Abstract
Poxviruses belonging to the Orthopoxvirus, Capripoxvirus and Parapoxvirus genera share common host species and create a challenge for diagnosis. Here, we developed a novel multiplex PCR method for the simultaneous detection and differentiation of eight poxviruses, belonging to 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 assay is based on high-resolution melting curve analysis (HRMCA) of PCR amplicons produced using genus specific primer pairs and dsDNA binding dye. Differences in fragment size and GC content were used as discriminating power. The assay generated three well separated melting regions for each genus and provided additional intra-genus genotyping allowing the differentiation of the eight poxviruses based on amplicon melting temperature. Out of 271 poxviral DNA samples tested: seven CPXV, 25 CMLV, 42 GTPV, 20 SPPV, 120 LSDV, 33 ORFV, 20 PCPV and two BPSV were detected; two samples presented co-infection with CMLV and PCPV. The assay provides a rapid, sensitive, specific and cost-effective method for the detection of pox diseases in a broad range of animal species and humans.
Highlights
Autonomy from its host[2,3]
The current study focuses on eight viruses belonging to three genera of the Chordopoxvirinae subfamily: 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 target region was identified by aligning the nucleotide sequences of the full genome of orthopoxviruses, capripoxviruses and parapoxviruses available in GenBank (Fig. 1)
Summary
Autonomy from its host[2,3]. Poxviral genomes vary significantly in their GC content, the evolutionary factors for this divergence are unknown. Capripoxviruses, parapoxviruses and orthopoxviruses are all able to infect ruminants, with the orthopoxviruses and parapoxviruses having in addition the potential to infect both camels and humans. Within the genus Orthopoxvirus, CMLV causes severe infections of economic importance only in camels, with rare incursions in humans[6]. Cowpox virus has a much wider host range, in addition to cattle. Capripoxvirus infections of ruminants cause significant morbidity and mortality in Africa, the Middle East and Asia. Their incursions into Eastern Europe have been reported[7,11,12]. Parapoxviruses infect a wide range of hosts worldwide and are of both economical and medical importance, infecting mainly persons working in close contact with animals. ORFV infects small ruminants, camels and humans[13,14,15], while BPSV and PCPV infect large ruminants[16], with the latter being able to infect camels[17] and humans[18,19]
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