First identification and isolation of Varicellovirus Equidalpha1 in aborted fetal lung tissues of donkeys

First identification and isolation of Varicellovirus Equidalpha1 in aborted fetal lung tissues of donkeys | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Case Report First identification and isolation of Varicellovirus Equidalpha1 in aborted fetal lung tissues of donkeys Panpan Tong, Juanjuan Pan, Yueyi Dang, Enhui Yang, Chenyang Jia, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3948282/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Background: Varicellovirus equidalpha1 (formerly Equid alphaherpesvirus 1, EqAHV-1) is commonly linked to horse abortion. Currently, there are no reported cases of abortion resulting from EqAHV-1 infection in donkeys. Case presentation: This was the first survey-based study of Chinese donkeys, which identified EqAHV-1 in three aborted fetal lung tissues of donkeys via PCR technique. This survey was conducted in Chabuchar County, north Xinjiang, China, 2020. A donkey EqAHV-1 strain (Chabuchar/2020) was successfully isolated in MDBK cells. Moreover, the ORF33 sequence of the donkey-origin EqAHV-1 Chabuchar/2020 showed a high level of similarity, both in nucleotide (nt) (99.7 to 100%) and amino acid (99.5 to 100%) sequences, with horse EqAHV-1 strains. The discovery of EqAHV-1 Chabuchar/2020 revealed a remarkably consistent region, leading to its classification within cluster 1 of horse EqAHV-1 strains. Further, analysis of the expected ORF30 nt sequence revealed that donkey EqAHV-1 strains contained guanine (G) at the 2254 position of nt, leading to the presence of aspartic acid (D) at the 752 positions of the viral DNA polymerase. Therefore, these strains were classified as horse neuropathogenic strains. Lastly, a phylogenic network model was constructed via partial ORF68 nt sequences. This model disclosed that newly identified donkey EqAHV-1, and China's EqAHV-1, which was found in aborted Yili horses, comprised a novel independent VIII group. Conclusion: This study showed the first detection and isolation of EqAHV-1 as an etiological agent of abortions in donkeys. Further analysis of the ORF33, ORF30, and ORF68 sequences indicated that the donkey EqAHV-1 contained the neuropathogenic genotype in the VIII group. It is important to pay attention to EqAHV-1 infection in the donkey population, even though the virus has only been identified in donkey abortions in China. Donkey Abortion Equid herpesvirus 1 Neuropathogenicity VIII group Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Varicellovirus equidalpha1 formerly know as Equid alphaherpesvirus 1 belongs to the family Herpesviridae , subfamily Alphaherpesvirinae , and genus Varicellovirus [ 1 ]. It is a highly detrimental viral pathogen in horses due to its frequent association with respiratory complications, abortion, and ocular and neurological diseases. It has a substantial influence on the global equine industry as well as animal health [ 2 – 8 ]. Currently, there are no documented cases of abortion resulting from EqAHV-1 in donkeys. However, two studies have found evidence of this virus in nasopharyngeal swabs of both symptomatic and asymptomatic donkeys with respiratory disease [ 9 , 10 ]. EqAHV-1 genome is composed of 150 kbp of double-stranded DNA and can encode for ≥ 80 open reading frames (ORFs) [ 11 – 13 ]. One of these genes, ORF33, which encodes the envelope glycoprotein B (gB), contains a conserved segment that is widely used in PCR and phylogenetic examinations [ 14 ]. Similarly, the ORF30-encoded viral DNA polymerase displays a strong, but not unique, correlation with non-neuropathogenic and neuropathogenic EqAHV-1, depending on the presence of a G or A nt at 2254 position. Thus, these results revealed the substitution of an aspartic acid (D) or asparagine (N) at amino acid (aa) position 752 of the viral DNA polymerase [ 8 , 14 – 27 ]. Moreover, EqAHV-1 has been classified into VII groups as per the phylogenic analysis of ORF68 genes. These groups are associated with the geographical origin of the identified EqAHV-1 [ 14 , 16 , 28 ]. This was the first study conducted on EqAHV-1, the causative agent of abortions in donkeys. Importantly, this study is pioneering in its identification, examination, and isolation of EqAHV-1 associated with equine abortion. Case presentation In November 2020, a total of 80 donkeys from a farm located in Chabuchar County of Yili region (Northern Xinjiang, China), reported spontaneous abortions without showing any clinical symptoms. Out of a total population of 700 milk donkeys, 400 were jackass (≥ 4 years) and 200 of 300 were pregnant mares (≥ 4 years) at an estimated seven months of gestation. This farm was established in 2019, and all the milk donkeys were brought in from other farms in the Yili area of Northern Xinjiang without any scheduled vaccination. The veterinarian of this farm provided lung tissues from three aborted fetuses to identify viral pathogens as potential causes of these abortions. Viral nucleic acids were extracted (Geneaid Biotech Co.) from lung tissues by following the previously described protocol [ 8 ]. Based on the previous studies on the abortions of China's Yili mare and donkey [ 7 , 8 , 29 ], PCR was conducted using TIANSeq HiFi Amplification Mix (Tiangen Biotech) to detect the presence of Varicellovirus equidalpha 8 (EqAHV-8), EqAHV-1, EqAHV-4, EqAHV-2, and EqAHV-5. The primers used for the PCR analysis are mentioned in Table S1. The PCR protocol comprised an initial denaturation (94°C for 2 min), proceeded by 35 cycles, each consisting of denaturation (98°C for 10 sec), annealing, and extension (68°C for 30 sec) steps. Lastly, the final extension was also carried out (68°C for 5 min) and the PCR analysis confirmed the existence of EqAHV-1 (ON809533-ON809535) in the lung tissues of