Isolation of Tibet orbivirus from Culicoides and associated infections in livestock in Yunnan, China

Background Culicoides-borne orbiviruses, such as bluetongue virus (BTV) and African horse sickness virus (AHSV), are important pathogens that cause animal epidemic diseases leading to significant loss of domestic animals. This study was conducted to identify Culicoides-borne arboviruses and to investigate the associated infections in local livestock in Yunnan, China. Methods Culicoides were collected overnight in Mangshi City using light traps during August 2013. A virus was isolated from the collected Culicoides and grown using baby hamster kidney (BHK-21), Vero, Madin-Darby bovine kidney (MDBK) and Aedes albopictus (C6/36) cells. Preliminary identification of the virus was performed by polyacrylamide gel (PAGE) analysis. A full-length cDNA copy of the genome was amplified and sequenced. Serological investigations were conducted in local cattle, buffalo and goat using plaque-reduction neutralization tests. Results We isolated a viral strain (DH13C120) that caused cytopathogenic effects in BHK-21, Vero, MDBK and C6/36 cells. Suckling mice inoculated intracerebrally with DH13C120 showed signs of fatal neurovirulence. PAGE analysis indicated a genome consisting of 10 segments of double-stranded RNA that demonstrated a 3–3–3–1 pattern, similar to the migrating bands of Tibet orbivirus (TIBOV). Phylogenetic analysis of the viral RNA-dependent RNA polymerase (Pol), sub-core-shell (T2, and outer core (T13) proteins revealed that DH13C120 clustered with TIBOV, and the amino acid sequences of DH13C120 virus shared more than 98% identity with TIBOV XZ0906. However, outer capsid protein VP2 and outer capsid protein VP5 shared only 43.1 and 79.3% identity, respectively, indicating that the DH13C120 virus belongs to TIBOV, and it may represent different serotypes with XZ0906. A serosurvey revealed the presence of neutralizing antibodies with 90% plaque-reduction neutralization against TIBOV DH13C120 in local cattle (44%), buffalo (20%), and goat (4%). Four-fold or higher levels of TIBOV-2-neutralizing antibody titers were detected between the convalescent and acute phases of infection in local livestock. Conclusions A new strain of TIBOV was isolated from Culicoides. This study provides the first evidence of TIBOV infection in livestock in Yunnan, China, and suggests that TIBOV could be a potential pathogen in livestock.


Background
The viral genus Orbivirus within the family Reoviridae has a genome consisting of 10 segments of doublestranded (ds) RNA. It contains 22 recognized virus species and 10 unclassified viruses [1]. BTV and AHSV negatively impact animal husbandry and veterinary health. In addition, Equine encephalosis virus (EEV), Epizootic hemorrhagic disease virus (EHDV), Palyam virus (PALV) and Peruvian horse sickness virus (PHSV) are important pathogens causing epidemic diseases in animals. In these orbiviruses, although PHSV is mosquito-borne, most of these viruses are carried by Culicoides [2,3]. Therefore, Culicoides is considered a potent vector of important animal arbovirus diseases, which cause major economic losses in domestic animals [2,[4][5][6].
The TIBOV was first isolated from Anopheles maculatus mosquitoes collected in 2009 from Motuo County, Tibet, China [7]. Subsequently, TIBOV was isolated from Culex fatigan mosquitoes and Culicoides specimens collected from Guangdong and Yunnan, respectively [8].
Here, we report a new TIBOV named DH13C120, isolated from Culicoides. Sequence analysis of its entire genome and evidence of DH13C120 infection in local livestock were obtained from samples collected in the southwest border area of Yunnan Province in 2013.

Collection of Culicoides specimens
Specimens were collected during August 2013 from the suburb of Manbing Town in Mangshi City, Dehong Prefecture, Yunnan Province. Samples were collected overnight using light traps (12 V, 300 mA; Wuhan Lucky Star Environmental Protection Tech. Co., Ltd., Hubei, China). Captured Culicoides, killed by freezing at −20°C for 20 min and identified by using morphologic characteristics, were pooled into groups of approximately 100 insects and stored in liquid nitrogen until viral isolation.

