The family Orthomyxoviridae
consists of seven genera: Influenza A-D viruses, Isavirus, Quaranfil virus and the tick-transmitted thogotoviruses (1
). Thogotoviruses harbor a six-segmented negative-sense RNA genome (∼10 kb) encoding genes for the RNA-dependent polymerase, consisting of the polymerase basic protein 2 (PB2), PB1 and the polymerase acidic protein (PA), for a nucleoprotein (NP), a matrix protein (M) and a glycoprotein (GP) involved in virus attachment and fusion (3
). In 1960, the first thogotovirus was isolated from ticks collected from cattle in the eponymous Thogoto forest near Nairobi, Kenya (5
), and since then, 15 genetically distinct thogotoviruses have been described. Some of these isolates were initially not recognized as thogotoviruses and classified to this genus many years later as exemplified by Batken virus (BKNV) (6
) and Jos virus (JOSV) (7
). Based on the available genomic sequence information, multiple groups have shown that these pathogens can be subdivided into two clusters (7–9
), which we will here refer to as Thogoto-like (THOV-like) and Dhori-like (DHOV-like) thogotoviruses.
The majority of these pathogens have been isolated from ticks but serological surveys detected antibodies against thogotoviruses in various mammalian species, including cattle, sheep, camel, deer, opossum and rat (10–14
). In sheep it was reported that thogotovirus infections can cause a febrile illness and abortions (15
). However, their pathogenesis is best understood for laboratory mice infected with the prototypic isolates SiAr126 (THOV-like) and India/61 (DHOV-like). Upon infection these pathogens cause a severe, fatal disease with pathological lesions present in liver and lung leading to macroscopically detectable liver necrosis (16–18
). Besides their replication in hepatocytes (17
), it became apparent that thogotoviruses target myeloid dendritic cells and myeloid cells isolated from the peritoneal cavity (19
). Elevated inflammatory cytokines and aminotransferase levels are detectable in the blood and mice exhibit severe leukopenia, lymphopenia and thrombocytopenia (21
). Infections of humans are rare or only sporadically recognized as no pan-specific diagnostic tools are available. Historic reports described two cases in Nigeria (22
) and five accidental laboratory infections in the former USSR (23
). More recently, a novel thogotovirus, Bourbon virus (BRBV), was isolated from the blood of a fatal human case in Bourbon County (KS, USA) (24
) and 3 years later, in 2017, the same virus caused the death of a patient from the neighboring state Missouri (25
). Strong evidence supports the hypothesis that BRBV is a zoonosis likely transmitted by Amblyomma americanum
Of the 15 presently known thogotoviruses, only a few have been studied in more detail. Thus, basic questions about their phylogenetic relationships, morphology, transmission, and capacity to replicate and induce disease in mammals remain unanswered. In the present study, we addressed these questions by comparatively characterizing a panel of 10 distinct thogotovirus isolates and subsequently demonstrating the high genetic and phenotypic variability within the genus of tick-transmitted thogotoviruses.
(This work was conducted by Jonas Fuchs in partial fulfilment of the requirements for a Ph.D. degree  from the Faculty of Biology of the University of Freiburg, Germany.)
Similar to the influenzaviruses, thogotoviruses are enveloped and have a segmented single-stranded RNA genome of negative polarity (3
). However, they form a distinct genus within the family of Orthomyxoviridae
because they have only six segments and are transmitted by ticks.
Our comparative phylogenetic analysis of 10 thogotovirus isolates from different geographic areas revealed their classification into two clades, the THOV-like and the DHOV-like viruses confirming similar analyses in recent publications (7–9
). The most remarkable molecular difference between these two clades is the coding strategy of segment 6 encoding the matrix protein (M). In THOV-like virus-infected cells, M is expressed from a spliced transcript of segment 6 and the unspliced transcript encodes the matrix protein long (ML), a viral IFN antagonist (7
). However, the DHOV-like viruses encode the M from the unspliced transcript of segment 6 and the synthesis of a homologous IFN antagonist is not known (38
The phylogenetic relationships are very similar for all six segments with JOSV as outlier of the THOV-like group and OzV and BRBV of the DHOV-like clade (Fig. 1
). For most segments the two European THOV-like isolates SiAr126 and PoTi503 cluster together and are slightly distinct from the African Ken-IIA and Japanese Kami-25 as previously published (39
). Accordingly, in the DHOV-like clade the European isolate PoTi461 is closely related to the Asian India/61 and BKNV isolates.