three donkey mares' aborted fetuses. However, no evidence of EqAHV-4, EqAHV-2, EqAHV-5, and EqAHV-8 were detected. Thus, the results revealed that the abortion storm in the donkey might have been caused by EqAHV-1. EqAHV-1 isolation was done on Madin-Darby bovine kidney (MDBK) cells from aborted fetal lung tissue (1:10 PBS, filtered 0.45 mm). The cytopathic impact was visible 36 h after inoculating MDBK cells (nine passages) with the supernatant of an EqAHV-1-positive aborted fetal lung tissue (Fig. 1 ). The identity of the viral isolate (Chabuchar/2020 strain) was verified via transmission electron microscope (TEM) and whole ORF33 gene sequencing (accession no: ON584564) (Fig. 2 ). Horse EqAHV-1 and donkey EqAHV-1 Chabuchar/2020 strains have great sequence similarity, as evidenced by the whole ORF33 sequences of the two strains sharing a nt similarity level of 99.7 to 100% and an amino acid similarity level of 99.5 to 100%. Furthermore, this study also matched the identified sequence with the reference strains of horse EqAHV-1 found in different countries, such as China (ZS-01: MZ561483, ZS-2: MZ561484, ZS-5: MZ561485, ZS-22: MZ561493, ZS-24: MZ561495, ZS-25: MZ561496, etc), UK (Ab1: KU206468, Ab4: AY665713, and Army: KU206477), in Japan (NY03: KF644569, 01c1: KF644578), US (RacL11: MF975656), India (Meerut: MT077857), and Australia (717A-82: KT324733, 438 − 77: KT324734). The results for DNA and aa sequences showed a 100% similarity level. The findings suggest that the ORF33 genes of donkey and horse EqAHV-1 displayed significant genetic conservation. These ORF33 nt sequences were used to construct a phylogenic network, which demonstrated that the identified EqAHV-1 strains were grouped to cluster 1 of EqAHV-1 strains found in horses (Fig. 3 ). To determine whether the newly identified donkey EqAHV-1 Chabuchar/2020 carried neuropathogenic or non-neuropathogenic characteristics, a partial PCR amplification was carried out on the ORF30 gene (559 nt) of EqAHV-1 Chabuchar/2020 via respective primers (Table S1). The results demonstrated that the partial ORF30 sequence of the neuropathogenic EqAHV-1 Chabuchar/2020 (ON624152) shared 100% nt and amino acid sequences identity with the referenced strains of EqAHV-1 (OM047215-OM047257), thus suggesting a high degree of genetic conservation. Moreover, similar to the neuropathogenic EqAHV-1 strains ZS-01, ZS-2, ZS-5, ZS-8, ZS-10, ZS-11, ZS-16, and ZS-17 (OM047215-OM047222), the identified donkey EHV1 strain had a G nt at 2254 position (corresponding to D aa at 752 positions of the viral DNA polymerase) (Fig. 4 ). Therefore, the identified strain was confirmed to be neuropathogenic EqAHV-1. Furthermore, phylogenetic analysis of the ORF68 genes has categorized all EqAHV-1 strains into VII groups that have a close relationship to the strains' origin [ 14 , 16 , 28 ]. In this study, a phylogenetic tree was developed using ORF68 gene sequences. The analysis showed that the donkey EqAHV-1 in this study and the EqAHV-1 found in abortions of Yili mares in China (ON624153, ON637792-ON637805) formed a distinct branch. They were classed as a new group, designated as group VIII EqAHV-1 (Fig. 5 ). The results indicated that EqAHV-1, which was detected in abortions between Yili mares and donkeys, may propagate independently in the Yili region of Northern Xinjiang, China. Discussion and conclusions The milk donkey sector is developing rapidly as it significantly contributes to the Xinjiang economy. However, abortion, a detrimental condition, is negatively impacting the progress of the milk donkey industry. Wang et al. [ 29 ] found that EHV-8 was associated with donkey abortions in Shangdong province, China; however, the current study did not identify this association. EqAHV-1 is commonly linked to respiratory complications, equine abortion, etc [ 2 – 8 ]. In previous studies, Negussie et al. (2017) and Temesgen et al. (2021) identified EqAHV-1 in Ethiopian donkeys with and without respiratory diseases [ 9 , 10 ]. This study was the first to identify EqAHV-1 in aborted donkey samples, suggesting that there is cause for concern regarding EqAHV-1 infection in donkey abortions, even though the virus has only been detected in donkeys from China. A recent study [ 8 ] found that EqAHV-1 was responsible for a significant number of abortion storms among Yili mares in Zhaosu County, and the majority of milk donkeys originated from this county. Therefore, the detection of EqAHV-1 in aborted samples obtained from an unvaccinated donkey is not unexpected. The prevalence of EqAHV-1 in China's donkeys will be studied further to confirm the link between this virus and diseases affecting donkeys. Despite the initial identification of EqAHV-1 in nasopharyngeal samples from donkeys [ 9 , 10 ], there is currently no published information regarding the genetic composition or viral particles of EqAHV-1 in donkeys. In the present study, the donkey EqAHV-1 Chabuchar/2020 strain was successfully isolated and observed (Fig. 2 ), and the partial ORF33 sequences of three donkey EqAHV-1 were sequenced and submitted to GeneBank (ON809533-ON809535). The current study compared the sequences of ORF33, ORF30, and ORF68 of the EqAHV-1 Chabuchar/2020 strain and horse EqAHV-1 reference strains found in abortions of Yili mares at the Chinese State Studs of Zhaosu, China. The sequences (MZ561483-MZ561485, MZ561493, MZ561495-MZ561497, MZ561500-MZ561503, MZ561507-MZ561510, MZ561513, MZ561515, MZ561517, MZ561518, MZ561521-MZ561523, MZ561525, OM047215-OM047257, ON637792-ON637805) showed 100% nt and amino acid identity. Further, the donkey EqAHV-1 strain that caused abortion showed neuropathogenic characteristics, which aligns with the EqAHV-1 strains responsible for abortions in Yili mares. Moreover, based on phylogenetic analysis of ORF68 sequences, it has been shown that the donkey