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First Detection and Molecular Characterisation of Pseudocowpox Virus in a Cattle Herd in Zambia Maureen Wakwamba Ziba1* , Chanda Chitala1, Tirumala Bharani K

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First Detection and Molecular Characterisation of Pseudocowpox Virus in a Cattle Herd in Zambia Maureen Wakwamba Ziba1* , Chanda Chitala1, Tirumala Bharani K Ziba et al. Virol J (2020) 17:152 https://doi.org/10.1186/s12985-020-01426-7 RESEARCH Open Access First detection and molecular characterisation of pseudocowpox virus in a cattle herd in Zambia Maureen Wakwamba Ziba1* , Chanda Chitala1, Tirumala Bharani K. Settypalli2, Malama Mumba1, Giovanni Cattoli2, Paul Fandamu1 and Charles Euloge Lamien2 Abstract Background: Pseudocowpox virus (PCPV) of the genus Parapoxvirus in the family Poxviridae causes pseudocowpox in cattle worldwide and presents a zoonotic concern. Most poxviruses produce diseases of similar clinical signs in afected animals, which are impossible to diferentiate clinically or by serology. It is, therefore, vital to use molecular assays to rapidly identify the causative agents of poxvirus infections. This study aimed to detect, diagnose, and char- acterize the causative agent of pox-like skin lesions in a cattle herd in Zambia, initially suspected to be infected with Lumpy Skin Disease virus. Methods: We used a High-Resolution Melting (HRM) analysis assay to detect the PCPV genome and sequenced the major envelope protein (B2L gene) for comparative sequence and phylogenetic analysis. Results: Our feld investigations showed cattle presenting atypical skin lesions and high morbidity within the herd. The laboratory diagnosis, based on the HRM assay revealed PCPV DNA in the samples. Phylogenetic and comparative sequence analyses confrmed PCPV in the samples and revealed genomic diferences between samples collected in 2017 and 2018 from the same farm. Conclusion: Our work is the frst documented report of PCPV in Zambia. It shows the strength of molecular methods to diagnose pox-like infections in cattle and discriminate between diseases causing similar clinical signs. This rapid and accurate diagnosis improves the response time for more accurate veterinary interventions. Keywords: Pseudocowpox virus, HRM assay, B2L gene, Parapoxvirus, Zambia Background genera Capripoxvirus and Orthopoxvirus, can also afect Pseudocowpox is a pox-like disease of cattle caused by cattle. pseudocowpox virus (PCPV) of the genus Parapoxvirus Parapoxviruses cause papules and erosions on the (PPV) within the family Poxviridae [1]. Tis genus also muzzle, oral mucosa, and udder [6] and may cause high includes bovine papular stomatitis virus (BPSV) of cat- morbidity and loss of productivity [7]. Parapoxvirus tle and orf virus (ORFV) of sheep and goats. Additional infections may also be asymptomatic [8], and can infect PPVs afect red deer of New Zealand (PVNZ), reindeers, humans working in close contact with infected animals seals, and musk ox [2–5]. Other poxviruses, within the [7, 9]. Parapoxvirus infections can be clinically diagnosed, however, clinical signs may overlap with other diseases *Correspondence: [email protected] 1 Department of Veterinary Services Ministry of Fisheries and Livestock, such as lumpy skin disease (LSD), bovine herpes virus Central Veterinary Research Institute, P.O Box 33980, Lusaka, Zambia (BoHV), BPSV, and orthopoxvirus. In Zambia, LSD has Full list of author information is available at the end of the article been documented since 1929, but information on PPVs © The Author(s) 2020. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Ziba et al. Virol J (2020) 17:152 Page 2 of 8 and orthopoxvirus infections of cattle are lacking. Typi- scabs from a herd of cattle, initially presumed to have cally, cases presenting with pox-like lesions are treated LSD infection. Te cattle were a Zebu-Boran cross- as LSD, BoHV, or other skin diseases of cattle. Samples breed, of mixed dairy and beef, from Chiyuni veteri- are rarely sent to the laboratory for further diagnosis due nary camp, Chief Chitanda area, Mundu, Chibombo to a lack of diferential diagnostic tools. Recent advance- District, Central Province of Zambia (Fig. 1). ments have produced molecular assays that are fast, sen- Te afected farm is designated under zone 3 of the sitive, and ofer a means to discriminate parapoxviruses East Coast fever (ECF) control strategy in Zambia [13]. from other agents producing similar cutaneous