Arboviruses that infect humans and other mammals pose significant public health challenges worldwide, particularly in developing countries. The border region of Yunnan Province, a key entry point for imported arboviruses into China, is characterized by a subtropical climate and a rich diversity of mosquito and biting midge species, making it a high-risk area for arbovirus transmission. While some studies have focused on mosquito-borne viruses in this region, research on midge-borne viruses, particularly those with zoonotic relevance, remains limited. This study aimed to systematically investigate the diversity of arboviruses in both vector groups and to address critical gaps in the assessment of spillover risk. In July 2018, mosquitoes and biting midges were collected from Mangshi, Yingjiang, Lushui, and Tengchong in the border region of western Yunnan Province, China. Following morphological classification, specimens were pooled for virus isolation using BHK-21 and C6/36 cells. Viral isolates were identified by reverse transcription polymerase chain reaction (RT-PCR) using virus-specific primers, and sequence analyses were performed using Clustal X, DNASTAR, and MEGA-X. A total of 8581 mosquitoes (8 species in 4 genera) and 18,298 biting midges (19 species) were collected. Culex tritaeniorhynchus dominated the mosquito populations in Mangshi (92.11

Bluetongue (BT) virus (BTV) is an arthropod-borne pathogen that causes substantial economic losses in ruminants globally. Yunnan Province, located in China's tropical and subtropical border region, faces a high risk of BTV circulation. This study monitored BTV vectors, genetically characterized circulating BTV strains, and assessed seroprevalence to inform early warning efforts. In 2025, 46,000 Culicoides midges were collected from three counties, identified, and grouped into 460 pools. Additionally, 5934 cattle serum samples from 17 border counties were tested using C-ELISA. Midge pools were screened by RT-qPCR for BTV nucleic acids and then inoculated into C6/ 36 cells for virus isolation. Whole-genome sequencing and phylogenetic analysis of VP2 and VP5 genes were performed on the isolates. C. oxystoma and C. trithecoides were the dominant midge species at all sites. RT-qPCR identified 11 BTV-positive midge pools, primarily involving C. oxystoma, C. imicola, C. tainanus, and C. jacobsoni. Four BTV isolates were identified as serotypes 1, 4, 5, and 16. Phylogenetic analysis of VP2 and VP5 showed all four strains likely belong to the Eastern topotype and are closely related to historical BTVs from China, Japan, India, and Australia. The overall BTV seroprevalence in cattle was 41.37% (95% confidence interval [CI], 40.12%-42.62%), with significantly higher rates in autumn (58.07%, 95% CI, 56.29%-59.85%) than in spring (24.87%, 95% CI, 23.32%-26.42%). Several BTV serotypes have been detected and isolated from diverse Culicoides in the border regions of Yunnan. Additionally, BTV seroprevalence has been observed in local cattle populations, indicating that this area may represent a significant BTV endemic hotspot. These findings underscore the need for ongoing vector surveillance and early warning systems to prevent BT outbreaks in susceptible livestock.

Tibet orbivirus (TIBOV) is a poorly characterized orbivirus whose antigenic diversity and circulation in livestock remain incompletely defined. Here, we report the isolation of a TIBOV strain, MS18C217-16, from Culicoides spp. collected in Mangshi, Yunnan Province, China, in 2018. MS18C217-16 replicated efficiently in multiple mammalian cell lines and induced clear cytopathic effects. Phylogenetic analysis assigned the isolate to the TIBOV clade, with high amino acid conservation in VP1, VP3, and VP7, but marked divergence in the outer-capsid proteins VP2 and VP5. Structural modeling and predicted MHC class II-binding peptide profiles suggested distinctive antigenic features in these proteins, and neutralization assays provided evidence that MS18C217-16 is antigenically distinct from the reference TIBOV strain DH13C120. Serological surveys detected neutralizing antibodies in goats, swine, and cattle across four counties, with particularly high seropositivity in goats from Mengla. Together, these data identify MS18C217-16 as an antigenically divergent TIBOV strain with features consistent with a putative novel serotype, while highlighting the need for reciprocal cross-neutralization assays using strain-specific antisera for formal serotype confirmation.