Isolation of viruses
Culicoides pools, which included midges that were engorged or not engorged, were removed from liquid nitrogen and immediately homogenized and centrifuged as reported previously [9]. The supernatants were used to inoculate monolayers of baby hamster kidney (BHK-21) cells, Vero cells (from the China National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China), and Madin-Darby bovine kidney (MDBK) cells (From Yunnan biological pharmaceutical factory, Kunming, China) at 37°C; Aedes albopictus (C6/36) cells (from the National Institute for Viral Disease Control and Prevention) were inoculated at 28°C. The cells were observed daily (days 1-7 post-inoculation) for cytopathic effects (CPEs).

Assay of neurovirulence in suckling mice
Aliquots of 30 μL cell culture material (100 plaqueforming units, PFU/mL) were inoculated intracerebrally into 1-day-old suckling mice, and 30 μL of Dulbecco's modified Eagle's medium (DMEM) was similarly applied in the control group, with eight suckling mice in each group. Symptoms (or the time of death) in the virustreated neonatal mice were recorded each day for 14 days post-inoculation.

Polyacrylamide gel electrophoresis (PAGE)
To reveal the number of dsRNA segment and the genome pattern, viral RNA was extracted using RNAiso Plus (TaKaRa, Dalian, China) according to the manufacturer's protocols, and then separated on RNA-PAGE as described previously [9][10][11].

Full genome sequencing
The virus genome was amplified by full-length amplification of cDNA (FLAC), as described previously [9,12,13]. Briefly, viruses were propagated in BHK-21 cells, and total RNA was extracted using RNAiso Plus (TaKaRa) according to the manufacturer's protocols. Single stranded (ss) RNAs were removed by precipitation with 2 M LiCl (Sigma-Aldrich, St Louis, MO, USA), and dsRNAs were precipitated by the addition of 2.5 volumes of isopropanol and 1 volume of 7.5 M ammonium acetate. The dsRNAs were subjected to 1% agarose gel electrophoresis (AGE) (7 V/cm, for 1 h) in TAE buffer (40 mMTris-acetate, 1 mM EDTA, pH 8.0) and purified from the agarose gel using MinElute gel extraction kits (Qiagen, Hilden, Germany).
An anchor primer, PC3-T7 loop (synthesized by Sangon, Shanghai, China), similar to that described by Maan et al. [13], was ligated to the viral dsRNA. Ligation reactions were carried out in a total volume of 30 μL, as described previously [9,12]. The ligated dsRNA was purified using MicroElute RNA Cleanup kits (Omega Bio-Tek Inc., Norcross, GA, USA), denatured with dimethyl sulfoxide (DMSO) for a final concentration of 15% (v/v), heated in boiling water for 2 min, and snapfrozen in an ice-water slurry. The full-length complementary DNAs (cDNAs) of 10 viral dsRNA segments were synthesized in a total volume of 50 μL using a PrimeScript II high-fidelity reverse transcription polymerase chain reaction (RT-PCR) kit (TaKaRa) according to the manufacturer's recommendations. Amplification of the cDNA was carried out with primer PC2 in a total volume of 50 μL, as described previously [9,12]. The PCR products were viewed after separation on 1% agarose gels in 1× TAE buffer containing a nucleic acid dye, and purified using TaKaRa DNA fragment purification kits (ver. 2.0; TaKaRa). The gel-purified fragments were cloned into the pMD19-T vector (TaKaRa), and introduced into chemically competent Escherichia coli DHα5 cells (TaKaRa). Single colonies were cultured and sequenced by the Shenzhen Huada Genome Institute, Shanghai, China. We performed 2 sequencings from the same RNA extraction and these sequences (that include the differences) were cover 2.5 times.

