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.

Diversity and seasonal abundance of Culicoides (Diptera, Ceratopogonidae) in Shizong County, Yunnan Province, China.

Culicoides (Diptera, Ceratopogonidae) are small biting midges, some of which are vectors for animal associated arboviruses such as bluetongue virus (BTV) and Akabane virus (AKAV). BTV and AKAV are both pathogenic for livestock, with BTV in particular posing a major threat to domestic ruminants. Ongoing problems with BTV in ruminants in Shizong County of Yunnan Province, China, promoted a year-long investigation of the Culicoides in Shizong to determine relative abundance and seasonality of midges and to attempt to identify species that might be acting as vectors in the area. Culicoides were collected by UV light trap for one night per week for most weeks between May 2020 and May 2021. More than 21,000 specimens consisting of at least 21 species belonging to six subgenera and one unplaced group, including 5 species previously associated with BTV and one associated with AKAV, were collected. Culicoides tainanus dominated most collections throughout the year although C. sumatrae was often the dominant species over summer. Most species were abundant between May and October. These results indicate that C. tainanus, C. jacobsoni and C. oxystoma are the major midge pests of livestock in Shizong and should be considered in any disease investigation.

Title: Diversité et abondance saisonnière des Culicoides (Diptera, Ceratopogonidae) dans le comté de Shizong, province du Yunnan, Chine. Abstract: Les culicoïdes (Diptera, Ceratopogonidae) sont de petits moucherons piqueurs, dont certains sont des vecteurs d'arbovirus associés aux animaux tels que le virus de la fièvre catarrhale du mouton (FCMV) et le virus Akabane (AKAV). Le FCMV et l'AKAV sont tous deux pathogènes pour le bétail, le FCMV en particulier constituant une menace majeure pour les ruminants domestiques. Les problèmes persistants de FCMV chez les ruminants dans le comté de Shizong de la province du Yunnan, en Chine, ont encouragé une enquête d'un an sur les Culicoides à Shizong pour déterminer l'abondance relative et la saisonnalité des culicoïdes et tenter d'identifier les espèces qui pourraient agir comme vecteurs dans la région. Les culicoïdes ont été collectés par piège à lumière UV une nuit par semaine pendant la plupart des semaines entre mai 2020 et mai 2021. Plus de 21 000 spécimens comprenant au moins 21 espèces appartenant à six sous-genres et un groupe non placé, dont 5 espèces précédemment associées au FCMV et une associée à l'AKAV, ont été collectés. Culicoides tainanus a dominé la plupart des collectes tout au long de l'année bien que C. sumatrae ait souvent été l'espèce dominante pendant l'été. La plupart des espèces étaient abondantes entre mai et octobre. Ces résultats indiquent que C. tainanus, C. jacobsoni et C. oxystoma sont les principaux culicoïdes nuisibles pour le bétail à Shizong et devraient être pris en compte dans toute enquête épidémiologique.

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.

Detection of bluetongue virus in Culicoides spp. in southern Yunnan Province, China.

BACKGROUND: Culicoides (Diptera: Ceratopogonidae) are vectors for many arboviruses. At least 20 species are considered as vectors or potential vectors of bluetongue virus (BTV) which cause bluetongue disease in ruminants. A BTV prevalence of 30-50% among cattle and goats in tropical southern Yunnan Province, China, prompted an investigation of the potential BTV vectors in this area. METHODS: Culicoides were collected by light trapping at three sites in the tropical region of Yunnan Province. Species were identified based on morphology and DNA sequences of cytochrome c oxidase subunit 1 (cox1). PCR and quantitative PCR following reverse transcription were used to test for the presence of BTV RNA in these specimens. Phylogenetic analysis was used to analyze the cox1 sequences of Culicoides specimens infected with BTV. RESULTS: Approximately 67,000 specimens of Culicoides were collected, of which 748 were tested for the presence of BTV. Five specimens, including two of Culicoides jacobsoni, one of C. tainanus and two of C. imicola, were identified as infected with BTV. No specimens of C. (subgenus Trithecoides) or C. oxystoma tested were positive for BTV infection. CONCLUSIONS: To our knowledge this is the first report of C. jacobsoni as a potential BTV vector and the fourth report of an association between C. tainanus and BTV, as well as the first direct evidence of an association between BTV and C. imicola in Asia. A fourth potential cryptic species within C. tainanus was identified in this study. Further analysis is required to confirm the importance of C. jacobsoni and C. tainanus in BTV epidemiology in Asia.

Potential vectors of bluetongue virus in high altitude areas of Yunnan Province, China.

