Molekularna diferencijacija izolata bolesti kvrgave kože na terenu i cjepnih sojeva virusa Capripox u Egiptu 2018.

Lumpy skin disease virus is a member of the Capripoxvirus genus of the Poxviridae family, which affects cattle and causes a notifiable disease with significant economic losses. It is controlled by vaccination with capripox live attenuated vaccines. The aim of the study was the isolation and identification of the lumpy skin virus field virus strain during 2018. Nodular skin lesions were collected from clinically infected lumpy skin disease cattle that were used for the virus isolation on the chorioallantoic membrane of specific pathogen free embryonated chicken eggs and Madin Darby Bovine Kidney tissue culture. Polymerase chain reaction targeting the Capripoxvirus CaPV ORF103 gene was applied on the isolated virus and three Capripoxvirus vaccinal strains (Kenyan sheep pox virus, Held goat pox virus and Ismailia lumpy skin disease virus). The amplicons of the four strains of Capripoxvirus (one isolated and three vaccinal strains) were used for sequencing. Reference capripox viruses were obtained from GenBank to create the phylogenetic tree. The virus isolated from the collected nodular skin samples on chicken eggs showed clear typical pock lesions on the chorioallantoic membrane and on tissue cultures and showed a characteristic cytopathic effect. Positive samples of the isolated strain were identified by PCR for the CaPV ORF103 gene that yielded expected amplicon sizes of 570 bp. This was confirmed through gene sequence and analysed by BLAST, and submitted to GenBank under accession number MW 546997_LSD_Aziz_LSD. The phylogenetic tree revealed that the field isolate strain of LSDV had different identity percentages ranging from 98.2–99.8% with the tested vaccinal Capripoxvirus strains in Egypt. The amino acid sequence showed only different amino acid found in the field isolate strain and not in other tested vaccinal strains, and a maximum homology (100%) of the isolated strain nucleotide sequence was with two GenBank recorded strains. We recommend maintaining the routine lumpy skin disease vaccination programme in Egypt, frequent eradication of the insect population, and further genetic studies on the genomes of this virus strain and the Capripoxvirus vaccinal strains to reach the most related and homologous vaccinal strain given the massive genome of this disease.Virus bolesti kvrgave kože pripada rodu Capripox virusa porodice Poxviridae, koji pogađa stoku i uzrokuje bolest sa značajnim ekonomskih gubitcima, a koja se kontrolira cijepljenjem živim oslabljenim capripox cjepivima. Cilj studije bio je izolirati i identificirati soj virusa BKK na terenu tijekom 2018. godine. Prikupljene kvrge na koži u stoke klinički inficirane bolešću kvrgave kože (BKK) rabljene su za izoliranje virusa na korioalantoičnoj membrani (CAM) embrioniranog kokošjeg jaja (ECE) bez specifičnog patogena (SPF) i staničnoj kulturi bubrega goveda Madin Darby Bovine Kidney (MDBK stanična linija). Lančane reakcije polimerazom (PCR) usmjerene na ORF103 gen virusa Capripox (CaPV) primijenjene su na izolirani BKK virus i tri cjepna soja virusa Capripox (kenijski virus ovčjih boginja, Held virus kozjih boginja i Ismailia virus bolesti kvrgave kože). PCR amplikoni četiriju sojeva CaPV (jednog izoliranog i tri cjepna soja) rabljeni su za sekvenciranje i dobivanje pristupnog broja u banci gena te ilustriranje filogenetskog stabla u usporedbi s drugim referentnim virusima Capripox dobivenima iz banke gena. BKK virus izoliran iz prikupljenih uzoraka kvrga na koži na embrioniranom kokošjem jaju pokazao je jasne tipične pustula lezije na korioalantoičnoj membrani, a na staničnim kulturama (MDBK stanična linija) pokazao je karakterističan citopatski učinak. Pozitivni uzorci izoliranog soja BKK identificirani su PCR-om za CaPV ORF103 gen koji je dao očekivane veličine amplikona od 570 bp te potvrđeni sekvenciranjem gena uz analizu putem BLAST-a i su dostavljeni banci gena pod pristupnim brojem MW 546997_LSD_Aziz_LSD. Dizajnirano je filogenetsko stablo i otkriveno je da je soj izolata virusa BKK na terenu imao postotke različitog identiteta koji su se kretali od 98,2-99,8 % s testiranim cjepnim sojevima virusa Capripox u Egiptu. Sekvenca aminokiselina pokazala je samo jednu posebnu aminokiselinu koja je pronađena u soju izolata s terena, ali ne u ostalim testiranim cjepnim sojevima. Maksimalna podudarnost (100%) nukleotidne sekvence izoliranog BKK soja bila je s dva soja zabilježena u banci gena (MK342935_LSD-CPD/Menofiya1/18) i (MN792930_LSD/AHRI/_Wadi Elgdid/18). Zaključeno je da je izolirani soj virusa BKK imao velike postotke identiteta (98,2-99,8 %) s testiranim cjepnim sojevima virusa Capripox u Egiptu. Da bi se postigao najpovezaniji i homologni cjepni soj, jer je genom BKK virusa velik preporučujemo nastavak programa rutinskog cijepljenja za BKK u Egiptu, često uništavanje populacije insekata i provedbu dodatnih genetskih studija na genomima soja BKK virusa i cjepnih sojeva virusa Capripox da bi se postigao najpovezaniji i homologni cjepni soj, jer je genom BKK virusa golem