Serological investigations of antibodies against TIBOV isolate DH13C120 Sample collection
Buffalo serum samples were collected in Mangshi City, Dehong Prefecture, Yunnan Province in 2009. The separated serum samples were stored at −20°C until tested. In 2013, three collection sites were chosen, in Mangshi City, Jiangcheng County of Pu'er City, and Shizong County of Qiujing City. Sentinel animals were picked from and existing population if they were first shown to serologically naïve on the May. Ten cattle and five goats were chosen as sentinel animals for each collection site, and serum samples were collected monthly and stored at −20°C until tested.

Plaque-reduction neutralization tests
Serum samples were tested for neutralizing antibodies against TIBOV isolate DH13C120 by 90% plaquereduction neutralization tests (PRNT 90) using standard methods. Samples were tested with serial two-fold dilutions from 1:10 to 1:1,280. Diluted samples were mixed with equal volumes of culture medium 1× MEM (pH = 7.4) containing DH13C120 (100 PFU) and incubated at 37°C in a 5% CO 2 for 1 h. Six-well plates of confluent BHK-21 cells were inoculated with the serum-virus mixtures and incubated at 37°C in a 5% CO 2 incubator for 1 h. Plates were overlaid with 3 mL of the same medium containing 0.8% agarose, and again with 3 mL of a second overlay medium containing neutral red vital stain (Sigma-Aldrich). The neutralizing antibody titer was identified as the highest serum dilution that reduced the number of virus plaques in the test by ≥ 90%. The samples were considered to be positive when titers were ≥ 20. A ratio of TIBOV PRNT 90 titer between two samples of a same animal ≥ 4 was considered to confirm the presence of a TIBOV infection [14].

Assay of neurovirulence in suckling mice
Suckling mice inoculated intracerebrally with DH13C120 showed signs of tremor and stiff necks at 48 h, and died within 72 h.

Genome organization and characteristics of DH13C120 virus
A full length cDNA copy of the DH13C120 virus genome was obtained by FLAC and sequencing, with the length of segments 1-10 ranging between 3,740 nucleotides (nt) (Seg-1) to 788 nt (Seg-10) ( Table 1). The sequences of the 10 segments were deposited in the NCBI GenBank with the accession numbers KU754026-KU754035, as indicated in Table 1. Compared with the TIBOV XZ0906 strain, the DH13C120 virus has 12 nt insertions in the open reading frame and 1 nt insertion in the 3′ untranslated region (UTR) in Seg-2, and 5 nt insertions in the 5′ UTR and 1 nt insertion in the 3′ UTR in Seg-6. Conserved nt sequences of 6 nt were observed at the ends of the 5′ and 3′ UTRs (5′-GTAAAA A / T and C / A ACTTAC-3′), and 4 nt in the terminal sequences were reverse complementary. The 5′ and 3′ UTRs of DH13C120 comprised 3.80% of the total genome; the G + C content of DH13C120 was 42.59%, similar to Culicoides-borne Orbivirus species; and an Arg-Gly-Asp (RGD) motif [15][16][17] was observed at position 168-170 in the viral outer core protein T13 (VP7) of DH13C120.

Phylogenetics
Phylogenetic analysis of DH13C120 virus compared with TIBOV (XZ0906, Fengkai and YN12246) The sub-core-shell (T2) (VP3) protein of DH13C120 is encoded by segment 3. Phylogenetic analvysis showed that DH13C120 has a close relationship with TIBOV XZ0906, YN12246 (only partial nt sequence of the T2 protein) and Fengkai virus. Amino acid (aa) sequence analysis of the T2 protein revealed 96.3-99.9% sequence similarity between DH13C120 and the TIBOV species (XZ0906, YN1226 and Fengkai virus), which was above the threshold of 91% used for species determination by Attoui et al. [18]. This similarity confirmed that DH13C120 is a strain of TIBOV.
Sequence analysis of the outer capsid VP2 (encoded by segment 2) protein of TIBOV isolated from China (DH13C120, XZ0906 and the Fengkai virus) revealed that the DH13C120 virus isolated from Culicoides in Yunnan and the Fengkai virus isolated from mosquitoes in Guangdong (partial sequence) had sequence similarities of 96.8%/97.1% nt/aa, whereas XZ0906 isolated from mosquitoes in Tibet displayed similarities of 53.9%/43.1% nt/aa ( Table 2).

