Maedi-Visna disease is a chronic, wasting disease that affects several mammals in the genera Capra and Ovis (1). The disease is caused by Visna/Maedi virus (VMV, species Visna-Maedi virus), a member of the family Retroviridae, genus Lentivirus, which is collectively referred to as small ruminant lentiviruses (SRLVs) together with the Caprine Arthritis-Encephalitis Virus (2). VMV crosses species barriers and has been found in wild ruminants such as red deer, Alpine ibexes, Passirian goats, and Mouflon (3–7). The virus transmits primarily through body fluids such as respiratory exudates, colostrum and milk from infected ewes (1). Vertical transmission through the placenta and semen during pregnancy and mating is an alternative for animal infection with VMV (8–10). The VMV-infected animals manifest continuous and progressive pathological damage to joints, breasts, lungs, and the nervous system (11, 12). In addition, VMV causes lifelong subclinical infections, and only 30% of subclinical VMV infections present clinical symptoms following an incubation period of about 3–4 years (13). The disease causes direct economic losses, including reduced life length, less milk production, and shorter lactation duration (14, 15).

Maedi-Visna disease was initially described in Iceland in 1939 (16). The virus spread to Denmark, Finland, France, Hungary, and Norway through the inter-country trade of breeding sheep between 1960 and 1990 (17). VMV has spread worldwide except for New Zealand and Australia (13, 17). In China, Maedi-Visna disease was identified in 1985 (18), and it was believed to be an infectious disease with individual seroprevalence of 4.6%−50.0% and herd seroprevalence of 100% (19–21). The disease is listed as a Class II animal disease by the Ministry of Agriculture and Rural Affairs of the P.R. China and as a notified terrestrial animal disease by World Organization for Animal Health.

Visna/Maedi virus is an enveloped virus with double-stranded RNA of 8.4–10 kb in length. The nucleic acid encodes three structural protein genes: env, gag, and pol (22). Pol possesses the longest open reading frame, located in the middle of the genome, encoding a pol protein involved in replication and DNA integration (23). The gag gene encodes a structural protein comprised of three subunit proteins (capsid, matrix, and nucleocapsid proteins), which stimulates the production of antibodies in the host and protects genomic DNA (24). There are three non-structural protein genes (rev, tat, and vif ), and in genome 5′ and 3′ ends, a long terminal repeat (LTR) comprises U5. The R and U3 regions are responsible for host tropism for monocytes, macrophages, and dendritic cells (25).

The gag and gag-pol genes and LTR sequences are markers for dividing SRLVs into five genotypes (A–E) (26). Of which, Genotypes A and B are MVV-like and CAEV-like strains that dominated worldwide, respectively, while genotypes C, D and E were only found in Europe, i.e., Group C strains in Norwegian sheep and goats, genotype D strains in sheep and goats in Switzerland and Spain, and genotype E in Italy (27–29). VMV or VMV-like viruses are grouped into genotype A, divided into 22 subgenotypes, including A1–A22 (26, 30). The subgenotype presents a geographical distribution and is clustered into additional subgroups. For example, the USA has only A2 genotype strains to date; however, these are classified further into four subgroups based on a neighbor-joining tree of partial gag sequences (31).

Although VMV was identified in China nearly four decades ago with exceptionally high seroprevalence in Chinese sheep herds, little is known regarding VMV genomic characteristics. This study aimed to characterize two VMV strains isolated in 1985 and 1994 and determine the genotype and the genetic relationship with VMV outside China. We employed a next-generation sequencing method to obtain draft maps of two Chinese isolates. Cloning and Sanger sequencing were performed to obtain precise VMV genome sequences. We also conducted a detailed genomic comparison of Chinese isolates and related genomic sequences reported. The research results will provide a basis for VMV control in China.

Visna/Maedi virus is a high-risk infectious disease in animals in China, and its recessive and latent infections have caused health risks to the national sheep-breeding industry. In this study, two VMV strains isolated from Xinjiang, northwest China in 1985 and 1994 were retrospectively identified using Illumina sequencing and whole-genome cloning. Two precise genome sequences were obtained, which would be beneficial for tracing the source and developing diagnostic methods in China.

A total of 189 variable sites in the genomic sequences caused variations in three ORFs between CMV-1 and XM-MDV30. The altered ORFs encoded three proteins (gag, pol, and env). Of these, pol and env were highly variable, suggesting that these two proteins' diversities probably resulted from adaptive evolutionary pressure. Genomic comparisons of the Chinese VMV strains CMV-1 and XM-MDV30 with those in other countries showed the highest genomic similarity levels (88.7%−89.1%) with the USA strain, with only 78.3%−88.7% identities with other VMV strains. Phylogenetic analyses using whole-genome sequences indicated that CMV-1 and XM-MDV30 were most closely related to USA strain 1150, suggesting that these VMV strains originated from the same ancestor. More circulating VMV strains sequenced in China will help precisely trace the origin.

Visna/Maedi virus spread in sheep and goats worldwide, with about 22 subtypes of VMV genotype A identified and molecularly characterized. A study demonstrated that VMV subtypes were distributed within regions or countries (31), suggesting that the high homologous genetic variation might present in specific regions or countries. Fortunately, only subgenotype A2 was found in the early epidemic period in China, suggesting that the Chinese circulating strain is probably less complicated than those of other countries. However, VMV has been ignored for a long time in China. Currently, limited genomic sequences do not assist in-depth studies that produce eradication programs or diagnostic tests, resulting in challenges in obtaining insights into circulating and genetic diversity in naturally VMV-infected sheep and goats.

Combined with our results showing subgenotype A2 circulated and high seroprevalence presence in China, the findings suggest that implementing control programs would be necessary to block the further spread of VMV. Although current national technical standards and World Organization for Animal Health specifications are implemented to detect and eliminate VMV, limited genomic data remains on the VMV epidemic in China. Therefore, our results would be beneficial for source-tracing and developing a diagnostic method for controlling VMV.

Next-generation sequencing is used to determine the genome sequences of various viruses worldwide (38, 39). Before our study, we failed to directly amplify the whole-genome sequences using several primers referred to as the reference genome sequence in GenBank before combining Illumina sequencing and whole-genome cloning methods, which might be attributed to a low level of similarity between the Chinese VMV strains and strains outside of China. This situation suggests that next-generation sequencing technologies help determine the sequences of highly variable viruses.

Illumina sequencing and whole-genome cloning confirmed that the two Chinese VMV strains (CMV-1 and XM-MDV30) belong to subgenotype A2 and share 78.3%−89.1% identities with other strains. The two Chinese VMV strains were highly homologous, and were genetically related to USA strain 1150. This study's conclusions apply to carrying out epidemiological investigations and implementing control strategies. A comprehensive molecular epidemiological investigation is warranted to reveal the molecular characterization and viral evolution in Chinese VMV isolates in the future.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/Supplementary Material.

J-YW and Y-RW conceived and designed the experiments and finalized the manuscript. J-YW, X-YM, X-YY, JW, X-XM, HB, and Y-RW performed the experiments and analyzed the data. J-YW drafted the manuscript. All authors read and approved the contents of the manuscript.

This work was funded by the National Key Research and Development Program of China (2016YFD0500908) and the Xinjiang Project for Construction of Veterinary Microbiological Resources and Sharing Platform (PT1809).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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