and Yili mares EqAHV-1 of group VIII originate from the Yili area in Xinjiang. Thus, it can be concluded from the results that the EqAHV-1 Chabuchar/2020 strain may have originated in Yili horses. In conclusion, EqAHV-1 was for the first time identified in aborted fetuses of donkeys. It was revealed that EqAHV-1 might have been the causative agent of donkey abortions. Further analysis also showed evidence suggesting a potential involvement of the neuropathogenic strain of EqAHV-1 found in new group VIII. Thus, this study is suggested to raise awareness of the involvement of EqAHV-1 in donkey abortions, as well as encourage further research on EqAHV-1 in donkeys and the development of vaccinations targeting the virus. Abbreviations EqAHV-1 Varicellovirus equidalpha1 ORF Open reading frame G Guanidine A Adenine N Asparagine D Aspartic acid Declarations Acknowledgements Not applicable. Authors’ contributions P.T. and J.X. performed the research, analyzed the data, and drafted the manuscript. J.P., Y.D., E.Y., C.J., T.S., N.P., R.D., and L.K. contributed to the collection of samples and detection of PCR. P.T., C.W., G.L and J.X. revised the manuscript. J.X. conceived the study, carried out additional analyses and finalized the manuscript. All authors contributed to the revising of the manuscript. The authors read and approved the final manuscript. Funding This study was supported by the Natural Science Foundation of Xinjiang Uyghur Autonomous Region (2022D01A167), National Natural Science Foundation of China (32060808). Availability of data and materials All data generated or analyzed during this study are included in this published article and its additional files. Sequences of EqAHV-1 from aborted fetuses generated in this study have been submitted to GenBank under accession nos: ON809533-ON809535, ON584564, ON624152, ON624153 and ON637792-ON637805. Ethics approval and consent to participate All experimental procedures involving animals were approved by the Animal Care and Use Committee of Xinjiang Agricultural University, Urumqi, Xinjiang, China under animal protocol number: 2020012 and performed according to the Animal Ethics Procedures and Guide-lines of the Ministry of Agriculture of China. Lung tissues of aborted fetuses were collected by the farm’s veterinarian according to the approved procedures. 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Wang TT, Hu LY, Wang YH, Liu WQ, Liu GQ, Zhu MX, Zhang W, Wang CF, Ren HY, Li LL. Identifcation of equine herpesvirus 8 in donkey abortion: a case report. Virol J. 2022;19:10. Kumar S, Stecher G, Tamura K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33(7):1870–4. Additional Declarations No competing interests reported. Supplementary Files TableS1.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 09 Apr, 2024 Reviews received at journal 08 Apr, 2024 Reviewers agreed at journal 07 Mar, 2024 Reviewers invited by journal 20 Feb, 2024 Editor assigned by journal 14 Feb, 2024 Submission checks completed at journal 14 Feb, 2024 First submitted to journal 11 Feb, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3948282","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Case Report","associatedPublications":[],"authors":[{"id":272866420,"identity":"bb901bb7-136c-4832-a5a5-2e98d98b4936","order_by":0,"name":"Panpan Tong","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Panpan","middleName":"","lastName":"Tong","suffix":""},{"id":272866421,"identity":"dac8ea71-329b-4332-a7c3-399c1ca2460b","order_by":1,"name":"Juanjuan Pan","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Juanjuan","middleName":"","lastName":"Pan","suffix":""},{"id":272866422,"identity":"8e1b3415-ef5c-4846-b52f-0354fa9deb0a","order_by":2,"name":"Yueyi Dang","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yueyi","middleName":"","lastName":"Dang","suffix":""},{"id":272866423,"identity":"89653abb-ab74-464a-b27a-f9b178f9162d","order_by":3,"name":"Enhui Yang","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Enhui","middleName":"","lastName":"Yang","suffix":""},{"id":272866424,"identity":"45cf1fc7-2a04-4d13-91c3-be271e944334","order_by":4,"name":"Chenyang Jia","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Chenyang","middleName":"","lastName":"Jia","suffix":""},{"id":272866425,"identity":"23564e0a-6aa0-4a8d-90bf-251ae96100bc","order_by":5,"name":"Ruli Duan","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Ruli","middleName":"","lastName":"Duan","suffix":""},{"id":272866426,"identity":"f87bead1-b156-43a6-98af-226089728db0","order_by":6,"name":"Shuyao Tian","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Shuyao","middleName":"","lastName":"Tian","suffix":""},{"id":272866427,"identity":"46a7d85f-8998-476a-ad18-637fe7a67554","order_by":7,"name":"Nuerlan Palidan","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Nuerlan","middleName":"","lastName":"Palidan","suffix":""},{"id":272866428,"identity":"53259083-1e22-47e4-bfeb-6da6a5e137d2","order_by":8,"name":"Ling Kuang","email":"","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Ling","middleName":"","lastName":"Kuang","suffix":""},{"id":272866429,"identity":"8268b9f6-5df0-47a6-81f7-dcd05a5cb29d","order_by":9,"name":"Chuanfeng Wang","email":"","orcid":"","institution":"Yili Vocational and Technical College","correspondingAuthor":false,"prefix":"","firstName":"Chuanfeng","middleName":"","lastName":"Wang","suffix":""},{"id":272866430,"identity":"d590ddcc-fbe1-4b39-b224-afbc7c38abfd","order_by":10,"name":"Gang Lu","email":"","orcid":"","institution":"South China Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Gang","middleName":"","lastName":"Lu","suffix":""},{"id":272866431,"identity":"f31f7972-43bd-426b-9a6e-8f7fe54a4730","order_by":11,"name":"Jinxin