Emergence of human infection with Jingmen tick virus in China: A retrospective study

Abstract

Background: A tick-borne segmented RNA virus called Jingmen tick virus (JMTV) was recently identified, variants of which were detected in a non-human primate host and fatal patients with Crimean-Congo haemorrhagic fever. We investigated its infectivity and pathogenicity for humans.

Methods: We obtained skin-biopsy, blood and serum samples from patients with tick bites, and used high-throughput sequencing, in situ hybridisation, and serologic testing to diagnose and ascertain the cases of JMTV infection.

Findings: A JMTV strain was isolated from the tick Amblyomma javanense into an embryo-derived tick cell line. We obtained sustained passage of JMTV, and revealed that it was able to accumulate in salivary glands of experimentally infected ticks. Four JMTV-infected patients were identified by high-throughput sequencing of skin biopsies and blood samples. The virus replication in skin tissue was visualised by in situ hybridisation. The four patients all had an itchy or painful eschar at the site of tick bite, with or without lymphadenopathy. Immunohistochemical examination revealed remarkable local inflammation manifested as infiltration by neutrophils. Eight patients were identified by serological testing and showed more severe clinical manifestations. Two Ixodes persulcatus ticks detached from patients were positive for JMTV. All JMTV strains identified in this study formed a well-supported sub-lineage, distinct from those previously reported in China. Interpretation The public significance of JMTV should be highly concerning due to its potential pathogenicity for humans and efficient transmission by potential ticks. FUND: China Natural Science Foundation, State Key Research Development Programme, and United Kingdom Biotechnology and Biological Sciences Research Council.

Keywords: Human infection; Jingmen tick virus; Pathogenicity; Ticks.

Fig. 1

Biological features of JMTV in BME/CTVM23 cells. Growth curves of each of the four genome segments of JMTV in BME/CTVM23 cells. The copy numbers of each viral segment were calculated by a standard curve method using a linearised plasmid containing the corresponding segment (Panel A). Detection of positive strand and negative strand of segment 1 by strand-specific quantitative PCR with a tagged primer system during growth in BME/CTVM23 cells (Panel B). The dose-response curve for JMTV. The count of fluorescent-positive cell for representative segment 2 probe sets per 10⁴ cells is shown on the y axis, and the relative viral dilution is shown on the x axis (Panel C). Detection of each genome segment of JMTV in infected BME/CTVM23 cells by fluorescence in-situ hybridisation (FISH). Cells were fixed at 72 h (Panel D) and 144 h (Panel E) post infection respectively. Magnification×200. Detection segment 2 probe sets-JMTV FISH in salivary glands and midguts of male A. javanense experimentally-infected at 7 and 14 days post infection. Nuclei, DAPI (blue), Segment 2 probe, Quasar 570 (red). Magnification×400 (Panel F). Three biological replicates were run for experiments of Panel A to E. Five biological replicates were run for experiment of Panel F. Three technical replicates were run for all experiments. Estimates from the three technical replicates were averaged and error bars indicated standard error across biological replicates. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

In situ Hybridisation and Immunohistochemical Analysis of Skin-biopsies with JMTV Infection. In situ RNA hybridisation using JMTV-segment 2 probe sets (Quasar 570) on formalin-fixed, paraffin-embedded (FFPE) skin eschar of Patient 1. Nuclei were counterstained with DAPI (blue). Magnification×200, insert×400 (Panel A). Haematoxylin and eosin staining, and immunophenotyping of lymphocytes (CD3), neutrophils (CD15) and macrophages (CD68) on serial sections of Patients 1–3. Magnification×200, counterstained with haematoxylin, peroxidase immunostaining in brown (Panel B). No FFPE specimen was available for Patient 4. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Phylogenetic Analysis of JMTVs. Bayesian phylogeny of gene segment 1 (Panel A), segment 2 (Panel B), segment 3 (Panel C) and segment 4 (Panel D). The tree is midpoint rooted. Green Dot for isolate strain; Red triangle for human skin samples; Blue triangle for human blood samples; Yellow triangle for ticks detached from patients. The samples relating to four patients are indicated as follows: Case 1: S17; Case2: S24, A276; Case3: S28, A450, T118; Case4: S16, T59. Bayesian posterior probabilities (BPP) and Maximum likelihood (ML) bootstrap values are displayed at major nodes (BPP/ML). Bootstrap testing (1000 replicates) was performed, and the bootstrap values are indicated. Branches are drawn to the scale of substitutions per site. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)