Heparin sulfate is the attachment factor of duck Tembus virus on both BHK21 and DEF cells
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RESEARCH
Open Access
Heparin sulfate is the attachment factor of duck Tembus virus on both BHK21 and DEF cells Shaoxiong Wu1, Zhen Wu1, Yuanyuan Wu1, Tao Wang1, Mingshu Wang1,2,3, Renyong Jia1,2,3, Dekang Zhu1,2,3, Mafeng Liu1,2,3, Xinxin Zhao1,2,3, Qiao Yang1,2,3, Ying Wu1,2,3, Shaqiu Zhang1,2,3, Yunya Liu1, Ling Zhang1, Yanling Yu1, Leichang Pan1, Shun Chen1,2,3* and Anchun Cheng1,2,3*
Abstract Background: Duck tembusu virus (DTMUV, genus Flaviviruses, family Flaviviridae) is an emerging flavivirus that can infect a wide range of cells and cell lines in vitro, though the initial step of virus invasion remains obscure. Methods: In this study, drug treatments that including heparin, chondroitin sulfate, heparinase I, chondroitinase ABC and trypsin were applied to detect the influence of DTMUV absorption, subsequently, the copy number of viral genome RNA was analyzed by quantitative real-time PCR. The inhibition process of viral absorption or entry by heparin was determined by western blotting, and the cytotoxicity of drug treated cells was detected by cell counting kit-8. Results: We found that the desulfation of glycosaminoglycans (GAGs) with sodium chlorate had a significant effect on the adsorption of DTMUV in both BHK21 and DEF cells. Based on this result, we incubated cells with a mixture of DTMUV and GAGs competition inhibitors or pre-treated cells with inhibitors, after incubation with the virus, the NS5 expression of DTMUV and viral titers were detected. The data suggested that heparin can significantly inhibit the absorption of DTMUV in a dose dependent manner but not at the step of viral entry in BHK21 and DEF cells. Meanwhile, heparinase I can significantly inhibit DTMUV attachment step. Conclusions: Our results clearly proved that heparin sulfate plays an important role in the first step of DTMUV entry, viral attachment, in both BHK21 and DEF cells, which sheds light on the entry mechanism of DTMUV. Keywords: Glycosaminoglycans, Heparin sulfate, Chondroitin sulfate, Attachment factor, Entry
Background DTMUV is an arthropod-borne flavivirus responsible for the severe decline in egg production in ducks. Since 2010, a newly emerging disease characterized by egg-drop syndrome has occurred in ducks in China. Egg production in sick ducks was seriously decreased within 2 weeks after disease onset. The infected ducks mainly showed clinical symptoms such as depression, loss of appetite, growth retardation, even paralysis or death. The disease has caused serious economic losses to the duck industry. Additionally, DTMUV can * Correspondence: [email protected]; [email protected] 1 Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang District, Chengdu City 611130, Sichuan Province, China Full list of author information is available at the end of the article
exhibit pathogenicity to Kunming mice by intracerebral inoculation [1–5]. DTMUV is similar to other flaviviruses, consisting of an 11-kb positive-sense single-stranded RNA genome composed of on
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