Phylogenetic analysis of DH13C120 compared with other Orbivirus species
To clarify the taxonomic position of DH13C120 with other Orbivirus species, we conducted phylogenetic analyses of the predicted aa sequences of RNA-dependent RNA polymerase (Pol) (VP1), T2 (VP3), and T13 (VP7) (Fig. 2). The phylogenetic trees revealed that the phylogeny was often correlated with a particular arthropod vector and could be classified into two groups: Orbivirus species transmitted by Culicoides (including BTV, AHSV, EHDV, Wallal virus, Eubenangee virus, Warrego virus, and PALV), and those transmitted by ticks (such as Great island virus, GIV) or mosquitoes (such as Corriparta virus, Wongorr virus, PHSV, and Yunnan Orbivirus (YUOV). Phylogenetic analysis showed that DH13C120 has a close relationship and similar coding pattern to other Culicoides-borne Orbivirus species. The Pol, T2, and T13 proteins of DH13C120 share 58-71.8%, 57.6-77.8%, and 40.6-68.2% aa sequence identity with the Culicoidesborne Orbivirus species, respectively, and 47%, 38%, and 23.6% aa identity with mosquito-borne YUOV, respectively.

Prevalence of DH13C120 virus antibodies in local livestock
A total of 135 serum samples were collected from cattle, buffaloes and goats in Mangshi City, Jiangcheng and Shizong County of Yunnan. Neutralizing antibodies against DH13C120 virus were detected by PRNT 90 assays. Most of the positive samples were collected in Jiangcheng County, with the highest seroprevalence (90%) in cows. Additionally, positive sera were collected in Mangshi City (27%) and Shizong County (10%), but positivity rates in both areas were much lower than those in Jiangcheng County (Table 3).
To understand the dynamics of infection by this orbivirus, five goats and ten cows were chosen as target animals at each of the three monitoring sites. Seroconversion of neutralization antibodies were detected in the target cattle (Table 4), with 9/10 in Jiangcheng County, 3/10 in Mangshi City, and only 1/10 in Shizong County. However, no seroconversion was detected in the target sheep. The period of seroconversion was from June to September (Table 4). This result illustrated that cattle