lesions Tis is an epidemic area for ECF, which causes high [10–12]. animal mortalities in Zambia. For the frst time, we report the diagnosis of pseudoc- Te samples initially tested negative by real-time Pol- owpox in Zambia from LSD suspected cases. Tis paper ymerase Chain Reaction (PCR) for capripoxviruses. Te describes the clinical presentation, molecular detec- extracted DNA was stored and subsequently analyzed tion, and molecular characterization of the index cases using a recently developed High Resolution Melting of PCPV from clinical samples and discusses the subse- (HRM) assay for the simultaneous detection and difer- quent feld investigation in a herd of cattle. entiation of eight poxviruses of medical and veterinary importance [12]. Te results prompted a follow-up trip Methods to the farm in April 2018 to clinically examine the ani- Clinical and epidemiological investigations mals and obtain additional history and samples. In December 2017, the Central Veterinary Research Institute (CVRI) received samples of skin nodules and Fig. 1 Outbreak and sample collection site Ziba et al. Virol J (2020) 17:152 Page 3 of 8 Sample collection and DNA extraction Te reaction contained 200 nM of each primer Skin nodules were collected from eight cattle showing (Table 2), 1 X SsoFast™ EvaGreen® Supermix (Bio- recent fresh lesions (Table 1) and were transferred to the Rad, USA), and 2 µl of the template. Each run included CVRI laboratory on ice in a coolbox. Te nodules were positive control plasmids representing each of the ground, and 50 mg was homogenized in 2 ml phosphate- eight pathogens, and a negative control composed of bufered saline and centrifuged at 2500 rpm for 10 min nuclease-free water. Capripoxviruses (GTPV-Deni- at 4 °C. 200 µl of the supernatant was added to 800 µl of zli, SPPV-Denizli, and LSDV-Ismalia), orthopoxvi- lysis bufer RLT Plus (QIAGEN, Germany) and the total ruses (CMLV- Hadow/01/2012 and CPXV-72/93), and nucleic acid extracted using the RNeasy mini kit (QIA- parapoxviruses (ORFV- DZ C-1, PCPV- 2200/12 and GEN, Germany) as previously described [14]. BPSV- Stamm M1) were used to produce positive con- trol plasmids [12]. Te PCR reactions and melting curve Genetic identifcation using the HRM assay analysis were performed on a real-time PCR machine We tested the extracted DNA using the HRM assay for (CFX96™ Real-Time PCR Detection System, Bio-Rad, the simultaneous detection and diferentiation of eight USA), following the conditions previously described [12] poxviruses.[12]. Te method can diferentially detect with slight modifcations. Briefy, an initial denaturation members of three diferent genera of poxviruses; Capri- step at 95 °C for 4 min was followed by 40 cycles of 95 °C poxvirus, Orthopoxvirus, and Parapoxvirus, and addi- for 1 s, 59 °C for 5 s and 70 °C for 5 s. Te PCR prod- tionally discriminate the viruses within each of the ucts were then denatured at 95 °C for 30 s, cooled down three genera: cowpox virus (CPXV) and camelpox virus to 65 °C for 60 s, then melted from 65 to 85 °C with an (CMLV) [genus Orthopoxvirus]; goatpox virus (GTPV), 0.2 °C increments every ten seconds with continuous sheeppox virus (SPPV) and lumpy skin disease virus data acquisition. Data was analyzed using the CFX Man- (LSDV) [genus Capripoxvirus]; orf virus (ORFV), pseu- ager Software (Bio-Rad, USA), and the Precision Melt docowpox virus (PCPV) and bovine papular stomatitis Analysis Software (Bio-Rad, USA). virus (BPSV) [genus Parapoxvirus]. Sequencing of the B2L gene fragment Te primers used to amplify a fragment of the B2L gene Table 1 List of PCPV samples and animals from which of parapoxviruses [15] are indicated in Table 2. Te posi- specimens were collected for this study in Zambia, 2017– tive PCR products (1210 base pairs) were purifed using 2018 the Wizard® SV Gel and PCR Clean-Up System (Pro- Animal Name Age Sex Collection date Host mega, USA) and sequenced in both directions by LGC number Biosearch Technologies (Germany), using standard 1 Lungu 2 years M 20.12.17 Cattle Sanger sequencing methods. Te sequences were edited 2 Mary 2 years 2 mo F 20.12.17 Cattle and assembled using Vector NTI 11.5 software (Invitro- 3 Mazete 3 years F 20.12.17 Cattle gen). All sequences were submitted to GenBank under 4 Beenzu 5 years F 24.04.18 Cattle accession numbers MT448677 to MT448684. 5 Mary 2 years 6 mo F 24.04.18 Cattle 6 Mazuba 1 years M 24.04.18 Cattle Phylogenetic analysis 7 Mercy 5 years F 24.04.18
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