key: cord-0032032-v8a7xzrb authors: Hemida, Maged Gomaa; Alnaeem, Abdelmohsen A. title: Betaretrovirus infections in dromedary camels in Saudi Arabia date: 2022-02-03 journal: Vet Med Sci DOI: 10.1002/vms3.760 sha: 6e381832125c89c5dd62ac113b45011b293a351c doc_id: 32032 cord_uid: v8a7xzrb BACKGROUND: Retroviral infections have been reported in many species of animals, especially cattle, sheep and goats. However, there are no available reports about retrovirus infection in dromedary camels. Several dromedary camels showed visible tumor‐like lesions on and around the nostrils as well as around the eyes. OBJECTIVES: Following are the objectives: to identify the causative agents of these identified tumours in dromedary camels and to perform molecular characterization of the detected strains of the causative agent. METHODS: We extracted the nucleic acids from some fresh lesions out of these animals, and then amplified some key retrovirus genes. We amplified several regions of the rotavirus genome using the PCR technique. The obtained sequences were assembled and the phylogenetic trees were conducted per each target retrovirus gene. RESULTS: Our results revealed a high degree of identity to some retroviruses of sheep. Phylogenetic analysis based on some retrovirus genes revealed that the causative agents of these lesions are closely related to sheep retroviruses, particularly the Jaagsiekte sheep Retrovirus (JSRV) and the ENTV. CONCLUSIONS: To the best of our knowledge, this is the first report of retrovirus infections in dromedary camels in the Arabian Peninsula. This highlights the possible species jump for the retrovirus from sheep and goats to the dromedary camels, which live in close proximity with these animals in many parts of the world, especially the Arabian Peninsula. The scope into four groups (A, B, C and D). However, the most recent classification of the virus was mainly based on the genetic sequences of the full-length genome of these viruses or the partial sequences of some key retrovirus genes (2018b, 2019) . According to the recent classification, retroviruses are divided into two subfamilies (Orthoretrovirinae and Spumaretroviradae) (2018b, 2019) . The retrovirus genome is mainly composed of two copies of single-stranded (+ve) sense RNA molecules, each of which is around 7 to 12 kb in length. The viral genomes are flanked by 5′cap and poly(A) tails at their 5′ and 3′ ends, respectively. The typical genome organization of the retroviruses is as follows: 5′-R U5 gag pro pol env U3 R-3′. The three major proteins in the retrovirus structure and functions are Gag, Pol and Env. The Gag protein represents three layers of the retroviruses. This protein plays important role in the assembly and packaging of the new viruses. The Pol gene has the function of the RNA-dependent DNA polymerase enzyme. Any changes in the genetic materials of this gene are responsible for the diversity among retroviruses. The provirus is integrated into the host cells representing a continuous source of infection to other animals (Toma et al., 1990) . Integration of the retroviral genes into the host cell genome especially during the early embryonic stages resulted in the development of many endogenous retroviruses (ERVs (Chessa et al., 2009; Palmarini et al., 2004) . We examined several dromedary camels showing skin lesions in different regions of the head including some lesions around the nostrils, some other lesions inside the nasal passage starting from the external nasal orifices, as well as some lesions around the eyes. The main objective of the current study was to identify these reported lesions in camels as well as to perform molecular characterization of their causative agent. Five dromedary camels showing skin lesions around the nostrils, mouth and eyes were identified in Saudi Arabia during 2016 (Table 1) . These animals were subjected to the routine slaughtering protocol. Physical inspection of the animals was conducted. The body of each ani-mal was examined carefully for the presence of any signs of abnormal discharges from the eyes and nostrils. The vital signs of each animal were assessed particularly the temperature and the colour of the mucous membranes of the eyes and vagina (in case of female animals). Tissue specimens from the lesions and some adjacent normal tissues were collected immediately after the slaughtering of these animals. Tissue specimens from each animal were divided into two halves. One half was placed on 10% tissue formalin for downstream histology processing while the other half was collected on the RNA later (QIAGEN, Catalog #76104). Samples collected on the RNAlater were stored at (−80 • C) for further processing. Meanwhile, we collected similar tissues from the exact locations from two healthy dromedary camels (one from Magaheem and one from Magateer camel) as negative controls. Samples from these animals were collected in the slaughterhouse during the necropsy examination of some of the animals showed the characteristic lesions under testing. Processing of the tissue specimens was done as previously described elsewhere (Khalafalla et al., 2017) . Extraction of viral DNA Viral DNAs were extracted from the freshly excised tumour tissues as previously described (Yousif et al., 2010) . Simply, 10 grams per lesion was collected and washed several times with sterile saline. These lesions were crushed using sterile sand in clean mortars with a pestle. We prepared 10% tissue suspensions per lesion by adding 90 mL of sterile saline. We centrifuged the prepared tissue suspensions at 5000 rpm for 10 min at 4 • C. We collected the supernatants and stored them at (−80 • C) until further DNA extraction. We extracted the total viral RNAs from the prepared tissues using We used the designed oligonucleotides for the papillomavirus fulllength ORF-L1 as previously described (Bernard et al., 2010) and as listed in Table 2 . Meanwhile, we used the primer design free online tools to design the primers of the betaretrovirus target genes (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). Table 2 shows the primer sequences used for the amplification of various betaretrovirus genes including gag, env, pol and pro. We tried to