Discussion
The parameters recognized by the International Committee on Taxonomy of Viruses for the polythetic definition of individual Orbivirus species include: the reassortment of genome segments; genome segment migration patterns shown by 1% agarose gel electrophoresis; conserved terminal nt sequences; serological cross-reactions; comparison of homologous genome segments or proteins by sequence analysis or cross-hybridization; host and vector ranges; and the nature of clinical signs induced by infection [19,20]. In this study, some features of DH13C120 resembled the   Fengkaivirus Seg-2: analyse only using 2779 nt of 5′-end sequence XZ0906 strain of TIBOV isolated from mosquitoes in Tibet [7] in terms of CPE, genome segment migration patterns, and conserved terminal nt sequences of the 5′ and 3′ UTRs, suggesting that DH13C120 belongs to the TIBOV species of the Orbivirus genus. Conserved sequence from T2, Pol or T13 protein genes had previously been used for phylogenetic comparisons and taxonomic classification of Orbivirus [19,[21][22][23]. Among Orbivirus species, these proteins shared > 83%, > 73% and > 73% aa identity, respectively [18,19,22,24]. DH13C120 and TIBOV shared   97% aa identity in the above-mentioned conserved proteins. In contrast, they showed lower levels of aa identity (<77.8%) with T2, Pol and T13 protein in other Orbivirus species. These data confirmed that DH13C120 is closely related to TIBOV. However, DH13C120 and TIBOV shared only 43.1% aa identity in the outer capsid protein VP2, and 79.3% aa identity in the outer capsid protein VP5. The outer capsid protein VP2 of the Orbivirus genus is an outer capsid protein; outer capsid protein VP2 is highly variable and determines viral serotype [21,25,26]. A pairwise alignment of outer capsid protein VP2s from different serotypes of BTV, EHDV and AHSV indicated variations in the ranges of 28.3-64%, 31.1-76.7%, and 46-52% aa, respectively [26][27][28]. Based on these data, although the DH13C120 virus and TIBOV belong to the same species, they may represent different serotypes.
In terms of vectors, the genus Orbivirus contains Culicoides-borne, mosquito-borne and tick-borne viruses [21]. Although TIBOV has been isolated from both Culex and Culicoides, phylogenetic analysis of T2, polymerase proteins and T13 proteins reveals that TIBOV is more closely related with the Culicoidesborne Orbivirus. Furthermore, Pol, T2, T13 proteins of DH13C120 share higher aa sequence identities with the Culicoides-borne Orbivirus than the mosquito-or tickborne Orbivirus species, suggesting that Culicoides may be biological vectors of DH13C120 virus.
Previous studies have shown that the noncoding region (NCR) comprises 5.03-5.695% of Orbivirus genomes in the mosquito-borne group, 4.47-4.9% in the tick-borne group, and 3.5-4.1% in the Culicoides-borne group [22]. Analysis of the DH13C120 genome showed that the NCR comprises 3.80% of the genome, within the range of the Culicoides-borne group. In addition, the G + C content was 42.59% in DH13C120, which was within the range for Culicoides-borne Orbivirus species (from 39.89% in Chuzan virus to 45.89% in EEV) [19,22], but more than that in the mosquito-borne species (from 36.72% in PHSV to 41.55% in YUOV) and less than that in the tick-borne species (from 57.29% in GIV to 51.93% in St Croix River virus). These data suggested that DH13C120 virus is more or most related to viruses vectored by Culicoides spp.
In 1995, 17 strains of Orbivirus were first isolated from ticks, birds, rats, and livestock in the Yadong and Cuona areas of Tibet, with typical CPE in BHK-21 cells and lethal illness in suckling mice. Antibodies against the Orbivirus isolates were detected in local residents with a seroprevalence of 26%, suggesting that the new Orbivirus isolates could potentially be a novel animal pathogen in China [29,30]. Recently, a new species, TIBOV, which could also generate CPE in BHK-21 cells, was isolated from mosquitoes and Culicoides in Yunnan, Guangdong Province and Tibet [7,8]. In the present study, a new serotype of TIBOV was isolated from Culicoides in Yunnan, and high levels of neutralization antibodies were detected in cattle serum from three areas in Yunnan Province. Moreover, neutralization antibodies against DH13C120 were detected in target animals at all three monitoring sites, with the highest positivity rate being 92%. In addition, neutralization antibody titers that increased more than four-fold were detected in target cattle, and sheep inoculated with DH13C120 experienced fever (data not shown), suggesting that DH13C120 can infect livestock and is a potential pathogen leading to livestock disease.
In this study, DH13C120 was isolated from Culicoides collected in cattle corrals, and neutralization antibodies were detected in animals from the same areas. Seroconversion in animals occurred from June to September, which matched the seasonal peak of Culicoides numbers. These results suggest that Culicoides is the main vector of this new serotype of TIBOV, and cattle form the natural host reservoir. The cattle in these localities have high economic value, and most cattle are bred together in herds numbering over 100. The density of Culicoides was high in these cattle corrals, facilitating circulation of the new serotype. To understand the impact of this new TIBOV serotype, further studies are required to characterize its dissemination and pathogenicity.

Conclusions
This investigation represents the isolation of TIBOV from Culicoides, and provides the first evidence of TIBOV DH13C120 strain infections in local livestock bred in the southwest border area of Yunnan, China. The results suggest that TIBOV isolated from Culicoides in yunnan, might be a pathogen causing livestock disease.