BACKGROUND: Bluetongue disease of ruminants is a typical insect-borne disease caused by bluetongue virus (BTV) of the genus Orbivirus (family Reoviridae) and transmitted by some species of Culicoides (Diptera: Ceratopogonidae). Recently, the detection of BTV in yaks in high altitude meadows of the Shangri-La district of Yunnan Province, China, prompted an investigation of the Culicoides fauna as potential vectors of BTV. METHODS: A total of 806 Culicoides midges were collected by light trapping at three sites at altitudes ranging from 1800 to 3300 m. The species were identified based on morphology and the DNA sequences of cytochrome c oxidase subunit 1 (cox1). PCR and quantitative PCR following reverse transcription were used to test for the presence of BTV RNA in Culicoides spp. A phylogenetic analysis was used to analyze the cox1 sequences of some specimens. RESULTS: Four species dominated these collections and cox1 barcoding revealed that at least two of these appear to belong to species new to science. Culicoides tainanus and a cryptic species morphologically similar to C. tainanus dominated low altitude valley collections while C. nielamensis was the most abundant species in the high-altitude meadow. A species related to C. obsoletus occurred at all altitudes but did not dominate any of the collections. BTV RT-qPCR analysis detected BTV RNA in two specimens of C. tainanus, in one specimen closely related to C. tainanus and in one specimen closely related to C. obsoletus by barcode sequencing. CONCLUSIONS: This study suggests that BTV in high altitude areas of Yunnan is being transmitted by three species of Culicoides, two of which appear to be new to science. This research may be useful in improving understanding of the effects of global warming on arboviral disease epidemiology and further study is important in research into disease control and prevention.

The serologic investigation and viral isolation of bluetongue virus in Shangri-La in Southwest China.

Bluetongue is an arthropod-borne viral disease of ruminants caused by bluetongue virus (BTV). In China, BTV is relatively common in Yunnan Province with the exception of northern regions around Shangri-La, where the average altitude is approximately 3,450 metres. Recently, the seroprevalence of BTV has been measured in yaks in Shangri-La; therefore, this study investigated BTV infections in this area. The serological investigation in five villages in Shangri-La showed that there were sporadic BTV infections in yaks (20 of 507 positive) during 2014 to 2017, while the seroprevalence of BTV at three goat farms in a nearby river valley was 35%-65% in 2017. Subsequently, 20 sentinel goats were kept on two separate farms in the river valley and monitored for seroconversion between May and September of 2017. Five of the sentinel animals were tested positive for antibodies to BTV by C-ELISA during the study period, and 13 BTV isolates were isolated from ten sentinel animals. All isolates were identified as the same serotype, and the complete nucleotide sequence of one was determined. The genomic sequences showed that the isolated BTV strain belonged to serotype 21 and had approximately 99.8%-100% homology with three Indonesian BTV-21 strains (D151, RIVS-66 and RIVS-60) between their coding sequences (CDSs) except for Seg4 (99.5%). Besides, our data suggested that this BTV-21 strain might have also infected some local yaks and sheep.

The Susceptibility of Primary Dermis Fibroblasts from the Chinese Tree Shrew to Human Cytomegalovirus Infection.

As a universal pathogen leading to neonatal defects and transplant failure, human cytomegalovirus (HCMV) has strict species specificity and this has prevented the development of a suitable animal model for the pathogenesis study. The mechanism of cross-species barrier remains elusive and there are so far no non-human cell culture models that support HCMV replication. The Chinese tree shrew (Tupaia belangeri chinensis) is a small laboratory animal and evolutionary closely related with primates. We investigated the susceptibility of primary tree shrew dermis fibroblasts (TSDF) to HCMV infection. Infection with a GFP-expressing HCMV virus resulted in green fluorescence in infected cells with the expression of IE1, UL44 and pp28. The titers of cell-free viruses reached 103 PFU/mL at 96 hpi, compared to titers of 104 PFU/mL observed in primary human foreskin fibroblasts. Our results suggested that TSDF was semi-permissive for HCMV infection. The TSDF model could be further used to investigate key factors influencing cross-species multiplication of HCMV.

Filia Is an ESC-Specific Regulator of DNA Damage Response and Safeguards Genomic Stability.

