Characteristics of viral ovarian tumor domain protease from two emerging orthonairoviruses and identification of Yezo virus human infections in northeastern China as early as 2012

Abstract

Emerging tick-borne orthonairovirus infections pose a growing global concern, with limited understanding of the viral ovarian tumor-like cysteine proteases (vOTUs) encoded by novel orthonairoviruses. These vOTUs, a group of deubiquinylases (DUBs), disrupt the innate immune response. Yezo virus (YEZV), a recently discovered pathogenic orthonairovirus, was first reported in Japan in 2021. In this study, we successfully isolated and identified YEZV and a new orthonairovirus, Jiànchuān tick virus (JCTV), from Ixodes persulcatus and Haemaphysalis montgomeryi ticks, respectively, in China. We found that the vOTU domains encoded by YEZV and JCTV exhibited both DUB and deISGylase activities, though with potentially less broad deISGylation compared to that of Crimean-Congo hemorrhagic fever virus (CCHFV) during natural infection. Phylogenetic analysis of global vOTUs, including 83 new sequences, revealed a high diversity of this domain. Interestingly, retrospective screening of tick-bite patients from 2012 to 2016 in northeastern China traced YEZV infections as far back as 2012, identifying four cases. Additionally, YEZV primarily infected I. persulcatus (31.4%) and Dermacentor nuttalli (10.5%) in northern China, while JCTV exhibited high infection rates in H. montgomeryi (81.3%) in southern China. In summary, our work emphasizes the active surveillance of orthonairovirus infections and the imperative need for the development of vOTU domain-targeted anti-virals, offering potential therapeutic solutions for a broad spectrum of orthonairoviruses.IMPORTANCEThe vOTUs, a group of DUBs, mimic the functions of host DUBs to enhance viral infectivity and may serve as potential drug targets. vOTUs from different orthonairoviruses exhibit distinct preferences toward ubiquitin (Ub) and ubiquitin-like protein interferon stimulated gene 15 (ISG15). In this study, we investigated the deubiquitinase and deISGylase functions of various orthonairoviral vOTUs using both an overexpression system and natural viral infections in vitro. Our findings illustrate that the vOTUs from YEZV and JCTV can cleave both Ub and ISG15 in an overexpression system, but these viruses exhibit potentially narrower deISGylation capacity than CCHFV during natural infection. This suggests that the diversity of vOTUs may have a potential relationship with the pathogenesis.

Keywords: orthonairovirus; ovarian tumor-like cysteine protease; retrospective surveillance; tick-bite patients.

Fig 1

Characteristics of YEZV and JCTV detected in this study. (A–C). The phylogenetic trees of YEZV and JCTV based on L (A), M (B), and S (C) segments. The trees were constructed using the maximum-likelihood method with 1,000 bootstraps. The scale bar shows estimated evolutionary distance. (D and E) Fluorescence in situ hybridization (D) and indirect immunofluorescence assay (E) were used to detect YEZV and JCTV in BHK-21 cells, respectively. (F). Transmission electron microscopy showed YEZV and JCTV (arrows) in the cytoplasm of infected BHK-21 cells. Insets show negatively stained virions.

Fig 2

YEZV and JCTV infections in various cells. Light microscopy shows virus-induced cellular changes (cytopathic effect) in YEZV-infected HepG2 cells (A) and JCTV-infected L929 cells (B). Growth curves of YEZV (C) and JCTV (D) in HUVEC, BHK-21, Vero 81, Huh7, and L929 cells over 120 h, respectively. Titers of progeny viruses in the supernatants were measured by TCID₅₀ assay. Error bars represent the standard deviation of the mean.

Fig 3

vOTU activity in transfected cells. (A) DUB activity of the vOTU of CCHFV, YEZV, and JCTV, respectively. 293T cells were co-transfected with plasmids expressing vOTUs and Ub. (B) DeISGylase activity of the vOTU of CCHFV, YEZV, and JCTV, respectively. 293T cells were co-transfected with plasmids expressing vOTUs, ISG15, Ube1L, UbcH8, and HERC5. Cell lysates were harvested 2 days post-transfection, and proteins were analyzed for Ub or ISG15-conjugates.

Fig 4

General levels of ubiquitinated/ISGylated proteins in Huh7 cells infected with YEZV and JCTV, respectively. Huh7 cells were infected with YEZV (1 × 10⁸ copies/μL) or JCTV (1 × 10⁷ copies/μL) viruses. (A) Western blot of ubiquitinated proteins in infected cells. Cell lysates were harvested 48 h post-infection, separated by SDS-PAGE, and probed for total Ub or K48- and K63-linked poly-Ub chains. (B) Western blot of ISGylated proteins in infected cells. Interferon-β (1.5 µg/mL) was added to the Huh7 cell culture media 4 h post-infection. Conjugation of ISG15 was visualized.

Fig 5

Phylogeographic analysis of diverse vOTU domains of orthonairoviruses. (A) The maximum-likelihood phylogenetic tree was built with vOTU domains from 93 global public sequences and 83 new ones from our work. vOTU domains were annotated in the Conserved Domains Database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). A total of 1,000 bootstraps were repeated to construct the tree. The scale bar shows estimated evolutionary distance. The orthonairoviruses of newfound sequences were indicated with red squares. The orthonairoviruses of public sequences that can infect humans are indicated with orange circles, and others are indicated with blue circles, respectively. Current species groupings are indicated by different colors, and the representative species are denoted. (B) The sequences of orthonairovirus vOTUs were visualized by ESPript 3.0. The catalytic triad is boxed in black. CCHFV, Crimean-Congo hemorrhagic fever virus; HNTV, Hénán tick virus; HPTV-1, Huángpí tick virus 1; JCTV, Jiànchuān tick virus; NSDV, Nairobi sheep disease virus; SGLV, Sōnglǐng virus; SXTV-2, Shǎnxī tick virus 2; WZTV, Wēnzhōu tick virus; YBTV-1, Yánbiān tick virus 1; YEZV, Yezo virus; YSTV-1, Yùshù nairo tick virus 1 .