Thogotoviruses have the potential to reassort their genome segments (40
). However, the similarity of the phylogenetic trees for all genomic segments suggests that reassortment did not occur between our analyzed isolates and is, if at all, a rare event.
We confirmed the classification of the thogotoviruses into two clades by their antigenic relationship and by the cross-reactivity of virus specific antibodies. In Western blot analyses antisera of postinfectious mice recognized the viral NP of isolates from the same but not from the other clade (Fig. 6
and Fig. S2). The lack of cross-reactivity correlates with the distant relationship of THOV-like and DHOV-like viruses on the sequence level. Accordingly, the amino acid sequences of the GP, NP and M proteins of the prototypic THOV-like SiAr126 compared to DHOV-like India/61 show only 32%, 41% and 22% sequence identities, respectively (Fig. S1) (30
). For the group of the three classical THOVs, Ken-IIA, PoTi503 and SiAr126, which were isolated at distant locations, their close antigenic similarity was described before (44
). Likewise, antisera directed against the classical PoTi461, India/61 and BKNV isolates in the DHOV-like clade were cross-reacting only in the DHOV-clade, confirming the absence of antibody cross-reactivity between the two clades (45
). However, there were some exceptions: sera from JOSV and BRBV infected animals recognized solely their respective viral antigens as reported previously (7
The morphology of the virus particles also supported the separation into the two clades. Kosoy et al. described spherical and filamentous appearances for the Bourbon virions (24
). We confirmed these two phenotypes by electron microscopy (Fig. 2
). A quantitative analysis of the EM images revealed a higher rate and an enhanced elongated phenotype of the filamentous virions for the DHOV-like isolates compared to the members of the THOV-like clade. It is known from filamentous influenzaviruses that they lose their elongated phenotype during passages in cells culture (47–49
). However, in the case of thogotoviruses that were partially passaged for years the passage history seems to not gravely affect the morphology of the virions, as the phenotypic differences between the multiple passaged prototypic SiAr126 and India/61 isolates were comparable with the differences between the rarely passaged JOSV and BRBV.
Thogotoviruses were mostly isolated from ticks, but the detection of virus-specific antibodies implicates their replication in diverse mammalian hosts, including humans (10
). Among them, rodents could represent a reservoir species in their enzootic cycle as discussed for other arboviruses. We therefore compared the organ tropism and virulence of the 10 isolates in mice. With the exception of OzV and BRBV, all thogotoviruses showed an early tropism for the spleen (Fig. 3
). Accordingly, previous immunohistochemistry and in situ
hybridization revealed viral proteins and RNA in spleen macrophages, alveolar macrophages as well as myeloid cells and hepatocytes in the liver (17–19
). One of the major differences between both clades was that THOV-like viruses additionally showed an early replication in the liver whereas DHOV-like viruses showed an early replication to high titers in the lung. The lack of any detectable replication of BRBV is in line with recent studies showing a remarkable strong interferon sensitivity of BRBV both in cell culture and in vivo
). It is tempting to speculate that also OzV is a highly IFN sensitive virus not able to replicate in hosts with a functional IFN system (8
). For THOV-SiAr126 it is assumed that cells of the hematopoietic compartment, namely, CD11b+ cells with a myeloid/macrophage phenotype, are the primary target of the virus upon intraperitoneal infection (20
) and play a crucial role in the further systemic dissemination to the peripheral organs (19
The replication to high virus titers in the mice correlated with the pathology of the infected animals within few days (Fig. 5
) as reported before (6
). The most virulent isolates, Ken-IIA, SiAr126, India/61, BKNV, and PoTi461, killed the animals with extremely low doses of 1 PFU within five to 7 days. Interestingly, animals with a lethal THOV-like infection showed a constant decline in body weight whereas DHOV-like viruses caused a moderate reduction in body weight despite severe clinical manifestations. Such differences in the pathogenesis might be caused by the primary tropism of the THOV-like viruses for the liver and the transient preference of DHOV-like viruses for the lung.
