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9 March 2022

Comparative Study of Ten Thogotovirus Isolates and Their Distinct In Vivo Characteristics


Thogotoviruses are tick-borne arboviruses that comprise a unique genus within the Orthomyxoviridae family. Infections with thogotoviruses primarily cause disease in livestock with occasional reports of human infections suggesting a zoonotic potential. In the past, multiple genetically distinct thogotoviruses were isolated mostly from collected ticks. However, many aspects regarding their phylogenetic relationships, morphological characteristics, and virulence in mammals remain unclear. For the present comparative study, we used a collection of 10 different thogotovirus isolates from different geographic areas. Next-generation sequencing and subsequent phylogenetic analyses revealed a distinct separation of these viruses into two major clades, the Thogoto-like and Dhori-like viruses. Electron microscopy demonstrated a heterogeneous morphology with spherical and filamentous particles being present in virus preparations. To study their pathogenicity, we analyzed the viruses in a small animal model system. In intraperitoneally infected C57BL/6 mice, all isolates showed a tropism for liver, lung, and spleen. Importantly, we did not observe horizontal transmission to uninfected, highly susceptible contact mice. The isolates enormously differed in their capacity to induce disease, ranging from subclinical to fatal outcomes. In vivo multistep passaging experiments of two low-pathogenic isolates showed no increased virulence and sequence analyses of the passaged viruses indicated a high stability of the viral genomes after 10 mouse passages. In summary, our analysis demonstrates the broad genetic and phenotypic variability within the thogotovirus genus. Moreover, thogotoviruses are well adapted to mammals but their horizontal transmission seems to depend on ticks as their vectors.
IMPORTANCE Since their discovery over 60 years ago, 15 genetically distinct members of the thogotovirus genus have been isolated. These arboviruses belong to the Orthomyxovirus family and share many features with influenza viruses. However, numerous of these isolates have not been characterized in depth. In the present study, we comparatively analyzed a collection of 10 different thogotovirus isolates to answer basic questions about their phylogenetic relationships, morphology, and pathogenicity in mice. Our results highlight shared and unique characteristics of this diverse genus. Taken together, these observations provide a framework for the phylogenic classification and phenotypic characterization of newly identified thogotovirus isolates that could potentially cause severe human infections as exemplified by the recently reported, fatal Bourbon virus cases in the United States.


The family Orthomyxoviridae consists of seven genera: Influenza A-D viruses, Isavirus, Quaranfil virus and the tick-transmitted thogotoviruses (1, 2). 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, 4). 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 (79), 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 (1014). 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 (1618). Besides their replication in hepatocytes (17, 18), it became apparent that thogotoviruses target myeloid dendritic cells and myeloid cells isolated from the peritoneal cavity (19, 20). 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 ticks (26, 27).
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 [2020] from the Faculty of Biology of the University of Freiburg, Germany.)


Phylogenetic relationships of the thogotovirus isolates.

Over the last years, several historical specimen and new isolates were classified to the genus Thogotovirus within the Orthomyxoviridae. For the present comparative study we collected 10 different isolates of thogotoviruses from four continents that were kindly provided by colleagues all over the world (Table 1). All these virus isolates were propagated in our lab under comparable conditions in Vero or BHK cells to titers of 106 to 107 PFU/ml.
TABLE 1 Overview over the different thogotovirus isolates
GenusIsolateOriginIsolated fromReplication in miceGenbank acc.
THOV-likeJOSVNigeriaCattleYes (7)HM627170-75
HI-Kamigano-25JapanHaemaphysalisYes (54)MT628434-39
Ken-IIAKenyaRhipicephalusYes (5)MT628404-09
PoTi503PortugalRhipicephalusYes (16)MT628446-51
SiAr/126/72SicilyRhiphicephalusYes (17, 60)MT628440-45
DHOV-likeOzVJapanAmblyommaYes (8)LC320123-28
BRBV-KSUSAHumanNo (9, 25, 28)MT628410-15
India/1313/61IndiaHyalommaYes (21, 29)MT628427-33
BKNVKirghiziaHyalommaYes (6)MT628416-21
PoTi461PortugalHyalommaYes (52)MT628422 -26
For many thogotovirus isolates only partial or no sequence information is available in public databases. To confirm the authenticity of our virus stocks, we analyzed their genomes by next-generation sequencing (NGS) using ultracentrifugation concentrated preparations of the 10 isolates. We determined the full-length genome sequences by ultradeep Illumina sequencing of the extracted RNAs generating 10 to 15 million reads per virus resulting in a 30.000–80.000-fold coverage depending on the individual isolate. The full-length genomes were de novo assembled and deposited in GenBank (accession numbers see Table 1). The genome of Oz virus (OzV) was not included in this NGS analysis and for further analyses the sequences from a reference genome (8) were used. Based on the deduced amino acid sequences we constructed maximum likelihood trees for each segment. The phylogenetic relationships between the isolates remained largely consistent in all six segments and showed a clear separation into two groups: The THOV-like and the DHOV-like thogotoviruses. This was furthermore, reflected by the amino acid sequence identities of the individual open reading frames (orf) that are high within (40–100%) but low (20–60%) between both clades (Fig. S1). Moreover, the phylogenetic analysis showed that JOSV (THOV-like) as well as OzV and BRBV (DHOV-like) are more distantly related members of the two clusters. Furthermore, we observed a distant relationship between OzV and BRBV in segment 4 and 6 (Fig. 1D and F) as previously suggested (8).
FIG 1 Next-generation sequencing and phylogenetic analysis of thogotovirus isolates. Analysis of the deduced full-length amino acid sequences determined by next-generation sequencing of the viral genomic RNAs. The sequences were aligned for each segment using MAFFT (75). Based on these multiple sequence alignments maximum likelihood trees were constructed using PhyML 3.1 (76). The Smart Model Selection (SMS) (77) was utilized to determine the best fitting substitution model. The analyses were performed under the LG substitution model with 1.000 bootstrap replicates and the trees visualized using Geneious 10. (A) Segment 1 (PB2), (B) Segment 2 (PB1), (C) Segment 3 (PA), (D) Segment 4 (GP), (E) Segment 5 (NP) and (F) Segment 6 (M). The coding sequences of Influenza virus A/California/07/2009 (H1N1) (NC_026435 - NC_026438, NC_026431) were used to root the trees, with the exception of GP. For segment 4 (GP) analysis, the GP46 (YP_473216) sequence of the alpha-baculoviruses Hyphantria cunea nucleopolyhedrovirus was used. For Oz virus (OzV) the sequences of a previous publication by Ejiri et al. (8) were used (accession numbers: LC320123–LC320128).

