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Identification of a new orthonairovirus associated with human febrile illness in China

Abstract

The genus Orthonairovirus, which is part of the family Nairoviridae, includes the important tick-transmitted pathogens Crimean–Congo hemorrhagic fever virus and Nairobi sheep disease virus, as well as many other poorly characterized viruses found in ticks, birds and mammals1,2. In this study, we identified a new orthonairovirus, Songling virus (SGLV), from patients who reported being bitten by ticks in Heilongjiang Province in northeastern China. SGLV shared similar genomic and morphological features with orthonairoviruses and phylogenetically formed a unique clade in Tamdy orthonairovirus of the Nairoviridae family. The isolated SGLV induced cytopathic effects in human hepatoma cells in vitro. SGLV infection was confirmed in 42 hospitalized patients analyzed between 2017 and 2018, with the main clinical manifestations being headache, fever, depression, fatigue and dizziness. More than two-thirds (69%) of patients generated virus-specific antibody responses in the acute phase. Taken together, these results suggest that this newly discovered orthonairovirus is associated with human febrile illness in China.

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Fig. 1: Discovery and characterization of SGLV.
Fig. 2: Timeline of patients with SGLV infection and serological investigation.
Fig. 3: Phylogenetic analysis of SGLV from humans and ticks.

Data availability

The authors declare that all viral sequences used in the analyses of this study are available within the article and its Supplementary Information. SLGV sequences have been deposited in the Genbank Nucleotide database under accession codes MT328775 (M segment), MT328776 (L segment) and MT328777 (S segment). To protect patient privacy and confidentiality, clinical data are not made publicly available in the Supplementary Material of the article but will be made available upon reasonable request to the corresponding authors. Source data are provided with this paper.

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Acknowledgements

We thank J.-F. Wang and S.-D. Wang at Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, for electron microscopic analysis of virus. This work was supported by the National Key R&D Program of China (project no. 2017YFD0501700, to Q.L.), the National Natural Science Foundation of China (project nos. 31672542 and 31372430, to Q.L.), the Pearl River Talent Plan in Guangdong Province of China (project no. 2019CX01N111, to Q.L.) and the ‘Three Major’ scientific research projects of Sun Yat-sen University in 2020 (project no. 50000-31143406, to J.Q.).

Author information

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Authors

Contributions

Conceptualization was provided by Q.L., J.Q. and W.W. The methodology was developed by J.M., Z.-D.W., L.L., J.-W.S. and L.-N.L. Investigations were carried out by J.M., X.Z., Z.-D.W., L.L., J.-W.S., C.C., Y.-H.Z., L.S. and L.-N.L. The original draft of the manuscript was written by J.M. and Z.-D.W. Review and editing of the manuscript were carried out by Q.L., J.Q. and W.W. Funding acquisition was performed by Q.L. and J.Q. Resources were provided by X.-L.L., S.-Z.H., W.W., H.-T.S., L.-X.M. and Z.-L.C. Q.L. provided supervision.

Corresponding authors

Correspondence to Jun Qian or Wei Wang or Quan Liu.

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The authors declare no competing interests.

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Peer review information Alison Farrell is the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Strategy of sequencing the genome of SGLV.

The genome of SGLV includes the large (L), medium (M), and small (S) segments, which are indicated by dot lines. Sequences obtained by metagenomic analysis are shown by gray boxes and marked with the mapped positions in the genome of Wēnzhōu tick virus. The complete genome was obtained by overlap RT-PCR (blue line) and RACE (red line), and the partial sequences for phylogenetic analysis were obtained by nested RT-PCR (orange line). The corresponding primers are listed in Supplementary Tables 13 and 14.

Extended Data Fig. 2 Genomic characteristics of SGLV and other orthonairoviruses.

a, Genomic illustration of SGLV and other orthonairoviruses. The predicted ORFs are annotated with different colors: large protein encoded by L segment is shaded with yellow, glycoprotein precursors (GPC) encoded by M segment is indicated with light blue, and nucleoproteins encoded by S segment is presented with gray. b, The terminal sequences of SGLV and other orthonairoviruses. The typical terminal sequences of orthonairoviruses are marked by red boxes and the putative terminal sequences are marked with yellow boxes. The sequences that appeared in the terminals of different segments or have a reverse-complementary sequence at the other terminal in the same segment are regarded as putative terminal sequences. L, Large segment; M, Medium segment; S, Small segment; CCHFV, Crimean-Congo hemorrhagic fever virus (L, HM452307; M, HM452306; S, HM452305); HUGHV, Hughes virus (L, KP792738; M, KP792739; S, KP792740); ERVEV, Erve virus (L, JF911697; M, JF911698; S, JF911699); TDYV, Tamdy virus (L, MK757580; M, MK757581; S, MK757582); TTV1, Tǎchéng tick virus 1 (L, MK554672; M, MK554676; S, MK554680); HTV1, Huángpǐ tick virus 1 (L, NC_031135; M, NC_031136; S, NC_031137); WTV, Wēnzhōu tick virus (L, KM817685; M, KM817718; S, KM817745); SGLV, Sōnglǐng virus (L, MT328776; M, MT328775; S, MT328777).

