A novel mosquito-borne reassortant orbivirus isolated from Xishuangbanna, China

Dear Editor,

The genus Orbivirus, within the family Reoviridae, includes 22 virus species (Kinget al., 2011). They are distributed globally, but are particularly prevalent in Europe, Asia, and Africa. In addition, they can be transmitted by ticks or other hematophagous insect vectors, including Culicoides, mosquitoes, and sandflies (Belaganahalliet al., 2015).

Orbiviruses contain double-stranded RNA as their genetic material, which includes 10 segments (S1–S10) of various lengths. Owing to this segmented structure, genetic reassortment occurs frequently among orbiviruses. Reassortment occurs when different segmented viruses of the same vector type infect a single host cell, and ge-nomic segments of the "parental" viruses are exchanged and repackaged during progeny formation (Simon-loriere and Holmes, 2011). Reassortment can result in novel viral genotypes and subsequent phenotypes, which can alter the mechanism of immune escape, the potential range of hosts or vectors, and the mechanism of virulence or pathogenicity (Mcdonald and Patton, 2011). In general, reassortment represents a critical mechanism in the evolu-tion of segmented viruses. Accordingly, to understand viral diversity, it is important to identify and characterize reassortant taxa.

To identify novel reassortant orbiviruses, specimens were collected during the active mosquito season (from June to September) in Xishuangbanna, Yunnan Province in southwestern China in 2007. A sample was virus-positive if it had a cytopathic effect in three successive cell passages (Zuoet al., 2014). Supernatants containing the virus were identified by reverse transcription-polymerase chain reaction (RT-PCR) with genus-or species-specific primers designed for potential mosquito-borne viruses (Leiet al., 2014). Virus-containing samples that could not be identified by RT-PCR were identified by high-throughput sequencing (HTS) (Patel and Jain, 2012). Complete nucleotide sequences for segments 1–10 (Seg1–10) of a virus, referred to as Banna orbivirus (BAOV), were determined. The sequences have been submitted to GenBank, with sequential accession numbers KX455487–KX455496. The full-length sequence genome map and the protein sequence of the 10 BAOV segments are shown in Figure 1A.

Figure 1.  (A) The full-length sequence genome map and protein sequence of 10 BAOV segments. (B) Similarity among BAOV, Fengkai orbivirus, and XZ0906. The red line represents TIBOV (XZ0906); the green line represents Fengkai orbivirus; the horizontal axis indicates the degree of sequence similarity; the vertical axis indicates the position of genomic S1–10 sequences.

Recently, several novel orbiviruses have been isolated. Among the most common orbiviruses, Tibet orbivirus (TIBOV) (XZ0906) was first isolated from a pool of Anopheles maculatus mosquitoes in Tibet, China in 2009. TIBOV (YN12246) was isolated from Culicoides spp. in Yunnan, China, and Fengkai orbivirus, which also belongs to TIBOV, was isolated from a pool of Culex fatigan mosquitoes in Guangdong Province, China in 2008. A comparison of gene and protein sequences of BAOV segments to those of Fengkai orbivirus and XZ0906 is summarized in Table 1. The complete ge-nomes of BAOV, Fengkai orbivirus, and XZ0906 were 19270, 19349, and 19235 bp, respectively, and had similar GC contents–42.4%, 42.7%, and 42.5%, respectively. Each genomic segment encodes a single major open reading frame. S1–10 encode seven structural proteins: RNA-dependent RNA polymerase (RdRp), outer capsid protein one (OC1), inner sub-core shell 'T2' protein, Cap, Tup, Hel, and OC2, and three non-structural proteins (NS), NS2, NS4, and NS3 (Mosset al., 1992).

To determine the phylogenetic relationships among BAOV and other viruses in the genus Orbivirus, multiple sequence alignments were generated using Mega v6.06 with the complete sequences of BAOV and 15 other orbiviruses (Tamuraet al., 2013) (Supplementary Table S1). Phylogenetic trees based on all 10 viral genome segments were constructed from the nucleotide sequence alignments by using the maximum likelihood method with the GTR+G+I model. The robustness of the tree was established by a bootstrap analysis with 1, 000 replicates. To determine similarity, Simplot 3.5 (http://www.med.jhu.edu/deptmed/sray/download/; provided by S. Ray) was used to generate similarity plots based on the Cluster alignment (Mosset al., 1992). To better understand the genetic relationships among BAOV and other orbiviruses, 10 phylogenetic trees were constructed based on S1–10 (Supplementary Figure 1A–1J). BAOV, Fengkai orbivirus, and XZ0906 were grouped into a single cluster, which was distinct from other orbiviruses. S3 from BAOV had a higher amino acid sequence homology with S3 from YN12246 (94%) than with S3 from Fengkai orbivirus (80%) or S3 from XZ0906 (80%). However, sequences for only two segments (S1 and S3) from YN12246 are available in the GenBank database. The remaining BAOV segments had high identities to those of Fengkai orbivirus and XZ0906. S2 and S6 from BAOV exhibited high sequence similarities with S2 and S6 from Fengkai orbivirus. S10 from BAOV exhibited high sequence similarity with S10 from XZ0906 (Supplementary Figure 1A–Supplementary Figure S1). The degrees of nucleotide and amino acid sequence homology between the 10 segments in BAOV and those of other orbiviruses were also examined using BLASTn and BLASTp, respectively (Supplementary Table S2). S2 and S6 from BAOV had higher nucleotide and amino acid sequence homology with S2 and S6 from Fengkai orbivirus (97% and 98%) than with S2 and S6 from Tibet orbivirus (70% and 71%). S10 from BAOV had higher nucleotide and amino acid sequence homology with S10 from XZ0906 (99%) than with S10 from Fengkai orbivirus (85%). Simplot and Bootscan analyses revealed the similarity among BAOV, Fengkai orbivirus, and XZ0906 genomic sequences in each region (Figure 1B). These results are consistent with those of the BLASTn analysis, as shown in Supplementary Table S2. S2 and S6 from BAOV were more similar to S2 and S6 from Fengkai orbivirus than to S2 and S6 from XZ0906. S10 from BAOV was more similar to S10 from XZ0906 than to S10 from Fengkai orbivirus.

Reassortment is a common event in the evolution of viruses. In this study, we report a novel mosquito-borne reassortant Orbivirus species, which we named Banna orbivirus (BAOV). The virus was isolated from a Culex tritaeniorhynchus pool collected in Xishuangbanna, Yunnan Province, China in 2007. Whole genome and phylogenetic analyses revealed that BAOV is a reassortant of several Tibet orbivirus strains. It has a double-stranded RNA genome of 19, 270 bp, containing ten segments (S1–S10) of various lengths. The 10 segments of BAOV had high nucleotide and amino acid sequence similarity with segments of either Fengkai orbivirus or Tibet orbivirus (TIBOV, XZ0906 strain). Phylogenetic analyses indicated that the novel BAOV is a reassortant derived from XZ0906 and Fengkai orbiviruses, and is a Tibet orbivirus. This is the first report of a novel genomic reassortment of Tibet orbivirus isolated in China; these findings contribute to our understanding of the diversity of orbiviruses.