Xie","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/UlEQVRIiWNgGAWjYDACZgYGgwQGBh4GZuaDDz5U1MixsbcfIFILO1uy4Ywzx4z5eM4kEGkdP4+ZNGcbc+I8CQcDvAoNjjMfKHi447CMOTOPsTHDGbb0NgmGBIYfFdtwapFsZkswSDyTxmPZzFb4uKBCJrdNuvEAY8+Z27idw8xjYJDYZsNjcJh5s/GMM2y5bTIHEpgZ23BrYWPm/wDUIgHUwmAmzdvGnM4mkWCAVwvQFgaoLSxgLQkEtQD9AnJYGlALJJAN24CBfBCfXwzOH35m+LPtsD2QAY5Kefn29oMPflTg1gLyDmY0HMCnHgiYHxBQMApGwSgYBSMdAABCzFKxMXw+fgAAAABJRU5ErkJggg==","orcid":"","institution":"Xinjiang Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Jinxin","middleName":"","lastName":"Xie","suffix":""}],"badges":[],"createdAt":"2024-02-11 11:22:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3948282/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3948282/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51210842,"identity":"58562c55-46c7-4c0f-b47c-f7cb61dc5aa4","added_by":"auto","created_at":"2024-02-16 05:04:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":34461092,"visible":true,"origin":"","legend":"\u003cp\u003eMDBK cells were allowed to grow for 72 h till nine passages. Cells were initially inoculated with EqAHV-1-positive aborted fetal lung tissue lysate from donkeys (right panel) or left untreated (left panel).\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-3948282/v1/c297de05ae0ef103c8b2f442.png"},{"id":51210838,"identity":"84490b2f-5444-446a-9947-3ec8df342817","added_by":"auto","created_at":"2024-02-16 05:04:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6266281,"visible":true,"origin":"","legend":"\u003cp\u003eThe virions of donkey EqAHV-1 Chabuchar/2020 display distinct morphologies.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3948282/v1/9ce4f49ad61991c1a217de6e.png"},{"id":51210836,"identity":"16ce1358-7e9a-4a34-b027-f1a12c5bb95b","added_by":"auto","created_at":"2024-02-16 05:04:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2128055,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenic network representation using the maximum-likelihood method based on the Tamura-Nei model (MEGA7) and EqAHV-1 ORF33 gene sequences. The newly identified EqAHV-1Chabuchar/2020 strain was detected and shown by the black circle.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3948282/v1/c49c6fe455741d203ad3ef79.png"},{"id":51210839,"identity":"2936a5ea-fce5-4053-81f8-bbdf037a82a9","added_by":"auto","created_at":"2024-02-16 05:04:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":207192,"visible":true,"origin":"","legend":"\u003cp\u003eThe ORF30 gene sequence was analyzed. All EqAHV-1 strains examined in this study displayed the G2254 polymorphism, as depicted in Fig 4A. The presence of the D752 polymorphism in all EqAHV-1isolates evaluated in this study is shown in Fig 4B. When comparing the alignment to the EqAHV-1 ZS-01 isolate (CLC Sequence Viewer 8), sequence identity is indicated by dots.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3948282/v1/a6dbe2d533ac5bc31cdac8b8.png"},{"id":51210841,"identity":"81d53264-6dff-4251-af1f-f0a43aa1a79a","added_by":"auto","created_at":"2024-02-16 05:04:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2332515,"visible":true,"origin":"","legend":"\u003cp\u003eA phylogenetic network is constructed \u003cem\u003evia \u003c/em\u003ethe Neighbor-Joining method (MEGA7) as per the sequences of the EqAHV-1 ORF68 gene. The filled circle represents the newly identified EqAHV-1Chabuchar/2020, while the open circles depict the EqAHV-1 detected in abortions of Yili mares.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3948282/v1/1f94c79a615016f8b311f99d.png"},{"id":51211221,"identity":"4daec073-24f1-4703-b7ad-955decb98627","added_by":"auto","created_at":"2024-02-16 05:12:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3491645,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3948282/v1/f8eaef24-bd17-4681-88e4-a6c9ad378ff3.pdf"},{"id":51210840,"identity":"4a03614b-8b44-4de0-8525-6cca6ebd8e45","added_by":"auto","created_at":"2024-02-16 05:04:50","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":14600,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3948282/v1/ecdc813a3727dc5e0a793bd2.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"First identification and isolation of Varicellovirus Equidalpha1 in aborted fetal lung tissues of donkeys","fulltext":[{"header":"Background","content":"\u003cp\u003e \u003cem\u003eVaricellovirus equidalpha1\u003c/em\u003e formerly know as Equid alphaherpesvirus 1 belongs to the family \u003cem\u003eHerpesviridae\u003c/em\u003e, subfamily \u003cem\u003eAlphaherpesvirinae\u003c/em\u003e, and genus Varicellovirus [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. It is a highly detrimental viral pathogen in horses due to its frequent association with respiratory complications, abortion, and ocular and neurological diseases. It has a substantial influence on the global equine industry as well as animal health [\u003cspan additionalcitationids=\"CR3 CR4 CR5 CR6 CR7\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Currently, there are no documented cases of abortion resulting from EqAHV-1 in donkeys. However, two studies have found evidence of this virus in nasopharyngeal swabs of both symptomatic and asymptomatic donkeys with respiratory disease [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEqAHV-1 genome is composed of 150 kbp of double-stranded DNA and can encode for \u0026ge;\u0026thinsp;80 open reading frames (ORFs) [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. One of these genes, ORF33, which encodes the envelope glycoprotein B (gB), contains a conserved segment that is widely used in PCR and phylogenetic examinations [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Similarly, the ORF30-encoded viral DNA polymerase displays a strong, but not unique, correlation with non-neuropathogenic and neuropathogenic EqAHV-1, depending on the presence of a G or A nt at 2254 position. Thus, these results revealed the substitution of an aspartic acid (D) or asparagine (N) at amino acid (aa) position 752 of the viral DNA polymerase [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15 CR16 CR17 CR18 CR19 CR20 CR21 CR22 CR23 CR24 CR25 CR26\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Moreover, EqAHV-1 has been classified into VII groups as per the phylogenic analysis of ORF68 genes. These groups are associated with the geographical origin of the identified EqAHV-1 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis was the first study conducted on EqAHV-1, the causative agent of abortions in donkeys. Importantly, this study is pioneering in its identification, examination, and isolation of EqAHV-1 associated with equine abortion.