based PCR method to differentiate sheeppox virus field isolates from vaccine strains

Figure S1. Gel picture of PCR products for the remaining capripoxvirus samples. These sample were tested in this study, but not presented in Fig. 2 of the manuscript. The PCR products of 218 bp, 302 bp and 338 bp represent SPPV vaccine strains, SPPV field isolates/GTPVs, and LSDVs respectively. First row: MM = 50 bp DNA ladder; a = positive control plasmid of the SPPV field isolates; b = positive control plasmid of the SPPV vaccine strain; c = Negative control; Lane 5 to 15 (sample 23 to 33 in Table 1 of the manuscript). Second row: MM = 50 bp; Lane 2 to 14 (sample 34 to 46 in Table 1 of the manuscript). (PDF 104 kb

Additional file 4: of A gel-based PCR method to differentiate sheeppox virus field isolates from vaccine strains

Figure S3. Gel picture of PCR for the non-capripoxvirus samples tested in this study. MM = 50 bp DNA ladder; a = positive control plasmid of the SPPV field isolates; b = positive control plasmid of the SPPV vaccine strain; c = Negative control; 1–5 (ORF viruses); 6 (BPSV); 7–8 (Mccp); 9 (cDNA, PPRV); 10 (BOHV-1); 11 (BOHV-2). (PDF 45 kb

Additional file 1: of A gel-based PCR method to differentiate sheeppox virus field isolates from vaccine strains

Table S1. Non-capripoxvirus samples tested for specificity study. (DOCX 16 kb