Extended Data Fig. 3 Alignment of the conserved domains in large protein of SGLV.

a, Amino acid sequence alignments of RNA-directed RNA polymerase motifs in orthonairoviruses. Motifs from pre-A to E are marked with black boxes. b, Alignment of the N-terminal of large protein. Ovarian tumor domain (OTU)-like cysteine proteases motif of CCHFV is underlined with gray line, and the N-terminal toposisomerase motif of CCHFV is marked in black box. c, Alignment of the zinc finger motif. The zinc finger motif of CCHFV is marked with black box. d, Alignment of leucine zipper motif. The leucine zipper motif of CCHFV is marked with black box. CCHFV, Crimean-Congo hemorrhagic fever virus; HUGHV, Hughes virus; ERVEV, Erve virus; TDYV, Tamdy virus; TTV1, Tǎchéng tick virus 1; HTV1, Huángpǐ tick virus 1; WTV, Wēnzhōu tick virus.

Extended Data Fig. 4 Alignment of the conserved C-terminal domain present in nucleoprotein of SGLV.

Residues implicated in nucleoprotein function are highlighted in black boxes. The sequences were visualized by ESPript 3.0 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). Conserved residues are marked in black boxes. The amino acid positions within the N proteins are displayed for each virus. CCHFV, Crimean-Congo hemorrhagic fever virus; HUGHV, Hughes virus; ERVEV, Erve virus; TDYV, Tamdy virus; TTV1, Tǎchéng tick virus 1; HTV1, Huángpǐ tick virus 1; WTV, Wēnzhōu tick virus.

Extended Data Fig. 5 Phylogenetic analysis of SGLV.

Phylogenetic tree of SGLV and related nairoviruses were constructed based on the aa sequences of RNA-directed RNA polymerase (a, aa 2182–2446), glycoprotein precursor (b, aa 258–617) and nucleoprotein (c, aa 59–296) with maximum likelihood method. Phylogenetic analysis was performed with Artashat, Chim, Crimean-Congo hemorrhagic fever (CCHF), Dera Ghazi Khan (DGK), Dugbe, Estero Real (ER), Hazara, Hughes, Kasokero, Keterah, Nairobi sheep disease (NSD), Qalyub, Sakhalin, Tamdy, and Thiafora orthonairoviruses. SGLV isolated from patients in this study are marked with a black dot. Detailed information of nairoviruses are shown in Supplementary Tables 4 and 11.

Extended Data Fig. 6 Similarity plot of the complete genome of SGLV.

Similarity plot of the large (a), medium (b) and small (c) sequence of SGLV strain HLJ1202 (L, MT328776; M, MT328775; S, MT328777) is shown. Full-length genome sequences of TDYV (Tamdy virus, L, MK757580; M, MK757581; S, MK757582); TTV1 (Tǎchéng tick virus 1, L, MK554672; M, MK554676; S, MK554680); HTV1 (Huángpǐ tick virus 1, L, NC_031135; M, NC_031136; S, NC_031137); WTV (Wēnzhōu tick virus, L, KM817685; M, KM817718; S, KM817745) were used as reference sequences.

Extended Data Fig. 7 Development of real-time RT-PCR for SGLV detection.

a, Amplification plot of plasmids containing the SGLV nucleoprotein gene as template for real-time RT-PCR, which was serially diluted from 108 to 101 copies was used as template. b, The standard curve for the real-time RT-PCR. The used primers and reaction condition are shown in the Methods.

Extended Data Fig. 8 The geographic distribution of patients with SGLV in northeastern China.

Areas where surveillance of tick-borne virus was presented with gray. Black dots indicate the locations of the patients with laboratory-confirmed SGLV infection in the study.

Extended Data Fig. 9 SGLV infection in different cells.

SGLV could grow in SMMC-7721, BHK-21, and Vero cells. Images are shown 4 days after infection and the uninfected cells were used as control. SGLV infection in different cells were tested by indirect immunofluorescence assay (IFA). Different cell lines were inoculated onto 24-well plates containing cell slides. After incubation for 12 h, the cells were infected with SGLV in triplicate at a MOI of 5. The infected cell slides at 12 h post-infection were collected for IFA; anti-SGLV sheep sera or human convalescent sera were used as the first antibody. Infected cells appeared green, and nuclear appeared blue due to DAPI staining. Images are representative of at least two independent experiments.

Extended Data Fig. 10 Recombinant nucleoprotein of SGLV used in ELISA assay.

a, Sodium dodecyl sulphate-polyacrylamide gel electrophoresis showing purity of his-tagged nucleoprotein of SGLV (lane 2) and albumin from bovine serum (lane 1). b, Western-blot analysis of recombinant nucleoprotein using anti-His monoclonal antibody (lane 2). Lane M is protein molecular weight marker. Biochemistry experiments are representative of at least two independent experiments. Source data

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Supplementary Information

Supplementary Tables 1–14.

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Source Data Extended Data Fig. 10

Unprocessed western blots and/or gels.

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Ma, J., Lv, XL., Zhang, X. et al. Identification of a new orthonairovirus associated with human febrile illness in China. Nat Med 27, 434–439 (2021). https://doi.org/10.1038/s41591-020-01228-y

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