EM analysis reveals heterogeneity in the virion morphology.

To investigate the morphology of thogotovirus virions, we analyzed virus-infected cells and supernatants of infected cell cultures by transmission electron microscopy. Representative images of ultrathin-sections show spherical particles with electron-dense walls budding from the surface of infected Vero cells (Fig. 2A and B). Extracellular and protruding particles contained electron dense material representing densely packaged viral proteins beneath the lipid envelope (Fig. 2A and B, insets). A detailed analysis of the clarified supernatants of virus-infected cells showed spherical particles with a diameter of around 100 nm (Fig. 2C to F). Interestingly, a substantial number of filamentous virions were observed as described previously for BRBV (9). The analyses of the four different isolates suggest that the frequency and size of these filamentous particles is higher for DHOV-like viruses (Fig. 2C to F left panel).
FIG 2 Electron micrographs of thogotoviruses. Vero cells were infected (MOI of 5) with the different thogotoviruses for 16 h. (A-B) Ultrathin sections of infected cells processed for negative staining were imaged with a transmission electron microscope. The images display numerous budding and extracellular virions of JOSV (A) and BRBV (B). Insets show boxed areas at higher magnification. (C-F) Supernatants of infected cells were fixed at 24 h.p.i. and processed for negative staining using phosphotungstic acid. Images show filamentous and spherical particles of SiAr126 (C), JOSV (D), India/61 (E) and BRBV (F) with distinct surface projections. Filamentous particles often show spherical head-like structures at one tip (arrowheads). Scale bars: 100 nm. For quantitative analyses of virion size and morphology 70 to 100 virions were counted to estimate the percentage of filamentous particles.

Replication of thogotoviruses in vivo.

So far, the capacity of different thogotoviruses to replicate and cause disease in mammals has not been comparatively analyzed. To address this, we studied the replication of the different thogotovirus isolates in mice. C57BL/6 mice were intraperitoneally infected with 100 PFU of the THOV-like (Fig. 3) and DHOV-like isolates (Fig. 4). Only for the infection with the Ken-IIA and the SiAr126 isolates, we chose a 100-fold reduced infectious dose of one PFU to delay their excessive pathogenic effects until day 6. The amount of viral progeny was analyzed in liver, lung, spleen, kidney, serum and brain after two, four and 6 days postinfection. Interestingly, upon infection with the THOV-like isolates we detected viral titers in almost all organs, except the brain, and already observed infectious particles in liver and spleen 2 days postinfection. Overall, the highest titers were present in the livers of Ken-IIA and the SiAr126 infected animals, whereas JOSV, Kami-25 and PoTi503 replicated to 100 to 1000-fold reduced titers (Fig. 3). The DHOV-like isolates India/61, BKNV and PoTi461 also showed efficient replication in liver, lung and spleen (Fig. 4C to E). However, OzV and BRBV infections did not lead to measurable viral titers in all analyzed organs (Fig. 4A and B).
FIG 3 Organ tropism of the THOV-like isolates in C57BL/6 mice. C57BL/6 mice (n = 4 to 6) were infected intraperitoneally with (A) JOSV (100 PFU), (B) Kami-25 (100 PFU), (C) Ken-IIA (1 PFU), (D) PoTi503 (100 PFU) and (E) SiAr126 (1 PFU). After 2, 4 and 6 d.p.i the mice were sacrificed. Liver, lung, spleen, kidney, serum, and brain were harvested and the viral titers determined by plaque assay. Shown are the geometric means. Statistical analyses were performed with a one-way ANOVA on log-transformed values (Tukey’s multiple-comparison test, *P < 0,05, **P < 0,01, ***P < 0.001, ns – not significant).
FIG 4 Organ tropism of the DHOV-like isolates in C57BL/6 mice. C57BL/6 mice (n = 4 to 6) were infected intraperitoneally with (A) OzV (100 PFU), (B) BRBV (100 PFU), (C) India/61 (100 PFU), (D) BKNV (100 PFU) and (E) PoTi461 (100 PFU). After 2, 4 and 6 d.p.i the mice were sacrificed. Liver, lung, spleen, kidney, serum, and brain were harvested and the viral titers determined by plaque assay. Shown are the geometric means. Statistical analyses were performed with an one-way ANOVA on log-transformed values (Tukey’s multiple-comparison test, *P < 0,05, **P < 0,01, ***P < 0.001, ns – not significant).
Overall, the spleen is one of the first organs affected by all thogotoviruses upon intraperitoneal infection. Remarkably, there was a clear difference in the organ tropism of both clusters as in contrast to the more hepatotropic THOV-like isolates (Fig. 3) the viral replication of DHOV-like isolates peaked in the lung (Fig. 4).

The pathogenicity in mice differs between the isolates.