amplify the papillomavirus ORF-L1 regions from the extracted DNAs of all samples. The PCR amplification procedure and conditions were carried out as previously described (Forslund et al., 1999) . The cDNAs were synthesised by using random hexamer as previously described (He et al., 2017) which have been used as a template for the downstream PCR reactions. We used four pairs of primers to amplify the betaretrovirus target genes ( Table 2 ). The PCR reactions were performed as described elsewhere (He et al., 2017) . The nucleotide sequences for each target genes were assembled into one contig using the Lasergene version 7.1 software sequencing analysis (DNASTAR Inc., Madison, WI). We used the GCG programs Clustal-W and the Cluster to calculate the nucleotide identity of the reported geg, env, pol and pro genes. The maximum likelihood, pairwise nucleotide identities were established by the Mega-7 software as previously described (Kumar et al., 2016) . Five dromedary camels (three Magaheem and two Magateer) were examined during the ante-mortem inspection in the regional abattoir in eastern Saudi Arabia. Animals showed a decrease in body weight and loss of appetite. Animals showed opened nares with forced breathing with serous to mucopurulent discharges from the nose. Physical examination of animals revealed the presence of a cauliflower-like mass of tumours in the left nostrils ( Figure 1 ). This tumour connected to the ethmoid bone and extended upwards toward the nasal turbinate bones resulting in their damage. These tumours partially blocked the left nasal meatuses (Figure 1) . Examination of the excised tumour tissues revealed the presence of a large, dark solid area of necrosis. Meanwhile, the nasal septum was penetrated by these tumours (Figure 1 ). Our results show that all the tested tissue specimens were negative for the camel papillomavirus. Five out of the tested tissue specimens from animals were positive for the retrovirus using the gag, env, pol and pro genes of retroviruses. The bioinformatics and the phylogenetic analysis, the obtained sequences from these genes revealed a high degree of identity with other members of the betaretroviruses particularly the JSRV and the enzootic nasal tumour virus (ENTV) (Figures 2 and 3) . We deposited all the developed sequences from the current study in the Genbank under the accession numbers (MK205419, MK205420, MK205421 and MK205422) for the gag, env, pol and pro genes, respectively. In many regions in the world, such as the Arabian Peninsula and the Horn of Africa, dromedary camels, sheep and goats are usually kept together especially in the open grazing area (Hemida et al., 2017) . There are a large number of viral infections reported in dromedary camels and sheeps such as the bluetongue virus (BTV), the pest des petite ruminants and the foot and mouth disease virus (FMDV), (Larska et al., 2009; Touil et al., 2012; Zakian et al., 2016) . Although these viral infections cause diseases of various severities in sheep and goats, some of them are found to cause no clinical signs of diseases in dromedary camels such as BTV and FMDV (Larska et al., 2009; Zakian et al., 2016) . There is no available data about retrovirus infections in vari- (1000) is showing next to branches. The phylogenetic analysis was conducted using Mega-X software. Sequences reported in this study are marked with black triangles 1995). Camel papillomavirus causes wart-like skin lesions on the head of the affected animals, and also particularly around the lips, eyes and nostrils (Munz et al., 1990) . However, retrovirus infection in dromedary camels was never reported. We observed several dromedary camels We detected retrovirus infection in some dromedary camels in Saudi Arabia for the first time. These viruses showed a high degree of similarities with other retroviruses of sheep. This study points out the importance of studying retroviruses in various species of animals in the Arabian Peninsula. The authors declare there is no conflict of interest. Data available on request from the authors. The peer review history for this article is available at https://publons. com/publon/10.1002/vms3.760. Maged Gomaa Hemida https://orcid.org/0000-0003-1663-5820 International Committee on Taxonomy of Viruses (ICTV) Orf (Ecthyma Contagiosum) transmitted from a camel to a human: A case report Immune responses of the camel (Camelus dromedarius) to contagious ecthyma (Orf) virus infection Classification of papillomaviruses (PVs) based on 189 PV types and proposal of taxonomic amendments Revealing the history of sheep domestication using retrovirus integrations Camelpox virus A broad range of human papillomavirus types detected with a general PCR method suitable for analysis of cutaneous tumours and normal skin Endogenous retroviruses in domestic animals Full-length genome sequence analysis of enzootic nasal tumor virus isolated from goats in China Dromedary camels and the transmission of middle east respiratory syndrome coronavirus (MERS-CoV) Investigation on papillomavirus infection in dromedary camels in Al-Ahsa, Saudi Arabia MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets Differences in the susceptibility of dromedary and Bactrian camels to foot-and-mouth disease virus Camel papillomatosis in Somalia Endogenous betaretroviruses of sheep: Teaching new lessons in retroviral interference and adaptation Molecular characterization of a novel Camelus dromedarius papillomavirus. Comparative Immunology, Microbiology and Infectious Diseases Animal diseases caused by retroviruses: Enzootic bovine leukosis, equine infectious anaemia and caprine arthritis-encephalitis Emerging viral diseases in dromedary camels in the Southern Morocco Viruses and tumours. Textbook of Medical Virology Recovery and molecular characterization of live Camelpox virus from skin 12 months after onset of clinical signs reveals possible mechanism of virus persistence in herds Rapid non-enzymatic extraction method for isolating PCR-quality camelpox virus DNA from skin The first report of peste des petits ruminants (PPR) in camels (Camelus dromedarius) in Iran. Tropical Animal Health and Production Betaretrovirus infections in dromedary camels in Saudi Arabia