Genome and evolution of Tibet orbivirus, TIBOV (genus Orbivirus, family Reoviridae).

Tibet orbivirus (TIBOV) was first isolated from Anopheles maculatus mosquitoes in Xizang, China, in 2009. In recent years, more TIBOV strains have been isolated in several provinces across China, Japan, East Asia, and Nepal, South Asia. Furthermore, TIBOVs have also been isolated from Culex mosquitoes, and several midge species. Additionally, TIBOV neutralizing antibodies have been detected in serum specimens from several mammals, including cattle, sheep, and pigs. All of the evidence suggests that the geographical distribution of TIBOVs has significantly expanded in recent years, with an increased number of vector species involved in its transmission. Moreover, the virus demonstrated infectivity towards a variety of animals. Although TIBOV is considered an emerging orbivirus, detailed reports on its genome and molecular evolution are currently lacking. Thus, this study performed the whole-genome nucleotide sequencing of three TIBOV isolates from mosquitoes and midges collected in China in 2009, 2011, and 2019. Furthermore, the genome and molecular genetic evolution of TIBOVs isolated from different countries, periods, and hosts (mosquitoes, midges, and cattle) was systematically analyzed. The results revealed no molecular specificity among TIBOVs isolated from different countries, periods, and vectors. Meanwhile, the time-scaled phylogenetic analysis demonstrated that the most recent common ancestor (TMRCA) of TIBOV appeared approximately 797 years ago (95% HPD: 16-2347) and subsequently differentiated at least three times, resulting in three distinct genotypes. The evolutionary rate of TIBOVs was about 2.12 × 10-3 nucleotide substitutions per site per year (s/s/y) (95% HPD: 3.07 × 10-5, 9.63 × 10-3), which is similar to that of the bluetongue virus (BTV), also in the Orbivirus genus. Structural analyses of the viral proteins revealed that the three-dimensional structures of the outer capsid proteins of TIBOV and BTV were similar. These results suggest that TIBOV is a newly discovered and rapidly evolving virus transmitted by various blood-sucking insects. Given the potential public health burden of this virus and its high infectious rate in a wide range of animals, it is significant to strengthen research on the genetic variation of TIBOVs in blood-feeding insects and mammals in the natural environment and the infection status in animals.

Evaluation of vector susceptibility in Aedes aegypti and Culex pipiens pallens to Tibet orbivirus.

Mosquito-borne viruses cause various infectious diseases in humans and animals. Tibet orbivirus (TIBOV), a newly identified arbovirus, efficiently replicates in different types of vertebrate and mosquito cells, with its neutralizing antibodies detected in cattle and goats. However, despite being isolated from Culicoides midges, Anopheles, and Culex mosquitoes, there has been a notable absence of systematic studies on its vector competence. Thus, in this study, Aedes aegypti and Culex pipiens pallens were reared in the laboratory to measure vector susceptibility through blood-feeding infection. Furthermore, RNA sequencing was used to examine the overall alterations in the Ae. aegypti transcriptome following TIBOV infection. The results revealed that Ae. aegypti exhibited a high susceptibility to TIBOV compared to Cx. p. pallens. Effective replication of the virus in Ae. aegypti midguts occurred when the blood-feeding titer of TIBOV exceeded 105 plaque-forming units mL-1. Nevertheless, only a few TIBOV RNA-positive samples were detected in the saliva of Ae. aegypti and Cx. p. pallens, suggesting that these mosquito species may not be the primary vectors for TIBOV. Moreover, at 2 dpi of TIBOV, numerous antimicrobial peptides downstream of the Toll and Imd signaling pathways were significantly downregulated in Ae. aegypti, indicating that TIBOV suppressed mosquitos' defense to survive in the vector at an early stage. Subsequently, the stress-activated protein kinase JNK, a crucial component of the MAPK signaling pathway, exhibited significant upregulation. Certain genes were also enriched in the MAPK signaling pathway in TIBOV-infected Ae. aegypti at 7 dpi.IMPORTANCETibet orbivirus (TIBOV) is an understudied arbovirus of the genus Orbivirus. Our study is the first-ever attempt to assess the vector susceptibility of this virus in two important mosquito vectors, Aedes aegypti and Culex pipiens pallens. Additionally, we present transcriptome data detailing the interaction between TIBOV and the immune system of Ae. aegypti, which expands the knowledge about orbivirus infection and its interaction with mosquitoes.