To further compare the virulence of the different thogotovirus isolates, C57BL/6 mice were intraperitoneally infected with serially diluted virus inoculums and daily monitored. The isolates Ken-IIA, SiAr126, India/61, BKNV and PoTi461 which grew to high titers caused severe lethal disease at a low viral dose of one PFU within five to 7 days (Fig. 5C, E, H to J). Interestingly, mice infected with the THOV-like viruses Ken-IIA and SiAr126 dramatically dropped in body weight within five to 6 days (Fig. 5C and E) and displayed severe symptoms, including lethargy, ruffed fur and hunched posture similar to the previously published clinical score (28). In contrast, mice infected with the DHOV-like isolates India/61, BKNV and PoTi461 got terminally ill in the absence of a dramatic weight loss (Fig. 5H to J). JOSV, Kami-25 and PoTi503 that did not grow to high titers (Fig. 3) and BRBV and OzV that were strongly attenuated in vivo (Fig. 4) did not cause severe disease even at the highest infection doses of 100,000 PFU per animal (Fig. 5A, B, D, F and G). Notably, mice infected with JOSV and PoTi503 (Fig. 5A and D) transiently dropped in weight at high infection inoculums, indicating a mild self-limiting disease.
FIG 5 Pathogenicity of the Thogoto- and Dhori-like virus isolates in C57BL/6 mice. Mice (n = 4/group) were infected intraperitoneally with the indicated viral doses of (A) JOSV, (B) Kami-25, (C) Ken-IIA, (D) PoTi503, (E) SiAr126, (F) OzV, (G) BRBV, (H) India/61, (I) BKNV or (J) PoTi461. Weight and survival were monitored. The mice were euthanized if severe symptoms were observed or weight loss reached 25%.
In summary, these data indicate that the course of disease caused by thogotovirus infections correlates with their capacity to replicate in vivo (Table 2).
TABLE 2 Pathogenicity of the different thogotovirus isolates in C57BL/6 micea
GenusIsolateReplicationLethal dose
THOV-likeJOSVYes>105 pfu (LD50)
HI-Kamigano25Yes>105 pfu (LD50)
Ken-IIAYes1 pfu (LD100)
PoTi503Yes>105 pfu (LD50)
SiAr/126/72Yes1 pfu (LD100)
DHOV-likeOzVNo>105 pfu (LD50)
BRBV-KSNo>105 pfu (LD50)
India/1313/61Yes1 pfu (LD100)
BKNVYes1 pfu (LD100)
PoTi461Yes1 pfu (LD100)
LD, lethal dose, infectious dose in plaque forming units (pfu) when 50% or 100% of i.p. infected animals were killed due to severe disease symptoms or their body weight loss reaching 25%.

Polyclonal antisera are cross-reactive between related thogotoviruses.

In order to evaluate the humoral immune response against thogotoviruses, we isolated and pooled postinfectious antisera from at least four C57BL/6 mice. In case of the highly pathogenic viruses, we infected congenic Mx1+/+ mice that can potently restrict thogotovirus replication (17, 29). We subsequently investigated whether these sera are able to recognize viral antigens present in the lysates of infected cells by Western blotting analyses (Fig. 6). The antibody responses of all viruses were primarily directed against GP (58 kDa) and NP (52 kDa) with NP showing the strongest signal in all cases (Fig. 6). Importantly, the GP was reported to have a high molecular weight of 75 kDa due to its glycosylation (4, 30). Antibodies against the M protein (31 kDa) were only detected in sera of OzV infected animals (Fig. 6F). Interestingly, antisera from mice infected with closely related virus isolates showed a high degree of cross-reactivity. This was observed between the isolates within the THOV-like (Fig. 6A to E) or within the DHOV-like clusters (Fig. 6F to J) but not between the two groups. However, postinfectious sera of JOSV and BRBV, both distantly related members of the two clusters (Fig. 1), were not cross-reactive (Fig. 6A and G). Curiously, postinfectious sera of the other THOV-like isolates, except SiAr126, recognized JOSV NP, suggesting that during a JOSV infection the humoral response is exclusively directed against strain specific epitopes. In contrast, the antiserum from OzV infected animals recognized antigens of all DHOV-like viruses (Fig. 6F) but sera from India/61, BKNV and PoTi461 showed comparable cross-reaction with each other but not with antigens of OzV and BRBV (Fig. 6H to J). The variations in the specificity of the immune sera against the different viruses that sometimes recognize only NP, sometimes additionally GP or M might be due to differences in the intensity of viral protein production in the infected cells as well as the presentation of the viral antigens in the infected animals. To confirm the specificity of the antigen recognition, we performed Western blot analysis of lysates of transfected cells expressing recombinant GP, NP and M proteins of THOV SiAr126 and DHOV India/61, respectively. The recombinant proteins of SiAr126 were best recognized by the antisera directed against Ken-IIA and to a lesser extent against SiAr126 but both sera did not recognize the India/61 proteins (Fig. S2B, C). Accordingly, the anti-India/61 and anti-BKNV antisera strongly recognized the India/61 NP but there was no cross-recognition of the SiAr126 proteins (Fig. S2E, F), confirming the clade specificity shown with the lysates of infected cells (Fig. 6). Only faint bands of SiAr126 GP and NP were recognized by the anti-JOSV antiserum (Fig. S2A), confirming the more distant position of JOSV in the THOV-like clade. The cross-reactivity of the anti-OzV antiserum recognizing GP and M but not NP in the lysates of DHOV-like viruses infected cells (Fig. 6F) could be confirmed for the recombinant India/61 proteins (Fig. S2D).
FIG 6 Cross reactivity of postinfectious sera. C57BL/6 mice which survived an infection with the THOV-like or DHOV-like isolates during the analysis of the lethal dose (see fig. 5) were boosted with a high infection dose (10.000 PFU) 12 days after the initial infection for further 10 days. Due to the lethality of SiAr126, Ken-IIA, India/61, BKNV, and PoTi461 in C57BL/6 the infections were performed in B6.A2G-Mx1 mice (Mx1+/+) mice, which can potently restrict thogotoviruses. Afterwards whole blood was harvested by cardiocentesis and the sera were prepared. Vero cells were infected with the different THOV-like and DHOV-like isolates (MOI = 1) and lysed after 24 h. The lysates were subjected to Western blot analysis. As a primary antibody, the postinfectious sera of (A) JOSV, (B) Kami-25, (C) Ken-IIA, (D) PoTi503, (E) SiAr126, (F) OzV, (G) BRBV, (H) India/61, (I) BKNV or (J) PoTi461 were used at 1:250 dilutions. Actin (J) was stained as a loading control.
Furthermore, the postinfectious antisera were tested for virus neutralization using a set of selected thogotoviruses, THOV-like SiAr126 and JOSV as well as DHOV-like India/61 and OzV. These analyses confirmed the clear separation between the THOV-like and the DHOV-like clade. Group specific antisera were only able to neutralize the respective prototypic isolate of each group (SiAr126 or India/61) (Fig. 7A and C and Fig. S3A, C). However, these two viruses were not neutralized by the anti-JOSV and anti-OzV antisera. Accordingly, JOSV and OzV were selectively neutralized by anti-JOSV and anti-OzV antisera, respectively (Fig. 7B and D and Fig. S3B, D). Unexpectedly, SiAr126 was not well neutralized by the anti-SiAr126 serum (Fig. 7A, Fig. S3A). However, the Western blot analysis of the recombinant proteins suggests that this antiserum is much less potent than, e.g., the anti-Ken-IIA antiserum in recognizing the viral SiAr126 GP and NP (Fig. S2B, C). Therefore, we tested other batches of anti-SiAr126 antisera and also the neutralization capacity of these sera batches against THOV-like PoTi503 and Ken-IIA with similar results (data not shown). Because of the extremely high virulence of the SiAr126 isolate in C57BL/6 mice these sera were obtained from Mx1+/+-positive mice that dampen the replication and pathogenic effects of the SiAr126 infection (17). We speculate that under these restricting conditions, the cell tropism of the virus is modified (19) and the expression of viral antigens is hampered and therefore production of neutralizing antibodies attenuated. In conclusion, the main humoral response during a thogotovirus infection is directed against the viral NP and GP and with a few exceptions the produced antibodies are only cross-reactive with closely related viruses.
FIG 7 Neutralization capacity of postinfectious sera. Serial 2-fold dilutions of the postinfectious mouse sera were incubated with 100 PFU of (A) SiAr126, (B) JOSV, (C) India/61 or (D) OzV and analyzed by plaque assay. NT50 values were calculated with a four parameter logistic regression (Fig. S3). Displayed are the individual values for each experiment as dots and the geometric mean and geometric standard deviation as bars (n = 3).