Animals infected with low replicating viruses like JOSV, Kami-25, PoTi503, OzV, and BRBV showed no or only a transient reduction in body weight and survived the infection even with high doses of up to 100.000 PFU (Fig. 5
) as also reported by others (9
). However, the lack of in vivo
virulence might not influence their spread in their enzootic transmission cycle, because thogotoviruses are transmitted between ticks even by nonviremic mammals (53–55
The passage history of most of our isolates is unknown. However, it is obvious to speculate that the most aggressive thogotoviruses that led to early and severe disease symptoms were already adapted to mice during the isolation process, SiAr126 (56
), Ken-IIA (5
), PoTi503 (16), India/61 (10) and PoTi461 (52
). To address this question, we performed an adaptation experiment with two replicating thogotoviruses that showed an attenuated pathology. However, even after 10 consecutive passages the liver titers only slightly increased. The full-genome sequence analyses showed only minor genomic changes after 10 passages with only a few common mutations in the two simultaneous passages of the same virus (Fig. 9
). Notably, we cannot fully exclude that the single cell culture passage of the viruses upon re-isolation from the organs did influence the observed variant frequencies. Nevertheless, this high genomic stability was unexpected as related experiments with influenza A viruses show an intense genomic plasticity that results in the adaptation of the viruses during mouse passages (32–34
). Therefore, the genomic stability of thogotoviruses supports the hypothesis that thogotoviruses are already well adapted to rodents (17
Thogotoviruses belong to the group of arboviruses that are transmitted through the skin of the mammalian host during the tick blood meal (58
). However, a single report implicated aerosol transmission for DHOV/India/61 (23). To evaluate possible tick-independent routes of virus transmission, C57BL/6 mice were infected with the highly replicating THOV-like SiAr126 or DHOV-like India/61 isolates by either the intraperitoneal, subcutaneous, intranasal, or intravenous route. The different infection routes did not grossly influence the pathology in the infected animals (Fig. 8
), indicating that aerosol transmission is a possible route of infection, as discussed previously (18
). Therefore, we analyzed the risk of a tick independent contact transmission of thogotoviruses from infected to highly susceptible IFNAR−/−
sentinel animals (28
). Although the index animals showed the presence of infectious virus in the lung and the snout, there was no indication for contact transmission, suggesting that the tick vector is the preferred transmission route.
In summary, our comparative characterization of a set of different thogotovirus isolates demonstrates important features of this exceptional class of orthomyxoviruses: (i) the clear division of the genus thogotoviruses into the two clades of THOV-like and DHOV-like viruses according to their phylogenetic, morphological, and serological features as well as differences in mouse pathology. (ii) The genetic stability of the viruses during consecutive mouse passages. And finally, (iii) the lack of contact transmission and therefore the relevance of the arthropod vector for the spread of thogotoviruses in their mammalian reservoir.
MATERIALS AND METHODS
Biosafety and animal ethics.
All work with thogotoviruses was performed under biosafety level (BSL) 2 conditions, except for the human isolate of BRBV that was handled under BSL3 conditions. The animals were handled in accordance with guidelines of the Federation for Laboratory Animal Science Associations and the national animal welfare body. Animal experiments were performed in compliance with the German animal protection law and approved by the local animal welfare committee (Regierungspraesidium Freiburg, permit 35–9185.81/G-15/127).