\u003c/p\u003e"},{"header":"Case presentation","content":"\u003cp\u003eIn November 2020, a total of 80 donkeys from a farm located in Chabuchar County of Yili region (Northern Xinjiang, China), reported spontaneous abortions without showing any clinical symptoms. Out of a total population of 700 milk donkeys, 400 were jackass (\u0026ge;\u0026thinsp;4 years) and 200 of 300 were pregnant mares (\u0026ge;\u0026thinsp;4 years) at an estimated seven months of gestation. This farm was established in 2019, and all the milk donkeys were brought in from other farms in the Yili area of Northern Xinjiang without any scheduled vaccination.\u003c/p\u003e \u003cp\u003eThe veterinarian of this farm provided lung tissues from three aborted fetuses to identify viral pathogens as potential causes of these abortions. Viral nucleic acids were extracted (Geneaid Biotech Co.) from lung tissues by following the previously described protocol [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Based on the previous studies on the abortions of China's Yili mare and donkey [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], PCR was conducted using TIANSeq HiFi Amplification Mix (Tiangen Biotech) to detect the presence of \u003cem\u003eVaricellovirus equidalpha\u003c/em\u003e8 (EqAHV-8), EqAHV-1, EqAHV-4, EqAHV-2, and EqAHV-5. The primers used for the PCR analysis are mentioned in Table S1.\u003c/p\u003e \u003cp\u003eThe PCR protocol comprised an initial denaturation (94\u0026deg;C for 2 min), proceeded by 35 cycles, each consisting of denaturation (98\u0026deg;C for 10 sec), annealing, and extension (68\u0026deg;C for 30 sec) steps. Lastly, the final extension was also carried out (68\u0026deg;C for 5 min) and the PCR analysis confirmed the existence of EqAHV-1 (ON809533-ON809535) in the lung tissues of three donkey mares' aborted fetuses. However, no evidence of EqAHV-4, EqAHV-2, EqAHV-5, and EqAHV-8 were detected. Thus, the results revealed that the abortion storm in the donkey might have been caused by EqAHV-1.\u003c/p\u003e \u003cp\u003eEqAHV-1 isolation was done on Madin-Darby bovine kidney (MDBK) cells from aborted fetal lung tissue (1:10 PBS, filtered 0.45 mm). The cytopathic impact was visible 36 h after inoculating MDBK cells (nine passages) with the supernatant of an EqAHV-1-positive aborted fetal lung tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The identity of the viral isolate (Chabuchar/2020 strain) was verified \u003cem\u003evia\u003c/em\u003e transmission electron microscope (TEM) and whole ORF33 gene sequencing (accession no: ON584564) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHorse EqAHV-1 and donkey EqAHV-1 Chabuchar/2020 strains have great sequence similarity, as evidenced by the whole ORF33 sequences of the two strains sharing a nt similarity level of 99.7 to 100% and an amino acid similarity level of 99.5 to 100%. Furthermore, this study also matched the identified sequence with the reference strains of horse EqAHV-1 found in different countries, such as China (ZS-01: MZ561483, ZS-2: MZ561484, ZS-5: MZ561485, ZS-22: MZ561493, ZS-24: MZ561495, ZS-25: MZ561496, etc), UK (Ab1: KU206468, Ab4: AY665713, and Army: KU206477), in Japan (NY03: KF644569, 01c1: KF644578), US (RacL11: MF975656), India (Meerut: MT077857), and Australia (717A-82: KT324733, 438\u0026thinsp;\u0026minus;\u0026thinsp;77: KT324734). The results for DNA and aa sequences showed a 100% similarity level. The findings suggest that the ORF33 genes of donkey and horse EqAHV-1 displayed significant genetic conservation. These ORF33 nt sequences were used to construct a phylogenic network, which demonstrated that the identified EqAHV-1 strains were grouped to cluster 1 of EqAHV-1 strains found in horses (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo determine whether the newly identified donkey EqAHV-1 Chabuchar/2020 carried neuropathogenic or non-neuropathogenic characteristics, a partial PCR amplification was carried out on the ORF30 gene (559 nt) of EqAHV-1 Chabuchar/2020 \u003cem\u003evia\u003c/em\u003e respective primers (Table S1). The results demonstrated that the partial ORF30 sequence of the neuropathogenic EqAHV-1 Chabuchar/2020 (ON624152) shared 100% nt and amino acid sequences identity with the referenced strains of EqAHV-1 (OM047215-OM047257), thus suggesting a high degree of genetic conservation. Moreover, similar to the neuropathogenic EqAHV-1 strains ZS-01, ZS-2, ZS-5, ZS-8, ZS-10, ZS-11, ZS-16, and ZS-17 (OM047215-OM047222), the identified donkey EHV1 strain had a G nt at 2254 position (corresponding to D aa at 752 positions of the viral DNA polymerase) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Therefore, the identified strain was confirmed to be neuropathogenic EqAHV-1.