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

Tibet orbivirus (TIBOV) was identified as a novel orbivirus in 2014. Antibodies against TIBOV were detected in cattle, Asian buffalo, and goats, while all the sequenced TIBOV strains were isolated from mosquitos and Culicoides. The known TIBOV strains have been classified into four putative serotypes. In this study, two TIBOV strains isolated from Culicoides spp. in Shizong County of Yunnan Province, China, were fully sequenced. The phylogenetic analysis of outer capsid protein 2 (VP2) indicated that these two viral strains belong to two novel putative serotypes of TIBOV. The updated putative serotypes may help in an investigation of the distribution and virulence of TIBOV.

Isolation and characterization of two novel serotypes of Tibet orbivirus from Culicoides and sentinel cattle in Yunnan Province of China.

Tibet orbivirus (TIBOV), a new candidate of Orbivirus genus, was initially isolated from mosquitoes in Tibet in 2009 and subsequently from both Culicoides and mosquitoes in several provinces of China and Japan. Little is known about the origin, genetic diversity, dissemination and pathogenicity of TIBOV, although its potential threat to animal health has been acknowledged. In this study, two viruses, V290/YNSZ and V298/YNJH, were isolated from the Culicoides and sentinel cattle in Yunnan Province. Their genome sequences, cell tropism in mammalian and insect cell lines along with pathogenicity in suckling mice were determined. Genome phylogenetic analyses confirmed their classification as TIBOV species; however, OC1 proteins of the V290/YNSZ and V298/YNJH shared maximum sequence identities of 31.5% and 33.9% with other recognized TIBOV serotypes (TIBOV-1 to TIBOV-4) and formed two monophyletic branches in phylogenetic tree, indicating they represented two novel TIBOV serotypes which were tentatively designated as TIBOV-5 and TIBOV-6. The viruses replicated robustly in BHK, Vero and C6/36 cells and triggered overt clinical symptoms in suckling mice after intracerebral inoculation, causing mortality of 100% and 25%. Cross-sectional epidemiology analysis revealed silent circulation of TIBOV in Yunnan Province with overall prevalence of 16.4% (18/110) in cattle, 10.8% (13/120) in goats and 5.5% (6/110) in swine. The prevalence patterns of four investigated TIBOV serotypes (TIBOV-1, -2, -5 and 6) differed from each one another, with their positive rates ranging from 8.2% (9/110) for TIBOV-2 in cattle to 0.9% (1/110) for TIBOV-1 and TIBOV-5 in cattle and swine. Our findings provided new insights for diversity, pathogenicity and epidemiology of TIBOV and formed a basis for future studies addressing the geographical distribution and the zoonotic potential of TIBOV.

Characterization of a novel reassortment Tibet orbivirus isolated from Culicoides spp. in Yunnan, PR China.

Orbiviruses are arboviruses with 10 double-stranded linear RNA segments, and some have been identified as pathogens of dramatic epizootics in both wild and domestic ruminants. Tibet orbivirus (TIBOV) is a new orbivirus isolated from hematophagous insects in recent decades, and, currently, most of the strains have been isolated from insects in PR China, except for two from Japan. In this study, we isolated a novel reassortment TIBOV strain, YN15-283-01, from Culicoides spp. To identify and understand more characteristics of YN15-283-01, electrophoresis profiles of the viral genome, electron microscopic observations, plaque assays, growth curves in various cell lines, and bioinformatic analysis were conducted. The results indicated that YN15-283-01 replicated efficiently in mosquito cells, rodent cells and several primate cells. Furthermore, the maximum likelihood phylogenetic trees and simplot analysis of the 10 segments indicated that YN15-283-01 is a natural reassortment isolate that had emerged mainly from XZ0906 and SX-2017a.