The 10 different thogotovirus isolates (Table 1
) were kindly provided by our colleagues: THOV/SiAr/126/72 (isolate number 113.3) by Robert E. Shope, Emerging Infectious Diseases, University of Texas, Galveston, TX (60
); the African THOV/Ken-IIA (isolate number 82.1) by Patricia A. Nuttall, Institute of Virology, University of Oxford, UK (5
); THOV/PoTi503 (isolate number 106.1) by Armindo R. Filipe, Laboratory of Virology, NIH, Lisbon, Portugal (61
); JOSV (TVP10564, isolate number 105.1) by Robert B. Tesh, Emerging Infectious Diseases, University of Texas, Galveston, TX (7
); HI-Kamigamo-25 (strain KSU-25, isolate number 69.1) by Kentaro Yoshii, Laboratory of Public Health, Hokkaido University, Sapporo, Japan (36
); DHOV/India/1313/61 (isolate number 77.1) by Fred J. Fuller, Department of Microbiology, North Carolina (10
); BKNV (strain LEIV306K, isolate number 95.3p.1) by Robert E. Shope (6
); DHOV/PoTi461 (isolate number 100.1) by Armindo R. Filipe (62
); BRBV-KS (strain NR-50132, ATCC VR-1842, Kansas-KS isolate number 87.1) by Amy J. Lambert and Brandy Russell, Centers for Disease Control and Prevention, Fort Collins, Colorado (24
); and Oz virus (strain OzV, isolate number 264.1) by Kyoko Sawabe, National Institute of Infectious Diseases, Tokyo, Japan (8
). All THOV-like viruses code for a functional orf encoding the ML IFN antagonist in their segment 6 (7, 37).
Most strains were initially isolated from tick lysates by intracranial infection of new borne mice or by inoculating Vero cells, followed by several passages on BHK-21 or Vero cells. Viral titers were determined by plaque assay on Vero cells.
African green monkey kidney Vero cells (ATCC CCL-81) and Syrian golden hamster kidney BHK-21 cells (ATCC CCL-10) were cultivated in Dulbecco's Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal calf serum (FCS) at 37°C and 5% CO2. Cell infections were performed using DMEM with 0.1% FCS. For viral replication, DMEM with 2% FCS and 20 mM HEPES was used.
Wild-type C57BL/6 mice were purchased from Janvier Labs (France). Congenic B6.A2G-Mx1 mice with a functional Mx1 allele (Mx1+/+
) (for the generation of postinfectious sera) and IFNAR−/−
) (index animals for transmission experiments) were bred in-house. Animal experiments were performed in compliance with the German animal protection law and approved by the local animal welfare committee (Regierungspraesidium Freiburg, permit G-15/127). All experiments were performed with age and sex matched 7 to 10 weeks old animals. Depending on the experiment the animals were infected intraperitoneally (i.p.), subcutaneously (s.c.) or intravenously (i.v.) with a total volume of 100 μl PBS. In case of the intranasal (i.n.) infection the animals were mildly sedated with 5% isofluran and then 40 μl of PBS diluted virus was applied directly onto the snout. Animals were daily monitored for weight and clinical signs and killed by cervical dislocation at the indicated time points. Organs, liver, lung, spleen and kidney were harvested at day 4 postinfection and homogenized (FastPrep Homogenizer, MP Biomedicals) in PBS in a final volume of 1 ml. After centrifugation at 5,000 × g
for 10 min and 4°C the supernatants were analyzed by plaque assay on Vero cells.
Post-infectious sera were generated by challenging animals that survived the infection with 10,000 PFU/animal. The second infection did not result in any weight loss or disease symptoms. At 10 days after the second infection the blood was harvested by heart punctuation of anesthetized animals (Ketamine 100 mg/kg body weight and Rompun 5 mg/kg body weight) prior to cervical dislocation. The serum was prepared from the blood by incubation at 37°C for 10 min and centrifugation at 5,000g for 10 min. Due to the high lethality of the virus strains SiAr126, India/61, BKNV, and PoTi461 B6-Mx1+/+
mice were used to elicit specific, postinfectious antisera as described previously (29
For the determination of the lethal dose (LD) weight and disease symptoms were monitored daily. The mice were killed, if the weight loss reached 25% or the mice showed severe disease symptoms.
For transmission experiments C57BL/6 index mice (n
= 9) were infected i.p. with 1.000 PFU/animal. At 6 h postinfection the animals were cohoused for 4 days in a fresh cage with naive IFNAR−/−
contact mice (n
= 12) that are highly susceptible to virus infection. After 4 days the C57BL/6 index animals were sacrificed. Liver, lung, spleen, and kidney were harvested to confirm the successful infection. Bladder, ileum, snout, and feces (3 pellets/animal) were obtained to analyze transmission relevant organs for viral propagation. The weight and disease symptoms of the contact animals were monitored for additional 16 days. Afterwards sera were obtained from the infected animals and checked for seroconversion in a neutralization assay as described (28
) (data not shown).
