Two putative novel serotypes of Tibet orbivirus isolated from Culicoides spp. in Yunnan, China

Twenty-two virus species are currently recognized by the International Committee on Taxonomy of Viruses (ICTV) as members of the genus Orbivirus (family Sedoreoviridae) [1]. Well-known Orbivirus, bluetongue virus (BTV), and epizootic hemorrhagic disease virus (EHDV) have caused some outbreaks of diseases in ruminants, such as cattle, sheep, and deer [2]. The African horse sickness virus (AHSV) recently caused a serious epidemic in horses in Thailand [3]. A virus isolated from mosquitoes in Tibet, China, in 2009 was identified as a novel orbivirus and called Tibet orbivirus (TIBOV) by Li et al. [4]. Eight TIBOV strains isolated from mosquitoes and Culicoides spp. were reported and seven of them were completely sequenced [4, 5, 6, 7, 8, 9]. On the other hand, no TIBOV has been isolated from mammals, but antibodies against TIBOV were detected in the serum samples of cattle, Asian buffalo, and goats [6]. No serum neutralization experiment was performed to identify the serotypes, but phylogenetic analysis suggested that these TIBOV genomes represent several putative serotypes [9].

Virions in the supernatants of strains infected BHK-21 cells were observed by TEM. Spherical viral particles without an envelope were noted (Fig. 1). The viral diameters of the strain YNV/KM-1 and YNV/17-14 were estimated to be 70.9 nm (95% CI, 68.2–72.2 nm; n = 10) and 74.7 nm (95% CI, 68.5–76.5 nm; n = 5), respectively, according to the photographs (Fig. 1).

Fig. 1
TEM images of the two viral strains. The virions of strains YNV/KM-1 and YNV/17-14 were observed by TEM (scale bar = 100 nm).
TEM, transmission electron microscopy.

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Table 1 lists the data of the novel strains, and Supplementary Table 3 provides more details. All the segments possessed an identical six-based untranslated sequence at the 5’ end (5-GUAAAA) and 3’ end (ACUUAC-3), respectively (Supplementary Table 3). Seg2 and Seg6, which encoded the outer capsid proteins VP2 and VP5, respectively, had the most variable sequences in TIBOV; their best base identities between strains were only approximately 50% and 66%, respectively (Table 1, Supplementary Table 4). In addition, the nucleotide sequences of VP3, VP7, NS2, and NS3 of strain YNV/KM-1 differed from those of the published strains (Supplementary Table 4).

Table 1
Major information of 1–10 segments of the TIBOV strains YNV/KM-1 and YNV/17-14

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The novel strains were confirmed as TIBOV from a phylogenetic tree of VP1-containing strains belonging to 21 orbiviruses (i.e., RNA-dependent RNA polymerase, RdRP) (Fig. 2A, Supplementary Tables 1 and 2). The phylogenetic tree of VP2 suggested that existing TIBOV strains were temporarily classified into six putative serotypes (Fig. 2B). Such classification was supported by the NJ tree of VP5 (Fig. 2C). The VP2 genetic distances between the putative serotypes were more than 0.9, while such values between the type 2 strains were less than 0.03 (Supplementary Table 5). In the NJ tree of VP7, strain YNV/KM-1 was distinct from the other TIBOV strains (Supplementary Fig. 1). In phylogenetic trees of NS1, VP3, VP4, and VP6, the strains were clustered, but the genetic distances between strains were very short (Supplementary Fig. 2). Strain YNV/KM-1 was close to YNV/17-14 in VP2 and VP5 (Fig. 2B and C) but was close to Japanese strain KSB-8/C/09 in NS2 and NS3 (Supplementary Fig. 2D and F).

Fig. 2
Phylogenetic trees of VP1, VP2, and VP5. The trees were constructed using the NJ algorithm following the alignments of complete CDS. The two novel strains reported in this study were marked by cycles. Bootstrap values less than 50% were omitted. (A) NJ tree of VP1. Forty-seven strains belonging to 21 Orbivirus species were used. (B) NJ tree of VP2. (C) NJ tree of VP5. Putative serotypes of TIBOV are labelled in (B) and (C).
NJ, Neighbor-Joining; CDS, complete coding sequences.

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Strains YNV/KM-1 and YNV/17-14 were confirmed as TIBOV by phylogenetic analysis, and the shapes and diameters of these virions were in accordance with the TIBOV reported previously [5].

The VP2 of BTV and its homogenous proteins (e.g., TIBOV VP2) from other orbiviruses were used to determine serotypes of orbiviruses [9, 12]. According to the classification by Suda et al. [9], the prototype of TIBOV (XZ0906) [4] was classified as serotype 1. The other four Chinese strains [6, 7, 8] were placed in serotype 2, while the two Japanese strains [9] were placed in serotypes 3 and 4, respectively. Therefore, YNV/KM-1 isolated from Culicoides spp. in 2019 and YNV/17-14 isolated from Culicoides jacobsoni in 2020 [10] were placed respectively in serotypes 5 and 6 in this study (Fig. 2B).

Generally, orbiviruses are clustered phylogenetically according to their arthropod hosts (mosquitoes, midges/sandflies, and ticks) [13]. On the other hand, there was no clear correlation between genetic clusters and host species (mosquito or Culicoides) among the TIBOV strains (Fig. 2, Supplementary Figs. 1 and 2). Hence, TIBOV had a wider profile of arthropod hosts than most other orbiviruses.

Supplementary Table 1

The data and GenBank access numbers of TIBOV strains used in this study

Supplementary Table 2

The information of the Orbivirus species used in this study

Supplementary Table 3

Additional genome information of the two novel TIBOV serotypes

Supplementary Table 4

Percent identities of genes between the novel TIBOV strains and the published TIBOV strains

Supplementary Table 5

VP2 genetic distances between TIBOV strains

Supplementary Fig. 1

Phylogenetic tree of VP7. NJ tree was constructed using VP7 CDS of 9 TIBOV strains, 3 BTV strains, and 3 EHDV strains. The two novel strains reported in this study were marked by cycles. Bootstrap values less than 50% were omitted.

Supplementary Fig. 2

Six TIBOV phylogenetic trees. The TIBOV NJ trees of VP3 (A), VP4 (B), NS1 (C), NS2 (D), VP6 (E), and NS3 (F) were constructed, respectively. Novel strains reported in this study were marked by cycles and highlighted by blue and red colors, respectively. Bootstrap values less than 50% were omitted.