No horizontal transmission of the THOV- and DHOV-like viruses in mice.

To evaluate tick-independent routes of virus transmission and the possibility that the infection route might influence the disease progression of thogotoviruses, C57BL/6 mice were infected with the THOV-like SiAr126 or the DHOV-like India/61 isolate by either the intraperitoneal, subcutaneous, intranasal or intravenous route. For both viruses all infection routes with 100 PFU led to a severe and fatal disease (Fig. 8A and B). However, intranasal and subcutaneous application delayed the time of death by up to 3 days for the SiAr126 isolate (Fig. 8A) and by 1 day for the India/61 isolate (Fig. 8B) in comparison to the most potent intravenous route of infection.
FIG 8 Infection route and transmission of THOV/SiAr126 and DHOV/India/61. C57BL/6 mice (n = 4/group) were infected with the indicated doses of 100 PFU of (A) SiAr126 or (B) India/61 either intraperitoneally (i.p.), subcutaneously (s.c.), intranasally (i.n.) or intravenously (i.v.). Virus was diluted in 100 μl PBS for i.p., i.v. and s.c., for the intransal infection the animals were infected with a volume of 40 μl. Weight and survival were monitored. If severe symptoms were observed or weight loss reached 25%, the mice were euthanized. (C-F) Female C57BL/6 index mice were infected i.p. with 1.000 PFU (n = 9). After 6 h the index mice were co-housed in three separated groups (n = 3 for each group) with four naive C57BL/6 IFNAR−/−/IL28R−/− contact mice each (3 groups, n = 12) for 4 days. At day 4 the index mice were killed and the viral loads of liver, lung, spleen, kidney, bladder, ileum, snout, and feces determined by plaque assay (geometric means) (C, E). The weight and symptoms of the contact mice were monitored daily for 20 days. Displayed are the individual weight curves and their mean (+/-SEM) (D, F). After this time period the mice were sacrificed and the blood was collected. No seroconversion was observed (data not shown).
The successful infection via the intranasal route and the fact that both THOV and DHOV-like viruses efficiently replicated in the lung (Fig. 3 and 4) suggested the possibility of a contact or aerosol driven horizontal transmission between mice. To test for such transmission events, SiAr126 or India/61 virus infected C57BL/6 index mice were cohoused for 4 days with IFNAR−/−/IL28R−/− contact animals. Mice lacking these receptors are nonresponsive to type I and III interferon (IFN) and are therefore highly susceptible to viral infections and ideal sentinels to detect viral transmission (31). Virus progeny was detected in the bladder and snout of the index animals 4 days after infection, indicating a possible release of infectious material (Fig. 8C and E). The contact animals were monitored for weight and seroconversion for 19 days after onset of cohousing. During that time no weight loss greater than 10% was observed and none of the animals were symptomatic or seroconverted. In conclusion, no thogotovirus transmissions were observed, rendering such events rather unlikely.

High genome stability of low-pathogenic thogotovirus isolates during serial liver passages.

