Abstract
Background:
Tamdy Virus (TAMV) is a pathogenic nairovirus widely distributed in central Asia and northwestern China. However, the host range of TAMV remains unclear, which limits our understanding the transmission cycle and cross-species patterns of this virus.
Materials and Methods:
A total of 160 serum samples were collected from livestock animals of camels, cattle, and sheep in Xinjiang, China between 2018 and 2021. An indirect immunofluorescence assay for TAMV were developed in this study, and have been employed to test TAMV-specific antibodies in these serum samples.
Results:
TAMV IgG antibody was detectable in camel sera collected from Urumqi in 2018 (6/17, 35%) and also from the Alertai Region in 2021 (1/8, 12.5%).
Conclusion:
The serological results in this study provide the first evidence that TAMV is able to infect camels and that the pathogen is circulating in different regions of Xinjiang. These findings highlight the need to further increase clinical and epidemiological surveillance of TAMV in humans and livestock in northwestern China.
Introduction
Tamdy virus (TAMV) is an emerging zoonotic, tick-borne virus taxonomically classified within the order Bunyavirales, family Nairoviridae, and genus Orthonairovirus. The TAMV genome consists of three negative-sense, single-stranded RNA segments: small (S), medium (M), and large (L) segments, encoding the structural nucleoprotein, glycoprotein precursor, and the viral RNA-dependent RNA polymerase (RdRp), respectively. TAMV was first isolated in 1971 from the hard-bodied tick Hyalomma asiaticum collected in the Tamdinsky district of Uzbekistan (Lvov et al., 1976), and subsequently isolated in several other countries in central Asia (Armenia, Azerbaijan, Kazakhstan, Kyrgyzstan, and Turkmenistan) during 1971–1983 (Lvov et al., 2015). Significantly, TAMV was detected in a patient with fever in Kyrgyzstan in 1973 (Lvov et al., 1984). Previous studies have revealed TAMV pathogenicity in both suckling and 3-week-old mouse models by intracerebral inoculation (Lvov et al., 1976). While most strains were identified from H. asiaticum ticks, and, also, in febrile human cases, it should be noted for future surveillance efforts that isolates were also recovered from both bird and bat species (Lvov et al., 2015).
The first TAMV strain XJ01 from China was isolated from H. asiaticum infesting Bactrian camels in Xinjiang in 2018 (Zhou et al., 2019). A recent study also revealed serological evidence of human exposure to TAMV in northwestern China (Moming et al., 2021). However, ascertaining the host range is crucial to understand the transmission cycle and cross-species patterns of tick-borne viruses. Herein, we report a sero-epidemiological investigation of TAMV in livestock in Xinjiang in northwestern China.
Materials and Methods
Sample collection
A total of 160 serum samples were collected from livestock animals in Xinjiang, China between 2018 and 2021. A total of 146 camel sera were collected from Urumqi City (n = 113), Jimunai County (n = 8), and Yining City (n = 25), respectively. Three cattle sera and 11 sheep sera were also collected from Yining City. All samples were stored at −80°C until further processing.
Cell lines and viruses
RD cell line was maintained in Minimum Essential Medium (MEM, HyClone Laboratories, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Mexico) and 1% penicillin/streptomycin (Solarbio, China). Vero cell line was maintained in Dulbecco’s Modified Eagle Medium (DMEM, HyClone Laboratories, USA) supplemented with 10% FBS and 1% penicillin/streptomycin. Both cell lines were cultured at 37°C, 5% CO2. TAMV XJ01 (GenBank accession no. MK757580-MK757582) and YEZV T-JL01 (GenBank accession no. ON563280-ON563282) were propagated in RD and Vero cells, respectively. Cells were subjected to three freeze-thaws followed by centrifugation at 10,000 × g for 10 min at 4°C, and supernatant was harvested with virus titer determined by TCID50. All viruses were stored at −80°C until use (Supplementary Data S1).