For in vivo passaging C57BL/6 mice (n = 2) were i.p. infected with 1.000 PFU for 4 d. The livers were homogenized and the viral load determined. Diluted liver homogenates were used for the next passage by i.p. infection with 1.000 PFU for 4 d. 10 consecutive liver passages were performed for each virus in two independent series. The 10th liver passages were used to grow virus stocks on Vero cells for next-generation sequencing.
Transmission electron microscopy.
Vero cells were infected with different thogotovirus isolates at an MOI of 5 and fixed 16 h postinfection in 4% paraformaldehyde (PFA) and 0.1% glutaraldehyde in a 100 mM PHEM buffer for 30 min at room temperature (64
). The method of in situ
prefixation allowed us to preserve the original shape of cells and virus particles in the process of budding. Then, cells were scrapped, pelleted, and incubated overnight with 4% PFA in the 100 mM PHEM buffer at 4°C. After washing with a 100 mM sodium cacodylate buffer, cells were post-fixed for 60 min with 1% osmium tetroxide and 50 mM potassium ferricyanide in a 100 mM sodium cacodylate buffer (pH 7.0), washed and treated with 0.1% tannic acid in HEPES buffer for 30 min. After washing, the cell pellets were incubated in 2% uranyl acetate. Then, the cells were dehydrated in grade ethanol, embedded in a mixture of Epon and Araldite, and polymerized at 60°C for 24 h. Ultrathin sections (60 to 90 nm) of the cells were cut with a Leica EM UC6 microtome. The sections were contrasted with uranyl acetate and lead citrate and analyzed with a JEM1400 transmission electron microscope at 120 kV. The images were acquired using a TVIPS TemCam F416 camera.
Viral supernatants from Vero-infected cells were fixed with 4% PFA. Formvar-coated 400-mesh grids were pretreated with 1% alcian blue to optimize adhesion of viral particles. The viral suspensions were applied onto EM grids using airfuge sedimentation of 80 μl viral suspension. All preparations were negatively stained with 2% phosphotungstic acid and analyzed with either a Zeiss 109 or a JEM1400 electron microscope. For quantification, particles in several independent fields of view of the EM preparations were analyzed. About 70 to 100 virions were counted for each isolate to determine the percentage of spherical versus filamentous particles as well as their diameter and length. Particles were classified as filamentous when their long axis was at least two times their diameter.
Western blot analysis and antibody neutralization assay.
Vero cells were infected with the indicated viruses (MOI of 1) for 24 h and lysed in T-PER tissue protein reagent (Thermo scientific). Proteins were denaturated in Laemmli buffer, separated by 12% SDS-polyacrylamid gel electrophoresis and transferred onto PVDF membrane (Merck Millipore). Viral proteins were detected using the postinfectious polyclonal mouse antisera. β-actin specific rabbit antiserum (Abcam) was used as an internal control. Primary antibodies were detected using fluorescent-labeled anti-mouse secondary antibodies (LI-COR). The fluorescent signals were detected using the ODYSSEYFc (Licor).
For the expression of recombinant viral proteins, pCAGGS-based expression plasmids (65
) encoding the viral GP, NP, and M of SiAr126 and India/61 were transfected into 293T cells (2 μg GP, 0.2 μg NP, 2 μg M plasmid per six well). At 48 h posttransfection the cells were lysed and analyzed by Western blotting as described above.
To test the postinfectious mouse sera for virus neutralization, serial 2-fold dilutions (1:32 to 1:16,384) were prepared and incubated with a fixed amount of 100 PFU of the appropriate virus strain for 1 h at room temperature (28
). As a control virus was incubated with PBS. The virus-serum mixture was transferred onto Vero cells and a plaque assay was performed. The PFU of the antibody-treated viruses were normalized to the PBS/virus control. For each experiment and sera a four parameter logistic regression with an upper (y = 100) and lower limit (y = 0) was calculated and based on the regression the neutralization titer NT50
About 60 ml of virus stock (about 1 × 107 PFU/ml) were subjected to ultracentrifugation through a 28% glycerin cushion in PBS for 2 h at 100,000 × g using a SW32 rotor (Beckman Coulter). The virus pellets were resuspended in 1 ml of PBS, virus titers were determined and about 2–5 × 108 PFU were subjected to RNA extraction. Subsequently, the viral RNAs were pooled for the library preparation (TruSeq RNA Library Prep kit v2) and sequenced with an Illumina HiSeq2500 (50 cycles, single end).