Pluripotent stem cells (PSCs) hold great promise in cell-based therapy, but the genomic instability seen in culture hampers their full application. A greater understanding of the factors that regulate genomic stability in PSCs could help address this issue. Here we describe the identification of Filia as a specific regulator of genomic stability in mouse embryonic stem cells (ESCs). Filia expression is induced by genotoxic stress. Filia promotes centrosome integrity and regulates the DNA damage response (DDR) through multiple pathways, including DDR signaling, cell-cycle checkpoints and damage repair, ESC differentiation, and apoptosis. Filia depletion causes ESC genomic instability, induces resistance to apoptosis, and promotes malignant transformation. As part of its role in DDR, Filia interacts with PARP1 and stimulates its enzymatic activity. Filia also constitutively resides on centrosomes and translocates to DNA damage sites and mitochondria, consistent with its multifaceted roles in regulating centrosome integrity, damage repair, and apoptosis.

Maintenance of large numbers of virus genomes in human cytomegalovirus-infected T98G glioblastoma cells.

UNLABELLED: After infection, human cytomegalovirus (HCMV) persists for life. Primary infections and reactivation of latent virus can both result in congenital infection, a leading cause of central nervous system birth defects. We previously reported long-term HCMV infection in the T98G glioblastoma cell line (1). HCMV infection has been further characterized in T98Gs, emphasizing the presence of HCMV DNA over an extended time frame. T98Gs were infected with either HCMV Towne or AD169-IE2-enhanced green fluorescent protein (eGFP) strains. Towne infections yielded mixed IE1 antigen-positive and -negative (Ag(+)/Ag(-)) populations. AD169-IE2-eGFP infections also yielded mixed populations, which were sorted to obtain an IE2(-) (Ag(-)) population. Viral gene expression over the course of infection was determined by immunofluorescent analysis (IFA) and reverse transcription-PCR (RT-PCR). The presence of HCMV genomes was determined by PCR, nested PCR (n-PCR), and fluorescence in situ hybridization (FISH). Compared to the HCMV latency model, THP-1, Towne-infected T98Gs expressed IE1 and latency-associated transcripts for longer periods, contained many more HCMV genomes during early passages, and carried genomes for a greatly extended period of passaging. Large numbers of HCMV genomes were also found in purified Ag(-) AD169-infected cells for the first several passages. Interestingly, latency transcripts were observed from very early times in the Towne-infected cells, even when IE1 was expressed at low levels. Although AD169-infected Ag(-) cells expressed no detectable levels of either IE1 or latency transcripts, they also maintained large numbers of genomes within the cell nuclei for several passages. These results identify HCMV-infected T98Gs as an attractive new model in the study of the long-term maintenance of virus genomes in the context of neural cell types. IMPORTANCE: Our previous work showed that T98G glioblastoma cells were semipermissive to HCMV infection; virus trafficked to the nucleus, and yet only a proportion of cells stained positive for viral antigens, thus allowing continual subculturing and passaging. The cells eventually transitioned to a state where viral genomes were maintained without viral antigen expression or virion production. Here we report that during long-term T98G infection, large numbers of genomes were maintained within all of the cells' nuclei for the first several passages (through passage 4 [P4]), even in the presence of continual cellular division. Surprisingly, genomes were maintained, albeit at a lower level, through day 41. This is decidedly longer than in any other latency model system that has been described to date. We believe that this system offers a useful model to aid in unraveling the cellular components involved in viral genome maintenance (and presumably replication) in cells carrying long-term latent genomes in a neural context.

Later passages of neural progenitor cells from neonatal brain are more permissive for human cytomegalovirus infection.

Congenital human cytomegalovirus (HCMV) infection is the most frequent infectious cause of birth defects, primarily neurological disorders. Neural progenitor/stem cells (NPCs) are the major cell type in the subventricular zone and are susceptible to HCMV infection. In culture, the differentiation status of NPCs may change with passage, which in turn may alter susceptibility to virus infection. Previously, only early-passage (i.e., prior to passage 9) NPCs were studied and shown to be permissive to HCMV infection. In this study, NPC cultures derived at different gestational ages were evaluated after short (passages 3 to 6) and extended (passages 11 to 20) in vitro passages for biological and virological parameters (i.e., cell morphology, expression of NPC markers and HCMV receptors, viral entry efficiency, viral gene expression, virus-induced cytopathic effect, and release of infectious progeny). These parameters were not significantly influenced by the gestational age of the source tissues. However, extended-passage cultures showed evidence of initiation of differentiation, increased viral entry, and more efficient production of infectious progeny. These results confirm that NPCs are fully permissive for HCMV infection and that extended-passage NPCs initiate differentiation and are more permissive for HCMV infection. Later-passage NPCs being differentiated and more permissive for HCMV infection suggest that HCMV infection in fetal brain may cause more neural cell loss and give rise to severe neurological disabilities with advancing brain development.