Clinical and epidemiological investigations
In December 2017, the Central Veterinary Research Institute (CVRI) received samples of skin nodules and scabs from a herd of cattle, initially presumed to have LSD infection. The cattle were a Zebu-Boran crossbreed, of mixed dairy and beef, from Chiyuni veterinary camp, Chief Chitanda area, Mundu, Chibombo District, Central Province of Zambia (Figure 1).
The affected farm is designated under zone 3 of the East Coast fever (ECF) control strategy in Zambia [13]. This is an epidemic area for ECF, which causes high animal mortalities in Zambia.
The samples initially tested negative by real-time Polymerase Chain Reaction (PCR) for capripoxviruses. The extracted DNA was stored and subsequently analyzed using a recently developed High Resolution Melting (HRM) assay for the simultaneous detection and differentiation of eight poxviruses of medical and veterinary importance [12]. The results prompted a follow-up trip to the farm in April 2018 to clinically examine the animals and obtain additional history and samples.
Sample collection and DNA extraction
Skin nodules were collected from eight cattle showing recent fresh lesions (Table 1) and were transferred to the CVRI laboratory on ice in a coolbox. The nodules were ground, and 50 mg was homogenized in 2 ml phosphate-buffered saline and centrifuged at 2500 rpm for 10 min at 4 oC. 200 µl of the supernatant was added to 800 µl of lysis buffer RLT Plus (QIAGEN, Germany) and the total nucleic acid extracted using the RNeasy mini kit (QIAGEN, Germany) as previously described [14].
Genetic identification using the HRM assay
We tested the extracted DNA using the HRM assay for the simultaneous detection and differentiation of eight poxviruses. [12]. The method can differentially detect members of three different genera of poxviruses; Capripoxvirus, Orthopoxvirus, and Parapoxvirus, and additionally discriminate the viruses within each of the three genera: cowpox virus (CPXV) and camelpox virus (CMLV) [genus Orthopoxvirus]; goatpox virus (GTPV), sheeppox virus (SPPV) and lumpy skin disease virus (LSDV) [genus Capripoxvirus]; orf virus (ORFV), pseudocowpox virus (PCPV) and bovine papular stomatitis virus (BPSV) [genus Parapoxvirus].
The reaction contained 200 nM of each primer (Table 2), 1 X SsoFastTM EvaGreen® Supermix (Bio-Rad, USA), and 2 µl of the template. Each run included positive control plasmids representing each of the eight pathogens, and a negative control composed of nuclease-free water. Capripoxviruses (GTPV-Denizli, SPPV-Denizli, and LSDV-Ismalia), orthopoxviruses (CMLV- Hadow/01/2012 and CPXV-72/93), and parapoxviruses (ORFV- DZ C-1, PCPV- 2200/12 and BPSV- Stamm M1) were used to produce positive control plasmids [12]. The PCR reactions and melting curve analysis were performed on a real-time PCR machine (CFX96TM Real-Time PCR Detection System, Bio-Rad, USA), following the conditions previously described [12] with slight modifications. Briefly, an initial denaturation step at 95 °C for 4 min was followed by 40 cycles of 95 °C for 1 sec, 59 °C for 5 sec and 70 °C for 5 sec. The PCR products were then denatured at 95 °C for 30 sec, cooled down to 65 °C for 60 sec, then melted from 65 °C to 85 °C with an 0.2 °C increments every ten seconds with continuous data acquisition. Data was analyzed using the CFX Manager Software (Bio-Rad, USA), and the Precision Melt Analysis Software (Bio-Rad, USA).
Sequencing of the B2L gene fragment
The primers used to amplify a fragment of the B2L gene of parapoxviruses [15] are indicated in Table 2. The positive PCR products (1210 base pairs) were purified using the Wizardâ SV Gel and PCR Clean-Up System (Promega, USA) and sequenced in both directions by LGC Biosearch Technologies (Germany), using standard Sanger sequencing methods. The sequences were edited and assembled using Vector NTI 11.5 software (Invitrogen). All sequences were submitted to GenBank under accession numbers MT448677 to MT448684.
Phylogenetic analysis
For comparative analysis, additional partial B2L gene sequences of other parapoxviruses were retrieved from GenBank and screened to remove short and duplicate sequences. The final data set for phylogenetic analyses comprised forty-five sequences, including eight PCPV sequences from this study, seven PCPV of cattle and reindeer, eleven camel contagious ecthyma virus (CCEV), fourteen ORFV, and five BPSV.
Sequences were aligned using the muscle (codon) option in MEGA 7. The aligned sequence file was saved in FASTA format, then converted to a nexus format using Seaview. We then performed the Bayesian phylogenetic inference with BEAST. First, a BEAST file was produced from the nexus file with the BEAUti module using the TN93 +G nucleotide substitution and a UPGMA starting tree. The Markov Chain Monte Carlo method was run with BEAST, for 10,000,000 generations with a sample taken each 10,000 generations. The TRACER program was used to inspect the log files and determine the optimum number of burn-in based on the Effective Sample Sizes (ESS > 200).
TreeAnnotator was used to generate the Maximum Clade Credibility (MCC) after discarding the 2% burn-in. The tree was visualized with the associated meta-data using the ggtree package in R version 3.5.2 [16].