The low replication capacity of some isolates suggested the possibility that these viruses might not be well adapted to the murine host. Influenza viruses are frequently adapted to mice by consecutive passages of lung or liver homogenates to increase their pathogenicity for mice (3235). Following this rationale, we chose the THOV-like isolates JOSV and Kami-25 for mouse adaptations because they showed a limited replication and a subclinical disease progression in vivo (Fig. 3A and B). For each isolate two independent series of 10 liver passages with consecutive intraperitoneal infections of 1000 PFU were performed in C57BL/6 mice. We chose an infection time of 4 days to avoid any effect of a possible up-coming adaptive immune response directed against viral antigens. Viral titers in the liver homogenates were determined and used for the next round of infection with 1000 PFU.
The viral titers of JOSV remained almost unchanged over the 10 passages (Fig. 9A). The titers of Kami-25 increased slightly about 10-fold over the 10 liver passages (Fig. 9B). Next, the viruses from the 10th mouse passages were amplified by a single passage in Vero cells and concentrated by ultracentrifugation to reach sufficient virus yield for deep sequencing and low variant frequency detection in the viral genomes. We mostly observed low frequency genome variants (<1%) apart from some variants that were present in a frequency above 10% (Fig. 9C and Fig. S4 and S5). Of these only a few resulted in amino acid substitutions (Fig. 9C). In comparison to the JOSV adaptations the Kami-25 adaptations showed more as well as higher frequency variants (Fig. 9C). The 10th passage of JOSV resulted in a relative uniform accumulation of variants per nucleotide (variants/nt) over all segments. However, segment 6 of both JOSV adaptations showed an increased variants/nt ratio (Fig. 9D). In contrast, the analysis of the 10th passage of Kami-25 indicated that segments 1 and 2 were highly preserved and variants were more frequently found in the other segments, arguing for a high structural conservation of PB2 and PB1 (Fig. 9E). For JOSV we only observed two low-frequency but nonsynonymous variations (∼1%) at position 40 and 49 of segment 3 that were present in both adaptations (Fig. S4). Interestingly, in segment 6 of Kami-25 we found two variants present in both adaptations at position 494 and 573 (Fig. S5). The variant at position 573 had a frequency of 15 and 3% but does not translate into an amino acid change. However, variant position 494 causes a proline to leucine exchange in codon 159 and was present at a high frequency of 20% and 46%, respectively. We also detected a synonymous mutation G855A in segment 4 in a frequency of 16% and 7%, respectively. This suggests a convergent selection for these variants during the adaptation to the mouse host. However, overall, the genomes of both viruses remained largely stable over the 10 mouse passages, implicating the absence of new selection pressure possibly due to a long history of coevolution of these viruses with rodents.
FIG 9 Mouse adaptations of two low pathogenic thogotovirus isolates. Serial liver passages of the low pathogenic THOV-like isolates, JOSV (A) and Kami-25 (B), were performed to adapt these viruses to the mouse host (two independent adaptions per virus). Initially, C57BL/6 mice (n = 2) were infected i.p. with 1,000 PFU. After 4 days the livers were harvested and the viral load titrated by plaque assay. For the nine consecutive infections, diluted liver homogenates of the previous passage containing 1,000 PFU were used for the next i.p. infection for 4 days. The trends of the viral titers are indicated as dotted lines. (C-E) The 10th passages were subjected to illumina sequencing and the reads mapped to the consensus sequence of the parental virus. The viral genome variants were called using loFreq. (C) Shown are the frequencies of all observed variants distributed over the whole genome. Black dots are variants that lead to synonymous and red dots that lead to nonsynonymous changes in the viral genome. (D, E) Variant/nt frequencies for JOSV (D) and Kami-25 (E) were calculated by the total amount of observed variants per segment divided by the length of the individual segment.


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 (79, 24, 36). 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, 37). 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, 4143). 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, 46). However, there were some exceptions: sera from JOSV and BRBV infected animals recognized solely their respective viral antigens as reported previously (7, 9, 28).
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 (4749). 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, 1214, 50, 51). 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 and 4). 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 (1719, 25, 29). 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 (25, 28). 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, 18, 29, 52). 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, 25, 28). 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 (5355).
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 (3234, 57). 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−/−/IL28R−/− sentinel animals (28, 59). 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.


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, 11); 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.

Mouse infections.

Wild-type C57BL/6 mice were purchased from Janvier Labs (France). Congenic B6.A2G-Mx1 mice with a functional Mx1 allele (Mx1+/+) (63) (for the generation of postinfectious sera) and IFNAR−/−/IL28R−/− mice (31) (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−/−/IL28R−/− 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 determined.

Next-generation sequencing.

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 (, 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 (74) was used with default parameters. In-house Python3 scripts were used to determine changes on amino acid level. The scripts are available at
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.

Phylogenetic analysis.

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.

Statistical analyses.

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.

Data availability.

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 (10.17605/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.