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, phylogenetic analysis of the ORF68 genes has categorized all EqAHV-1 strains into VII groups that have a close relationship to the strains' origin [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In this study, a phylogenetic tree was developed using ORF68 gene sequences. The analysis showed that the donkey EqAHV-1 in this study and the EqAHV-1 found in abortions of Yili mares in China (ON624153, ON637792-ON637805) formed a distinct branch. They were classed as a new group, designated as group VIII EqAHV-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The results indicated that EqAHV-1, which was detected in abortions between Yili mares and donkeys, may propagate independently in the Yili region of Northern Xinjiang, China.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion and conclusions","content":"\u003cp\u003eThe milk donkey sector is developing rapidly as it significantly contributes to the Xinjiang economy. However, abortion, a detrimental condition, is negatively impacting the progress of the milk donkey industry. Wang et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] found that EHV-8 was associated with donkey abortions in Shangdong province, China; however, the current study did not identify this association. EqAHV-1 is commonly linked to respiratory complications, equine abortion, etc [\u003cspan additionalcitationids=\"CR3 CR4 CR5 CR6 CR7\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In previous studies, Negussie et al. (2017) and Temesgen et al. (2021) identified EqAHV-1 in Ethiopian donkeys with and without respiratory diseases [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. This study was the first to identify EqAHV-1 in aborted donkey samples, suggesting that there is cause for concern regarding EqAHV-1 infection in donkey abortions, even though the virus has only been detected in donkeys from China. A recent study [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] found that EqAHV-1 was responsible for a significant number of abortion storms among Yili mares in Zhaosu County, and the majority of milk donkeys originated from this county. Therefore, the detection of EqAHV-1 in aborted samples obtained from an unvaccinated donkey is not unexpected. The prevalence of EqAHV-1 in China's donkeys will be studied further to confirm the link between this virus and diseases affecting donkeys.\u003c/p\u003e \u003cp\u003eDespite the initial identification of EqAHV-1 in nasopharyngeal samples from donkeys [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], there is currently no published information regarding the genetic composition or viral particles of EqAHV-1 in donkeys. In the present study, the donkey EqAHV-1 Chabuchar/2020 strain was successfully isolated and observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), and the partial ORF33 sequences of three donkey EqAHV-1 were sequenced and submitted to GeneBank (ON809533-ON809535).\u003c/p\u003e \u003cp\u003eThe current study compared the sequences of ORF33, ORF30, and ORF68 of the EqAHV-1 Chabuchar/2020 strain and horse EqAHV-1 reference strains found in abortions of Yili mares at the Chinese State Studs of Zhaosu, China. The sequences (MZ561483-MZ561485, MZ561493, MZ561495-MZ561497, MZ561500-MZ561503, MZ561507-MZ561510, MZ561513, MZ561515, MZ561517, MZ561518, MZ561521-MZ561523, MZ561525, OM047215-OM047257, ON637792-ON637805) showed 100% nt and amino acid identity. Further, the donkey EqAHV-1 strain that caused abortion showed neuropathogenic characteristics, which aligns with the EqAHV-1 strains responsible for abortions in Yili mares. Moreover, based on phylogenetic analysis of ORF68 sequences, it has been shown that the donkey and Yili mares EqAHV-1 of group VIII originate from the Yili area in Xinjiang. Thus, it can be concluded from the results that the EqAHV-1 Chabuchar/2020 strain may have originated in Yili horses.\u003c/p\u003e \u003cp\u003eIn conclusion, EqAHV-1 was for the first time identified in aborted fetuses of donkeys. It was revealed that EqAHV-1 might have been the causative agent of donkey abortions. Further analysis also showed evidence suggesting a potential involvement of the neuropathogenic strain of EqAHV-1 found in new group VIII. Thus, this study is suggested to raise awareness of the involvement of EqAHV-1 in donkey abortions, as well as encourage further research on EqAHV-1 in donkeys and the development of vaccinations targeting the virus.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEqAHV-1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eVaricellovirus equidalpha1\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eORF\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOpen reading frame\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGuanidine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAdenine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eN\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAsparagine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAspartic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eP.T. and J.X. performed the research, analyzed the data, and drafted the manuscript. J.P., Y.D., E.Y., C.J., T.S., N.P., R.D., and L.K. contributed to the collection of samples and detection of PCR. P.T., C.W., G.L and J.X. revised the manuscript. J.X. conceived the study, carried out additional analyses and finalized the manuscript. All authors contributed to the revising of the manuscript. The authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the Natural Science Foundation of Xinjiang Uyghur Autonomous Region (2022D01A167),\u0026nbsp;National Natural Science Foundation of China (32060808).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article and its additional files. Sequences of EqAHV-1 from aborted fetuses generated in this study have been submitted to GenBank under accession nos:\u0026nbsp;ON809533-ON809535, ON584564, ON624152, ON624153\u0026nbsp;and\u0026nbsp;ON637792-ON637805.