TAMV antibody-positive sera were prepared in six-week-old ICR female mice by subcutaneous injection at three different sites each with 50 μL of 40% formaldehyde-inactivated TAMV XJ01 (106.3 TCID50) in Freund’s complete adjuvant (Sigma, USA). After two weeks, the mice received booster injections of 150 μL 40% inactivated TAMV again in Freund’s incomplete adjuvant (Sigma, USA). Serum was collected 14 days post-booster immunization and stored at −20°C until use. YEZV antibody-positive sera were obtained using the same process.
Indirect immunofluorescence assay
RD cells infected with TAMV XJ01, or Vero cells infected with YEZV T-JL01 were fixed with 4% Paraformaldehyde Fix Solution (Sangon Biotech, China) for 10 min, and were then rinsed three times with phosphate-buffered saline (PBS) (Solarbio, China) 1-day post-inoculation. Cells were permeabilized in PBS containing 0.1% Triton X-100 (Sigma, USA) for 10 min, washed with PBS, and blocked with 5% BSA (Solarbio, China) for 1 h at 37°C. Sera were diluted 1:100 with 1% BSA in PBS, incubated overnight at 4°C with the above cells and washed with PBS. The primary antibodies used were mouse polyclonal anti-TAMV, and mouse polyclonal anti-YEZV. The cells were then stained with diluted fluorescence-conjugated secondary antibodies for 1 h at room temperature. The fluorescence-generating reagent was IgG conjugated with fluorescein isothiocyanate (IgG-FITC) (Invitrogen, USA; Solarbio, China). The nuclei were stained blue with DAPI (4′,6-diamidino-2-phenylindole) (Solarbio, China) for 10 min at room temperature. Representative images were obtained using ZEISS Axio Observer 7 microscope. Real-time reverse transcription PCR (qRT-PCR)-based virus neutralization assay (Supplementary Data S1).
Virus neutralization assay
RD cells were seeded in 96-well plates (2 × 104 cells/well) and incubated at 37°C with 5% CO2. 12 hours later, heat-inactivated camel sera were 2-fold serially diluted (1:8 to 1:1,024) in 2% MEM, and 50 μL of the diluted serum samples were incubated with 50 μL of the 200 TCID50 TAMV XJ01 strain for 1 h at 37°C. The mixtures were then added to RD cells and incubated at 37°C for 8 days. The infected cells were frozen and thawed three times, and centrifuged to obtain the virus supernatant. RNA was extracted using MagaBio plus Virus DNA/RNA Purification Kit (BioFlux, China). Complementary DNA (cDNA) synthesis was performed using Evo M-MLV RT kit (Accurate Biotechnology, Hunan, China). Viral RNA was quantified by TaqMan based qRT-PCR using Pro Taq HS Premix Probe qPCR Kit (Accurate Biotechnology, Hunan, China). The primer and probe sequences for TAMV L gene were as follows: forward primer, 5′-TTCCTCTCAAGGGACAACAA-3′; reverse primer, 5′-TCCTTAGTTAGCACATCAGTTCT-3′; probe, 5′-FAM-TAACTGAAGTYGAGCTACATGTRTTGC-BHQ1-3′. Real-time PCRs were carried out on the QuantStudio 1 Real-Time PCR System (Applied Biosystems, Carlsbad, CA) according to the following conditions: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 56°C for 60 s (Supplementary Data S1).