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McCauley JW, Hongo S, Kaverin NV, Kochs G, et al. 2012. Orthomyxoviridae, Ninth report of the International Committee on Taxonomy of Viruses. Virus taxonomy:ed King, Adams, Carstens, and Lefkowitz, Elsevier.
King AMQ, Lefkowitz EJ, Mushegian AR, Adams MJ, Dutilh BE, Gorbalenya AE, Harrach B, Harrison RL, Junglen S, Knowles NJ, Kropinski AM, Krupovic M, Kuhn JH, Nibert ML, Rubino L, Sabanadzovic S, Sanfacon H, Siddell SG, Simmonds P, Varsani A, Zerbini FM, Davison AJ. 2018. Changes to taxonomy and the International Code of Virus Classification and Nomenclature ratified by the International Committee on Taxonomy of Viruses (2018). Arch Virol 163:2601–2631.
Clerx JP, Fuller F, Bishop DH. 1983. Tick-borne viruses structurally similar to Orthomyxoviruses. Virology 127:205–219.
Portela A, Jones LD, Nuttall P. 1992. Identification of viral structural polypeptides of Thogoto virus (a tick-borne orthomyxo-like virus) and functions associated with the glycoprotein. J Gen Virol 73:2823–2830.
Haig DA, Woodall JP, Danskin D. 1965. Thogoto virus: a hitherto underscribed agent isolated from ticks in Kenya. J Gen Microbiol 38:389–394.
Frese M, Weeber M, Weber F, Speth V, Haller O. 1997. Mx1 sensitivity: batken virus is an orthomyxovirus closely related to Dhori virus. J Gen Virol 78:2453–2458.
Bussetti AV, Palacios G, Travassos da Rosa A, Savji N, Jain K, Guzman H, Hutchison S, Popov VL, Tesh RB, Lipkin WI. 2012. Genomic and antigenic characterization of Jos virus. J Gen Virol 93:293–298.
Ejiri H, Lim CK, Isawa H, Fujita R, Murota K, Sato T, Kobayashi D, Kan M, Hattori M, Kimura T, Yamaguchi Y, Takayama-Ito M, Horiya M, Posadas-Herrera G, Minami S, Kuwata R, Shimoda H, Maeda K, Katayama Y, Mizutani T, Saijo M, Kaku K, Shinomiya H, Sawabe K. 2018. Characterization of a novel thogotovirus isolated from Amblyomma testudinarium ticks in Ehime, Japan: a significant phylogenetic relationship to Bourbon virus. Virus Res 249:57–65.
Lambert AJ, Velez JO, Brault AC, Calvert AE, Bell-Sakyi L, Bosco-Lauth AM, Staples JE, Kosoy OI. 2015. Molecular, serological and in vitro culture-based characterization of Bourbon virus, a newly described human pathogen of the genus Thogotovirus. J Clin Virol 73:127–132.
Anderson CR, Casals J. 1973. Dhori virus, a new agent isolated from Hyalomma dromedarii in India. Indian J Med Res 61:1416–1420.
Lvov DK, Karas FR, Tsyrkin YM, Vargina SG, Timofeev EM, Osipova NZ, Veselovskaya OV, Grebenyuk YI, Gromashevski VL, Fomina KB. 1974. Batken virus, a new arbovirus isolated from ticks and mosquitoes in Kirghiz S.S.R. Arch Gesamte Virusforsch 44:70–73.
Darwish MA, Hoogstraal H, Omar FM. 1979. A serological survey for Thogoto virus in humans, domestic mammals, and rats in Egypt. J Egypt Public Health Assoc 54:1–8.
Filipe AR, Calisher CH, Lazuick J. 1985. Antibodies to Congo-Crimean haemorrhagic fever, Dhori, Thogoto and Bhanja viruses in southern Portugal. Acta Virol 29:324–328.
Jackson KC, Gidlewski T, Root JJ, Bosco-Lauth AM, Lash RR, Harmon JR, Brault AC, Panella NA, Nicholson WL, Komar N. 2019. Bourbon virus in wild and domestic animals, Missouri, USA, 2012-2013. Emerg Infect Dis 25:1752–1753.
Davies FG, Soi RK, Wariru BN. 1984. Abortion in sheep caused by Thogoto virus. Vet Rec 115:654.
Filipe AR, Peleteiro MC, Monath TM, Calisher EH. 1986. Pathological lesions in mice infected with Thogoto virus, a tick-borne Orthomyxovirus. Acta Virol 30:337–340.
Haller O, Frese M, Rost D, Nuttall PA, Kochs G. 1995. Tick-borne thogoto virus infection in mice is inhibited by the Orthomyxovirus resistance gene product Mx1. J Virol 69:2596–2601.
Xiao S-Y, Tesh RB, DA Rosa APAT, Mateo RI, Lei HAO. 2007. Dhori virus (Orthomyxoviridae: Thogotovirus) infection in mice: a model of the pathogenesis of severe Orthomyxovirus infection. Am J Trop Med Hyg 76:785–790.
Spitaels J, Van Hoecke L, Roose K, Kochs G, Saelens X. 2019. Mx1 in Hematopoietic cells protects against thogoto virus infection. J Virol 93.
Kochs G, Anzaghe M, Kronhart S, Wagner V, Gogesch P, Scheu S, Lienenklaus S, Waibler Z. 2016. In vivo conditions enable IFNAR-independent type i interferon production by peritoneal CD11b+ cells upon thogoto virus infection. J Virol 90:9330–9337.
Li G, Wang N, Guzman H, Sbrana E, Yoshikawa T, Tseng CT, Tesh RB, Xiao SY. 2008. Dhori virus (Orthomyxoviridae: Thogotovirus) infection of mice produces a disease and cytokine response pattern similar to that of highly virulent influenza A (H5N1) virus infection in humans. Am J Trop Med Hyg 78:675–680.
Moore DL, Causey OR, Carey DE, Reddy S, Cooke AR, Akinkugbe FM, David-West TS, Kemp GE. 1975. Arthropod-borne viral infections of man in Nigeria, 1964-1970. Ann Trop Med Parasitol 69:49–64.
Butenko AM, Leshchinskaia EV, Semashko IV, Donets MA, Mart'ianova LI. 1987. Dhori virus–a causative agent of human disease: 5 cases of laboratory infection. Vopr Virusol 32:724–729.
Kosoy OI, Lambert AJ, Hawkinson DJ, Pastula DM, Goldsmith CS, Hunt DC, Staples JE. 2015. Novel thogotovirus associated with febrile illness and death, United States, 2014. Emerg Infect Dis 21:760–764.
Bricker TL, Shafiuddin M, Gounder AP, Janowski AB, Zhao G, Williams GD, Jagger BW, Diamond MS, Bailey T, Kwon JH, Wang D, Boon ACM. 2019. Therapeutic efficacy of favipiravir against Bourbon virus in mice. PLoS Pathog 15:e1007790.
Savage HM, Burkhalter KL, Godsey MS, Jr, Panella NA, Ashley DC, Nicholson WL, Lambert AJ. 2017. Bourbon virus in field-collected ticks, Missouri, USA. Emerg Infect Dis 23:2017–2022.
Savage HM, Godsey MS, Jr, Panella NA, Burkhalter KL, Manford J, Trevino-Garrison IC, Straily A, Wilson S, Bowen J, Raghavan RK. 2018. Surveillance for tick-borne viruses near the location of a fatal human case of Bourbon virus (family Orthomyxoviridae: genus Thogotovirus) in Eastern Kansas, 2015. J Med Entomol.
Fuchs J, Straub T, Seidl M, Kochs G. 2019. Essential role of interferon response in containing human pathogenic Bourbon virus. Emerg Infect Dis 25:1304–1313.
Thimme R, Frese M, Kochs G, Haller O. 1995. Mx1 but not MxA confers resistance against tick-borne Dhori virus in mice. Virology 211:296–301.
Morse MA, Marriott AC, Nuttall PA. 1992. The glycoprotein of Thogoto virus (a tick-borne orthomyxo-like virus) is related to the baculovirus glycoprotein GP64. Virology 186:640–646.
Mordstein M, Kochs G, Dumoutier L, Renauld JC, Paludan SR, Klucher K, Staeheli P. 2008. Interferon-lambda contributes to innate immunity of mice against influenza A virus but not against hepatotropic viruses. PLoS Pathog 4:e1000151.
Brown EG, Liu H, Kit LC, Baird S, Nesrallah M. 2001. Pattern of mutation in the genome of influenza A virus on adaptation to increased virulence in the mouse lung: identification of functional themes. Proc Natl Acad Sci USA 98:6883–6888.
Gabriel G, Dauber B, Wolff T, Planz O, Klenk HD, Stech J. 2005. The viral polymerase mediates adaptation of an avian influenza virus to a mammalian host. Proc Natl Acad Sci USA 102:18590–18595.
Grimm D, Staeheli P, Hufbauer M, Koerner I, Martinez-Sobrido L, Solorzano A, Garcia-Sastre A, Haller O, Kochs G. 2007. Replication fitness determines high virulence of influenza A virus in mice carrying functional Mx1 resistance gene. Proc Natl Acad Sci USA 104:6806–6811.
Ilyushina NA, Khalenkov AM, Seiler JP, Forrest HL, Bovin NV, Marjuki H, Barman S, Webster RG, Webby RJ. 2010. Adaptation of pandemic H1N1 influenza viruses in mice. J Virol 84:8607–8616.