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eEthics\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eapproval and consent to participate\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll experimental procedures involving animals were approved by the Animal Care and Use Committee of Xinjiang Agricultural University, Urumqi, Xinjiang, China under animal protocol number: 2020012 and performed according to the Animal Ethics Procedures and Guide-lines of the Ministry of Agriculture of China. Lung tissues of aborted fetuses were collected by the farm\u0026rsquo;s veterinarian according to the approved procedures.\u0026nbsp;The owner gave informed written consent for the lung tissues of aborted fetuses to be included in the study. The study was carried out in compliance with the ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eLaboratory of Animal Etiology and Epidemiology, College of Veterinary Medicine, Xinjiang Agricultural University, Urumqi, Xinjiang, China. \u003csup\u003e2\u003c/sup\u003eKey laboratory for animal disease detection, College of Animal Sciences, Yili Vocational and Technical College,\u0026nbsp;Yili, Xinjiang, China.\u0026nbsp;\u003csup\u003e3\u003c/sup\u003eCollege of Veterinary Medicine, South China Agricultural University, Guangzhou, Guangdong, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGatherer D, Depledge DP, Hartley CA, Szpara ML, Vaz PK, Benkő M, Brandt CR, Bryant NA, Dastjerdi A, Doszpoly A, Gompels UA, Inoue N, Jarosinski KW, Kaul R, Lacoste V, Norberg P, Origgi FC, Orton RJ, Pellett PE, Schmid DS, Spatz SJ, Stewart JP, Trimpert J, Waltzek TB, Davison AJ. 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(In Chinese).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTong PP, Duan RL, Palidan NEL, Deng HF, Duan LY, Ren ML, Song XZ, Jia CY, Tian SY, Yang EH, Kuang L, Xie JX. Outbreak of neuropathogenic equid herpesvirus 1 causing abortions in Yili horses of Zhaosu, North Xinjiang, China. BMC Vet Res. 2022;18:83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNegussie H, Gizaw D, Tesfaw L, Li Y, Oguma K, Sentsui H, Tessema TS, Nauwynck HJ. Detection of equine herpesvirus (EHV) \u0026ndash;\u0026thinsp;1, -2, -4 and \u0026ndash;\u0026thinsp;5 in Ethiopian equids with and without respiratory problems and genetic characterization of EHV-2 and EHV-5 strains. Transbound Emerg Dis. 2017;64(6):1970\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTemesgen T, Getachew Y, Negussie H. Molecular identification of equine herpesvirus 1, 2, and 5 in equids with signs of respiratory disease in central Ethiopia. Vet Med (Auckl). 2021;12:337\u0026ndash;45.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBurton EA, Wechuck JB, Wendell SK, Goins WF, Fink DJ, Glorioso JC. Multiple applications for replication-defective herpes simplex virus vectors. Stem Cells. 2001;19(5):358\u0026ndash;77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatel JR, Heldens J. Equine herpesviruses 1 (EHV-1) and 4 (EHV-4)--epidemiology, disease and immunoprophylaxis: a brief review. Vet J. 2005;170(1):14\u0026ndash;23.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTelford EA, Watson MS, McBride K, Davison AJ. The DNA sequence of equine herpesvirus-1. Virology. 1992;189(1):304\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePreziuso S, Sgorbini M, Marmorini P, Cuteri V. Equid alphaherpesvirus 1 from Italian horses: evaluation of the variability of the ORF30, ORF33, ORF34 and ORF68 genes. Viruses. 2019;11(9):851.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNugent J, Birch-Machin I, Smith KC, Mumford JA, Swann Z, New-ton JR, Bowden RJ, Allen GP, Davis-Poynter N. Analysis of equid herpesvirus 1 strain variation reveals a point mutation of the DNA polymerase strongly associated with neuropathogenic versus nonneuropathogenic disease outbreaks. J Virol. 2006;80(8):4047\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eStasiak K, Dunowska M, Rola J. Outbreak of equid herpesvirus 1 abortions at the Arabian stud in Poland. BMC Vet Res. 2020;16(1):374.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVissani MA, Becerra ML, Olgu\u0026iacute;n Perglione C, Tordoya MS, Mi\u0026ntilde;o S, Barrandeguy M. Neuropathogenic and non-neuropathogenic genotypes of equid herpesvirus type 1 in Argentina. Vet Microbiol. 2009;139(3\u0026ndash;4):361\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSmith KL, Allen GP, Branscum AJ, Frank Cook R, Vickers ML, Timoney PJ, Balasuriya UB. The increased prevalence of neuropathogenic strains of EHV-1 in equine abortions. Vet Microbiol. 2010;141(1\u0026ndash;2):5\u0026ndash;11.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeon A, Fortier G, Fortier C, Freymuth F, Tapprest J, Leclercq R, Pronost S. Detection of equine herpesviruses in aborted foetuses by consensus PCR. Vet Microbiol. 2008;126(1):20\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMori E, Lara do Carmo CSH, Lara M, Cunha EMS, Villalobos EMC, Mori CMC, Soares RM, Brandao PE, Fernandes WR, Richtzenhain LJ. Molecular characterization of Brazilian equid herpesvirus type 1 strains based on neuropathogenicity markers. Braz J Microbiol. 2015;46(2):565\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTuran N, Yildirim F, Altan E, Sennazli G, Gurel A, Diallo I, Yilmaz H. Molecular and pathological investigations of EHV-1 and EHV-4 infections in horses in Turkey. Res Vet Sci. 2012;93(3):1504\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePerkins GA, Goodman LB, Tsujimura K, Van de Walle GR, Kim SG, Dubovi EJ, Osterrieder N. Investigation of the prevalence of neurologic equine herpes virus type 1 (EHV-1) in a 23-year retrospective analysis (1984\u0026ndash;2007). Vet Microbiol. 2009;139(3):375\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePronost S, Leon A, Legrand L, Fortier C, Miszczak F, Freymuth F, Fortier G. Neuropathogenic and non-neuropathogenic variants of equine herpesvirus 1 in France. Vet Microbiol. 