Development of a cost-effective method for capripoxvirus genotyping using snapback primer and dsDNA intercalating dye

Sheep pox virus (SPPV), goat pox virus (GTPV) and lumpy skin disease virus (LSDV) are very closely related viruses of the Capripoxvirus (CaPV) genus of the Poxviridae family. They are responsible for sheep pox, goat pox and lumpy skin disease which affect sheep, goat and cattle, respectively. The epidemiology of capripox diseases is complex, as some CaPVs are not strictly host-specific. Additionally, the three forms of the disease co-exist in many sub-Saharan countries which complicates the identification of the virus responsible for an outbreak. Genotyping of CaPVs using a low-cost, rapid, highly specific, and easy to perform method allows a swift and accurate identification of the causative agent and significantly assists in selecting appropriate control and eradication measures, such as the most suitable vaccine against the virus during the outbreaks. The objective of this paper is to describe the design and analytical performances of a new molecular assay for CaPV genotyping using unlabelled snapback primers in the presence of dsDNA intercalating EvaGreen dye. This assay was able to simultaneously detect and genotype CaPVs in 63 samples with a sensitivity and specificity of 100%. The genotyping was achieved by observing the melting temperature of snapback stems of the hairpins and those of the full-length amplicons, respectively. Fourteen CaPVs were genotyped as SPPVs, 25 as GTPVs and 24 as LSDVs. The method is highly pathogen specific and cross platform compatible. It is also cost effective as it does not use fluorescently labelled probes, nor require high-resolution melting curve analysis software. Thus it can be easily performed in diagnostic and research laboratories with limited resources. This genotyping method will contribute significantly to the early detection and genotyping of CaPV infection and to epidemiological studies. (Résumé d'auteur

Capripoxvirus tissue tropism and shedding: A quantitative study in experimentally infected sheep and goats

AbstractSheeppox virus and goatpox virus cause systemic disease in sheep and goats that is often associated with high morbidity and high mortality. To increase understanding of the pathogenesis of these diseases, we undertook quantitative time-course studies in sheep and goats following intradermal inoculation of Nigerian sheeppox virus or Indian goatpox virus in their respective homologous hosts. Viremia, determined by virus isolation and real-time PCR, cleared within 2 to 3 weeks post inoculation. Peak shedding of viral DNA and infectious virus in nasal, conjunctival and oral secretions occurred between 10 and 14 days post inoculation, and persisted at low levels for up to an additional 3 to 6 weeks. Although gross lesions developed in multiple organ systems, highest viral titers were detected in skin and in discrete sites within oronasal tissues and gastrointestinal tract. The temporal distribution of infectious virus and viral DNA in tissues suggests an underlying pathogenesis that is similar to smallpox and monkeypox where greatest viral replication occurs in the skin. Our data demonstrate that capripoxvirus infections in sheep and goats provide additional and convenient models which are suitable not only for evaluation of poxvirus-specific vaccine concepts and therapeutics, but also study of poxvirus–host interactions

Detection of capripoxvirus DNA using a field-ready nucleic acid extraction and real-time PCR platform

Capripoxviruses, comprising sheep pox virus, goat pox virus and lumpy skin disease virus cause serious diseases of domesticated ruminants, notifiable to The World Organization for Animal Health. This report describes the evaluation of a mobile diagnostic system (Enigma Field Laboratory) that performs automated sequential steps for nucleic acid extraction and real-time PCR to detect capripoxvirus DNA within laboratory and endemic field settings. To prepare stable reagents that could be deployed into field settings, lyophilized reagents were used that employed an established diagnostic PCR assay. These stabilized reagents demonstrated an analytical sensitivity that was equivalent, or greater than the established laboratory-based PCR test which utilizes wet reagents, and the limit of detection for the complete assay pipeline was approximately one log10 more sensitive than the laboratory-based PCR assay. Concordant results were generated when the mobile PCR system was compared to the laboratory-based PCR using samples collected from Africa, Asia and Europe (n = 10) and experimental studies (n = 9) representing clinical cases of sheep pox, goat pox and lumpy skin disease. Furthermore, this mobile assay reported positive results in situ using specimens that were collected from a dairy cow in Morogoro, Tanzania, which was exhibiting clinical signs of lumpy skin disease. These data support the use of mobile PCR systems for the rapid and sensitive detection of capripoxvirus DNA in endemic field settings