Yoshii K, Okamoto N, Nakao R, Klaus Hofstetter R, Yabu T, Masumoto H, Someya A, Kariwa H, Maeda A. 2015. Isolation of the Thogoto virus from a Haemaphysalis longicornis in Kyoto City, Japan. J Gen Virol 96:2099–2103.
Hagmaier K, Jennings S, Buse J, Weber F, Kochs G. 2003. Novel gene product of Thogoto virus segment 6 codes for an interferon antagonist. J Virol 77:2747–2752.
Clay WC, Fuller FJ. 1992. Nucleotide sequence of the tick-borne orthomyxo-like Dhori/India/1313/61 virus membrane protein gene. J Gen Virol 73:2609–2616.
Kuno G, Chang GJ, Tsuchiya KR, Miller BR. 2001. Phylogeny of Thogoto virus. Virus Genes 23:211–214.
Davies CR, Jones LD, Green BM, Nuttall PA. 1987. In vivo reassortment of Thogoto virus (a tick-borne influenza-like virus) following oral infection of Rhipicephalus appendiculatus ticks. J Gen Virol 68:2331–2338.
Peng R, Zhang S, Cui Y, Shi Y, Gao GF, Qi J. 2017. Structures of human-infecting Thogotovirus fusogens support a common ancestor with insect baculovirus. Proc Natl Acad Sci USA 114:E8905–E8912.
Weber F, Haller O, Kochs G. 1996. Nucleoprotein viral RNA and mRNA of Thogoto virus: a novel “cap-stealing” mechanism in tick-borne orthomyxoviruses? J Virol 70:8361–8367.
Kochs G, Weber F, Gruber S, Delvendahl A, Leitz C, Haller O. 2000. Thogoto virus matrix protein is encoded by a spliced mRNA. J Virol 74:10785–10789.
Calisher CH, Karabatsos N, Filipe AR. 1987. Antigenic uniformity of topotype strains of Thogoto virus from Africa, Europe, and Asia. Am J Trop Med Hyg 37:670–673.
Nuttall PA, Morse MA, Jones LD, Portela A. 1995. Adaptation of members of the Orthomyxoviridae family to transmission by ticks, p 416–425. In Gibbs AJ, Calisher CH, Garcia-Arenal F (ed), Molecular basis of virus evolution. Cambridge University Press, Cambridge, England.
Briese T, Chowdhary R, Travassos da Rosa A, Hutchison SK, Popov V, Street C, Tesh RB, Lipkin WI. 2014. Upolu virus and Aransas Bay virus, two presumptive bunyaviruses, are novel members of the family Orthomyxoviridae. J Virol 88:5298–5309.
Choppin PW, Murphy JS, Tamm I. 1960. Studies of two kinds of virus particles which comprise influenza A2 virus strains. III. Morphological characteristics: independence to morphological and functional traits. J Exp Med 112:945–952.
Kilbourne ED, Murphy JS. 1960. Genetic studies of influenza viruses. I. Viral morphology and growth capacity as exchangeable genetic traits. Rapid in ovo adaptation of early passage Asian strain isolates by combination with PR8. J Exp Med 111:387–406.
Dadonaite B, Vijayakrishnan S, Fodor E, Bhella D, Hutchinson EC. 2016. Filamentous influenza viruses. J Gen Virol 97:1755–1764.
Komar N, Hamby N, Palamar MB, Staples JE, Williams C. 2020. Indirect Evidence of Bourbon virus (Thogotovirus, Orthomyxoviridae) infection in North Carolina. N C Med J 81:214–215.
Lledo L, Gimenez-Pardo C, Gegundez MI. 2020. Epidemiological study of Thogoto and Dhori virus infection in people bitten by ticks, and in sheep, in an area of northern Spain. Int J Environ Res Public Health 17.
Filipe AR, Peleteiro MC, de Andrade HR. 1990. Dhori virus induced lesions in mice. Acta Virol 34:578–581.
Dessens JT, Nuttall PA. 1998. Mx1-based resistance to thogoto virus in A2G mice is bypassed in tick-mediated virus delivery. J Virol 72:8362–8364.
Talactac MR, Yoshii K, Hernandez EP, Kusakisako K, Galay RL, Fujisaki K, Mochizuki M, Tanaka T. 2018. Vector competence of Haemaphysalis longicornis ticks for a Japanese isolate of the Thogoto virus. Sci Rep 8:9300.
Godsey MS, Rose D, Burkhalter KL, Breuner N, Bosco-Lauth AM, Kosoy OI, Savage HM. 2021. Experimental Infection of Amblyomma americanum (Acari: Ixodidae) With Bourbon Virus (Orthomyxoviridae: Thogotovirus). J Med Entomol 58:873–879.
Albanese M, Bruno-Smiraglia C, Di di Cuonzo G, Lavagnino A, Srihongse S. 1972. Isolation of Thogoto virus from Rhipicephalus bursa ticks in western Sicily. Acta Virol 16:267.
Eiden S, Dijkman R, Zell R, Fuchs J, Kochs G. 2020. Using a mouse-adapted A/HK/01/68 influenza virus to analyse the impact of NS1 evolution in codons 196 and 231 on viral replication and virulence. J Gen Virol 101:587–598.
Nuttall PA, Jones LD, Labuda M, Kaufman WR. 1994. Adaptations of arboviruses to ticks. J Med Entomol 31:1–9.
Kochs G, Bauer S, Vogt C, Frenz T, Tschopp J, Kalinke U, Waibler Z. 2010. Thogoto virus infection induces sustained type I interferon responses that depend on RIG-I-like helicase signaling of conventional dendritic cells. J Virol 84:12344–12350.
Srihongse S, Albanese M, Casals J. 1974. Characterization of Thogoto virus isolated from ticks (Rhipicephalus bursa) in Western Sicily, Italy. Am J Trop Med Hyg 23:1161–1164.
Filipe AR, Calisher CH. 1984. Isolation of Thogoto virus from ticks in Portugal. Acta Virol 28:152–155.
Filipe AR, Casals J. 1979. Isolation of Dhori virus from Hyalomma marginatum ticks in Portugal. Intervirology 11:124–127.
Staeheli P, Dreiding P, Haller O, Lindenmann J. 1985. Polyclonal and monoclonal antibodies to the interferon-inducible protein Mx of influenza virus-resistant mice. J Biol Chem 260:1821–1825.
Kolesnikova L, Heck S, Matrosovich T, Klenk HD, Becker S, Matrosovich M. 2013. Influenza virus budding from the tips of cellular microvilli in differentiated human airway epithelial cells. J Gen Virol 94:971–976.
Niwa H, Yamamura K, Miyazaki J. 1991. Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108:193–199.
Xie Y, Wu G, Tang J, Luo R, Patterson J, Liu S, Huang W, He G, Gu S, Li S, Zhou X, Lam TW, Li Y, Xu X, Wong GK, Wang J. 2014. SOAPdenovo-Trans: de novo transcriptome assembly with short RNA-Seq reads. Bioinformatics 30:1660–1666.
Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q, Chen Z, Mauceli E, Hacohen N, Gnirke A, Rhind N, di Palma F, Birren BW, Nusbaum C, Lindblad-Toh K, Friedman N, Regev A. 2011. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 29:644–652.
Bushmanova E, Antipov D, Lapidus A, Prjibelski AD. 2019. rnaSPAdes: a de novo transcriptome assembler and its application to RNA-Seq data. Gigascience 8.
Fu L, Niu B, Zhu Z, Wu S, Li W. 2012. CD-HIT: accelerated for clustering the next-generation sequencing data. Bioinformatics 28:3150–3152.
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. Basic local alignment search tool. J Mol Biol 215:403–410.
Gurevich A, Saveliev V, Vyahhi N, Tesler G. 2013. QUAST: quality assessment tool for genome assemblies. Bioinformatics 29:1072–1075.
Kim D, Langmead B, Salzberg SL. 2015. HISAT: a fast spliced aligner with low memory requirements. Nat Methods 12:357–360.
Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, Genome Project Data Processing S. 2009. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25:2078–2079.
Wilm A, Aw PP, Bertrand D, Yeo GH, Ong SH, Wong CH, Khor CC, Petric R, Hibberd ML, Nagarajan N. 2012. LoFreq: a sequence-quality aware, ultra-sensitive variant caller for uncovering cell-population heterogeneity from high-throughput sequencing datasets. Nucleic Acids Res 40:11189–11201.
Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30:772–780.
Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O. 2010. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol 59:307–321.
Lefort V, Longueville JE, Gascuel O. 2017. SMS: smart model selection in PhyML. Mol Biol Evol 34:2422–2424.