2010;145(3):329\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTsujimura K, Oyama T, Katayama Y, Muranaka M, Bannai H, Nemoto M, Yamanaka T, Kondo T, Kato M, Matsumura T. Prevalence of equine herpesvirus type 1 strains of neuropathogenic genotype in a major breeding area of Japan. J Vet Med Sci. 2011;73(12):1663\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCastro ER, Arbiza J. Detection and genotyping of equid herpesvirus 1 in Uruguay. Rev Sci Tech. 2017;36(3):799\u0026ndash;806.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNegussie H, Gizaw D, Tessema TS, Nauwynck HJ. Equine herpesvirus-1 myeloencephalopathy, an emerging threat of working equids in Ethiopia. Transbound Emerg Dis. 2017;64(2):389\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDamiani AM, de Vries M, Reimers G, Winkler S, Osterrieder N. A severe equine herpesvirus type 1 (EHV-1) abortion outbreak caused by a neuropathogenic strain at a breeding farm in northern Germany. Vet Microbiol. 2014;172(3):555\u0026ndash;62.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalik P, B\u0026aacute;lint A, D\u0026aacute;n A, P\u0026aacute;lfi V. Molecular characterisation of the ORF68 region of equine herpesvirus-1 strains isolated from aborted fetuses in Hungary between 1977 and 2008. Acta Vet Hung. 2012;60(1):175\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang TT, Hu LY, Wang YH, Liu WQ, Liu GQ, Zhu MX, Zhang W, Wang CF, Ren HY, Li LL. Identifcation of equine herpesvirus 8 in donkey abortion: a case report. Virol J. 2022;19:10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar S, Stecher G, Tamura K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33(7):1870\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"virology-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"virj","sideBox":"Learn more about [Virology Journal](http://virologyj.biomedcentral.com/)","snPcode":"12985","submissionUrl":"https://submission.nature.com/new-submission/12985/3","title":"Virology Journal","twitterHandle":"@VirologyJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Donkey, Abortion, Equid herpesvirus 1, Neuropathogenicity, VIII group","lastPublishedDoi":"10.21203/rs.3.rs-3948282/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3948282/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e \u003cem\u003eVaricellovirus equidalpha1\u003c/em\u003e (formerly Equid alphaherpesvirus 1, EqAHV-1) is commonly linked to horse abortion. Currently, there are no reported cases of abortion resulting from EqAHV-1 infection in donkeys.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCase presentation:\u003c/strong\u003e This was the first survey-based study of Chinese donkeys, which identified EqAHV-1 in three aborted fetal lung tissues of donkeys via PCR technique. This survey was conducted in Chabuchar County, north Xinjiang, China, 2020. A donkey EqAHV-1 strain (Chabuchar/2020) was successfully isolated in MDBK cells. Moreover, the ORF33 sequence of the donkey-origin EqAHV-1 Chabuchar/2020 showed a high level of similarity, both in nucleotide (nt) (99.7 to 100%) and amino acid (99.5 to 100%) sequences, with horse EqAHV-1 strains. The discovery of EqAHV-1 Chabuchar/2020 revealed a remarkably consistent region, leading to its classification within cluster 1 of horse EqAHV-1 strains. Further, analysis of the expected ORF30 nt sequence revealed that donkey EqAHV-1 strains contained guanine (G) at the 2254 position of nt, leading to the presence of aspartic acid (D) at the 752 positions of the viral DNA polymerase. Therefore, these strains were classified as horse neuropathogenic strains. Lastly, a phylogenic network model was constructed \u003cem\u003evia\u003c/em\u003e partial ORF68 nt sequences. This model disclosed that newly identified donkey EqAHV-1, and China's EqAHV-1, which was found in aborted Yili horses, comprised a novel independent VIII group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eThis study showed the first detection and isolation of EqAHV-1 as an etiological agent of abortions in donkeys. Further analysis of the ORF33, ORF30, and ORF68 sequences indicated that the donkey EqAHV-1 contained the neuropathogenic genotype in the VIII group. It is important to pay attention to EqAHV-1 infection in the donkey population, even though the virus has only been identified in donkey abortions in China.\u003c/p\u003e","manuscriptTitle":"First identification and isolation of Varicellovirus Equidalpha1 in aborted fetal lung tissues of donkeys","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-16 05:04:44","doi":"10.21203/rs.3.rs-3948282/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-04-09T07:29:37+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-08T04:22:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"16b87fcd-7fb5-4921-9d02-5f93665d43a0","date":"2024-03-07T06:02:08+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-20T18:01:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-14T16:07:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-14T10:06:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"Virology Journal","date":"2024-02-11T11:19:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"virology-journal","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"virj","sideBox":"Learn more about [Virology Journal](http://virologyj.biomedcentral.com/)","snPcode":"12985","submissionUrl":"https://submission.nature.com/new-submission/12985/3","title":"Virology Journal","twitterHandle":"@VirologyJ","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6625dce3-2473-4309-a69d-edce74719388","owner":[],"postedDate":"February 16th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-14T17:52:57+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-16 05:04:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3948282","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3948282","identity":"rs-3948282","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}