Results
A total of 160 sera from captive Bactrian camels (n = 146), domesticated cattle (n = 3), and sheep (n = 11) were collected in Xinjiang, China between 2018 and 2021 (Fig. 1A and 1B). We developed an indirect immunofluorescence assay for TAMV to test these serum samples. Sera from immunized mice were tested with TAMV-specific indirect immunofluorescence (IFA), and yellow-green fluorescence was detected in the cytoplasm with no discernible signal observed in the nucleus and mock-infected mice (Fig. 1C). This indicated that the inoculated murine sera contained TAMV-specific antibodies and that we had established an assay suitable for TAMV antibody screening. Next, 17 sera collected from captive Bactrian camels from Urumqi, Xinjiang in the summer of 2018 were tested, six of which (samples 2018001, 2018004, 2018007, 2018009, 2018013, and 2018015) tested TAMV IgG positive with yellow–green fluorescence observed (35.3% IgG seropositivity) (Fig. 1C). Additional camel sera (n = 96) collected from Urumqi in the winter of 2020, however, were all negative for TAMV IgG. Of the 33 camel sera collected in the summer of 2021, one (12.5%; sample 2021003) of the eight samples collected from Jimunai County, Alertai Region tested IgG positive, while 25 samples collected from Yining at the same time were negative (Fig. 1C). Moreover, sera from cattle and sheep also collected from Yining were all negative for TAMV IgG. Although the in-house anti-TAMV IgG could also recognize YEZV with a cross-reactivity (Supplementary Fig. S1), the seven IFA-positive sera were evidently positive against TAMV confirmed by virus neutralization assay (VNA, Supplementary Fig. S2). The viral load of these samples at a dilution of 1:8 incubated with TAMV was 107.0–107.8 copies/μL, which were lower than that of cells incubated TAMV only (108.4 copies/μL). Therefore, TAMV IgG antibodies were intermittently detected in camel sera collected from different regions of Xinjiang and from different years.
Bactrian camel seropositivity for Tamdy virus in Xinjiang, China.
Discussions
Tick vectors are medically important as they can transmit numerous pathogens to humans and, also, to domesticated and wild animals (and birds) maintaining zoonotic cycles of infection with ongoing transovarial transmission during overwintering in arthropods (Chmelar et al., 2016). Notably, in recent years, the incidence of tick-borne diseases has been increasing worldwide and poses a serious threat to global public health which may be expected to be exacerbated by climate change (Zhou et al., 2023). Several members of the genus Orthonairovirus are known high-consequence human pathogens, which have been shown to cause fatal febrile illnesses in humans and other animals (Kodama et al., 2021). Therefore, the epidemiological surveillance of arboviruses within this genus is of vital importance.
In this study, we have detected TAMV IgG antibody in camel sera collected from Urumqi in 2018 (6/17, 35%) and also from the Alertai Region in 2021 (1/8, 12.5%). Antibody-positive sera against TAMV were further found to have neutralizing activity to TAMV by VNA. Because of potential cross-reactivity of orthonairovirus N antigens (Ward et al., 1992), we further detected lab-prepared polyclonal antibody anti-TAMV IgG with Yezo virus, a novel nairovirus associated with febrile illness previously identified in Japan and China (Kodama et al., 2021; Lv et al., 2023). IFA results showed that the anti-TAMV positive serum could efficiently recognize TAMV antigen, while its recognition of YEZV antigen was reduced. Thus, a neutralization test using TAMV should be performed in the future to exclude the possibility of infections of other nairoviruses antigenically cross-reactive to TAMV.
To our knowledge, this is the first report that TAMV antibody was detected from camel. Moreover, we provide evidence that Bactrian camels are likely mammalian hosts of H. asiaticum potentially amplifying TAMV. Owing to the limited number of serum samples collected from cattle and sheep, a large-scale investigation of various animal sera from Xinjiang is necessary to verify whether the animals have prior exposure to TAMV. Therefore, our findings support larger serological and molecular surveys to increase surveillance of TAMV in both domesticated and wild animals to identify potential reservoirs, and, also, investigations on febrile human cases of unknown etiology in Xinjiang and northwestern China, where H. asiaticum ticks are endemic.
Footnotes
Acknowledgments
The authors would like to acknowledge the support and care of the veterinarians and farmers involved in this study. Kind thanks also to Professor Zedong Wang (Jilin University, China) for providing us the YEZV strain T-JL01.
Authors’ Contributions
M.C.: Methodology, Writing—original draft. Y.B.: Writing—review and editing. M.G.: Resources. M.J.C.: Writing—review & editing. W.S.: Formal analysis, Writing—review and editing, Funding acquisition. Z.M.: Resources, Writing—review and editing. H.Z.: Formal analysis, Writing—review and editing, Funding acquisition.
Author Disclosure Statement
No conflicting financial interests exist.
Funding Information
This study was supported by grants from the
Supplementary Material
Supplementary Data S1
Supplementary Figure S1
Supplementary Figure S2
References
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