Information & Contributors


Published In

cover image Journal of Virology
Journal of Virology
Volume 96Number 59 March 2022
eLocator: e01556-21
Editor: Anice C. Lowen, Emory University School of Medicine
PubMed: 35019718


Received: 7 September 2021
Accepted: 26 December 2021
Accepted manuscript posted online: 12 January 2022
Published online: 9 March 2022


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  1. Orthomyxovirus
  2. thogotoviruses
  3. Thogoto virus
  4. Dhori virus
  5. Bourbon virus
  6. arboviruses
  7. zoonosis
  8. arbovirus



Institute of Virology, Medical Center-University of Freiburg, Freiburg, Germany
Kevin Lamkiewicz
Faculty of Mathematics and Computer Science, Friedrich Schiller University, Jena, Germany
European Virus Bioinformatics Center, Friedrich Schiller University, Jena, Germany
Larissa Kolesnikova
Institute of Virology, Philipps University Marburg, Marburg, Germany
Martin Hölzer
Faculty of Mathematics and Computer Science, Friedrich Schiller University, Jena, Germany
European Virus Bioinformatics Center, Friedrich Schiller University, Jena, Germany
Present address: Martin Hölzer, MF1 Bioinformatics, Robert-Koch-Institute, Berlin, Germany.
Manja Marz
Faculty of Mathematics and Computer Science, Friedrich Schiller University, Jena, Germany
European Virus Bioinformatics Center, Friedrich Schiller University, Jena, Germany
Fritz Lippmann Institute, Leibniz Institute of Age Research, Jena, Germany
Institute of Virology, Medical Center-University of Freiburg, Freiburg, Germany
Faculty of Medicine, University of Freiburg, Freiburg, Germany


Anice C. Lowen
Emory University School of Medicine


The authors declare no conflict of interest.

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