De novo assembly and variant calling.
Quality of raw sequencing data were assessed with FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/
, accessed on 13.07.2021), no adapter or low-quality sequences were observed. Assembly of the different samples was done with SOAPdenovo-Trans (version 1.03, default parameter) (66
), Trinity (version 2.8.4, default parameter) (67
) and rnaSPAdes (version 3.13.1, default parameter) (68
). Final assemblies were achieved by clustering the contigs of all individual assemblies using cd-hit-est (version 4.6.6, sequence identity threshold set to 0.95) (69
) and BLAST (version 2.7.1+, E-value threshold set to 1e-10) (70
). Visualization of the assemblies and their comparison was performed with QUAST (version 5.0.2, default parameter) (71
Raw sequencing data of the host adaptation experiments were mapped to the final assemblies with HISAT2 (version 2.0.4, default parameter) (72
). Mappings were processed with samtools (version 1.3) (73
). For SNP calling, loFreq (version 126.96.36.199) (74
) was used with default parameters. In-house Python3 scripts were used to determine changes on amino acid level. The scripts are available at https://github.com/klamkiew/thogotovirus_host_adaptation
Circular plots, as shown in Fig. S1 and S2, were created using circos (10.1101/gr.092759.109). Preparation of the data for circos configuration files was done with an in-house Python script, deposited in the mentioned github repository.
Multiple sequence alignments of the deduced amino acid sequences of the different THOV and DHOV-like isolates were generated using MAFFT (75
). Based on these alignments, maximum likelihood trees were constructed using PhyML 3.1 (76
). The Smart Model Selection (SMS) was utilized to determine the best fitting substitution model (77
). The analysis was performed under the LG substitution model with 1,000 bootstrap replicates and the tree visualized using Geneious 10.
Data were analyzed and statistically evaluated with GraphPad Prism 7. Viral titers of mouse organs were displayed on a log-scale (scatterplot, geometric mean) Statistics were computed by a one-way ANOVA with a Tukey’s multiple-comparison test on log-transformed values.
Full genome nucleotide sequences of the thogotoviruses were submitted to GenBank. For GenBank accession numbers see Table 1
Raw reads of the virus stocks sequencing are available under https://osf.io/zd2mx/
We thank Armindo R. Filipe, National Institute of Health, Lisbon, Portugal, for THOV/PoTi503 and DHOV/PoTi461; Frederick J. Fuller, North Carolina State University, Raleigh, NC, USA, for DHOV/India/1313/61; Patricia A. Nuttall, Institute of Virology, Oxford, UK, for THOV/SiAr/126/72 and THOV/Ken-IIA; Brandy Russell, CDC, Fort Collins, Colorado, USA, for BRBV-KS; Kyoko Sawabe, National Institute of Infectious Diseases, Tokyo, Japan, for Oz virus; Robert E. Shope, University of Texas, Galveston, TX, USA, for Batken virus; Robert B. Tesh, University of Texas, Galveston, TX, USA, for Jos virus; and Kentaro Yoshii, Hokkaido University, Sapporo, Japan, for HI-Kamigamo-25 virus.
We thank Valentina Wagner for excellent technical assistance and Martin Schauflinger and Elias Bendl for their critical reviews of our manuscript.
The work was funded by the German National Platform on Zoonoses Research and the Bundesministerium fuer Ernährung und Landwirtschaft (BMEL; German Federal Ministry of Food and Agriculture) through the Federal Office for Agriculture and Food (BLE), grant number 2816HS008, and by Deutsche Forschungsgemeinschaft (DFG; German Research Foundation) KO1579/12-1 and KO1579/9-2 in the SSP1596 DFG priority program to G.K. as well as FZT 118, 202548816 to M.M. We are thankful for the financial support of the Carl-Zeiss Stiftung (FKZ 0563-2.8/738/2) to M.M. The funders had no role in study design or data collection and interpretation.