Isolation of Tibet Orbivirus from Culicoides jacobsoni (Diptera, Ceratopogonidae) in China.

BACKGROUND: Tibet Orbivirus (TIBOV) is a recently discovered Orbivirus known to infect cattle, Asian buffalo and goats in south-western China. It was first isolated from mosquitoes and subsequently from biting midges (Culicoides spp.) in Yunnan, China, indicating that it is an arbovirus. Little is known of its potential to cause disease, but the economic importance of related viruses promoted an investigation of potential Culicoides spp. vectors of TIBOV. METHODS: Biting midges were collected approximately once per week between May and December 2020, at a cattle farm in Wulong village, Shizong County, Yunnan Province, China. Approximately 3000 specimens of nine species were subsequently used in attempts to isolate virus, and a further 2000 specimens of six species were tested for the presence of bluetongue virus (BTV) and TIBOV using a RT-qPCR test. RESULTS: Virus isolation attempts resulted in the isolation of three viruses. One isolate from a pool of Culicoides jacobsoni was identified as TIBOV, while the other two viruses from C. orientalis and C. tainanus remain unidentified but are not BTV or TIBOV. RT-qPCR analysis did not detect BTV in any specimens, but a single pool containing five specimens of C. jacobsoni and another containing five specimens of C. tainanus produced PCR quantification cycle (Cq) values of around 28 that may indicate infection with TIBOV. CONCLUSIONS: The isolation of TIBOV from C. jacobsoni satisfies one criterion required to prove its status as a vector of this virus. This isolation is supported by a low Cq value produced from a different pool of this species in the RT-qPCR test. The low Cq value obtained from a pool of C. tainanus suggests that this species may also be able to satisfy this criterion. Both of these species are widespread throughout Asia, with C. jacobsoni extending into the Pacific region, which raises the possibility that TIBOV may be more widespread than is currently known.

Isolation and Genome Phylogenetic Analysis of Arthropod-Borne Viruses, Including Akabane Virus, from Mosquitoes Collected in Hunan Province, China.

This study investigated the abundance of mosquitoes and circulation of mosquito-borne arboviruses from 16 villages in 8 cities of Hunan Province, China, in July-August of 2010 and in August of 2011. In total, 16,076 mosquitoes consisting of seven species from four genera were collected by ultraviolet-light trap. Culex quinquefasciatus was the most common species, accounting for 50.63% (8140/16,076) of the total. Anopheles sinensis (24.26%, 3900/16,076) made up the second most common species, followed by Culex tritaeniorhynchus (9.76%, 1569/16,076). The proportions of Culex pipiens pallens, Armigeres subalbatus, and Culex modestus were 6.7%, 5.2%, and 3.31%, respectively. Fourteen Aedes albopictus were detected. The mosquitoes were identified by morphologic characteristics and frozen in liquid nitrogen. The mosquitoes were pooled, triturated, and centrifuged. The clarified supernatant was used to inoculate monolayers of C6/36 and baby hamster kidney-21 cells. We obtained six virus isolates that caused cytopathic effects. Phylogenetic analysis revealed that two isolates were Akabane virus (AKAV, from A. sinensis and C. quinquefasciatus), two isolates were Japanese encephalitis virus (from C. pipiens pallens and C. quinquefasciatus), and two isolates were Tibet orbivirus (from C. quinquefasciatus and C. tritaeniorhynchus). This is the first report of AKAV isolated from A. sinensis and C. quinquefasciatus in nature in China. The detection of AKAV in these species confirms circulation of AKAV in Hunan province and suggests potential challenges to the prevention and control of arthropod-borne animal viruses in mainland China.

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.