Bluetongue is an insect-borne disease of ruminants caused by the Bluetongue virus (BTV). It is primarily transmitted through the bites of Culicoides midges and is widely distributed in tropical, subtropical, and temperate regions around the world. Due to its favorable geographical location and abundant population of BTV vector midges, Yunnan Province, China, has become a key region for research on BTV diversity. However, there are gaps in research on the genetic evolution of BTV in this region. Between January 2024 and December 2025, Culicoides midges were collected from three border counties in Yunnan Province. Researchers isolated BTV strains and sequenced the full-length segment 2 (Seg-2) gene. Subsequently, phylogenetic analysis, Bayesian evolutionary rate analysis, population dynamics analysis, and phylogeographic analysis were performed utilizing Seg-2 sequence data sourced from the NCBI database. Four BTV serotypes (BTV-1, BTV-4, BTV-5, and BTV-16) were identified. The estimated average evolutionary rate varied from 2.88 × 10−4 to 4.26 × 10−3 substitutions per site per year, with ancestral origins dating back to the period between 1872 and 1988. The effective population size displayed significant fluctuations over time. Phylogeographic reconstructions indicated that the pathogen entered Yunnan through various transcontinental transmission pathways, originating from Europe, South Asia, Oceania, and East Asia, with evidence of reciprocal transmission between Africa, Europe, and West Asia. The BTV serotypes circulating in Yunnan Province are highly diverse, and the population dynamics of different serotypes exhibit significant heterogeneity. Yunnan is simultaneously exposed to BTV introductions from multiple directions, including Europe, South Asia, Oceania, and East Asia.

Epizootic hemorrhagic disease (EHD) is a noncontagious viral infection transmitted by certain species of the genus Culicoides and has been included on the World Organization for Animal Health (OIE) list of diseases affecting domestic and wild ruminants. This study isolated four serotypes of EHDV strains (EHDV-1, -5, -6, and -7) from three Culicoides species collected in Yunnan Province, China. The phylogenetic analysis of Seg-3 revealed that EHDVs can be classified into two groups based on their geographical origins: Eastern and Western. The four isolates examined in this study belong to the Eastern group and are genetically most similar to EHDVs found in Japan and Australia. In the Seg-2 and Seg-6 phylogenetic trees, all EHDV serotypes can be classified into four clusters (Groups A-D). The four strains were categorized into Groups A, B, C, and D and belong to the EHDV-1, -5, -6, and -7 serotypes, respectively. Sero-epidemiological surveys were conducted on cattle in the border areas of China, Laos, Myanmar, and Vietnam using C-ELISA and neutralization tests. The rate of EHDV antibody positivity ranged from 29.22% to 58.86%, with an average of 43.09%. These surveys also revealed the presence of seven serotypes of EHDV circulating among cattle in these regions, resulting in diverse prevalence rates and spatial distributions. This finding indicates that EHDV circulates among natural vectors and infects local domestic cattle in these areas. The data gathered herein will help monitor the evolutionary characteristics of EHDV and its potential to cause outbreaks in China and neighboring countries. IMPORTANCE:EHDV is the causative agent of epizootic hemorrhagic disease (EHD), a viral illness with significant implications for animal welfare, society, and the economy. There are reports of EHDV-1, -6, -7, and -10 serotypes isolated from China, but no reports of EHDV-5 strains have been documented. Here, we report the isolation and identification of four serotypes of EHDV (EHDV-1, -5, -6, and -7) from three Culicoides species (C. tainanus, C. oxystoma, and C. jacobsoni) collected in Yunnan, China. Phylogenetic analysis revealed that the four strains of EHDV are most closely related to the Australian and Japanese strains. Sero-epidemiological survey results showed that in the border areas of China, Laos, Myanmar, and Vietnam, the rate of EHDV antibody positivity ranged from 29.22% to 58.86%, and seven serotypes of EHDVs circulating among cattle in these regions presented diverse prevalence rates and spatial distributions. This finding indicates that EHDV not only circulates among natural vectors but also infects local domestic cattle in these areas and that the EHDVs prevalent in the Asia-Pacific region may share a common gene pool.

Palyam serogroup virus (PALV) is a potential pathogen prevalent in Asia, Australia, and Africa that may cause abortion and teratogenesis in ruminants, especially cattle. In this study, we isolated three PALV strains (MY_C17, ML_C40, and SZ_C06) from Culicoides spp. in Yunnan, China, and obtained their complete genome sequences via next-generation sequencing. The phylogenetic analysis of Seg-5, Seg-7, and Seg-9 indicated that PALVs can be grouped based on their geographical origins: Asia, Australia, and Africa. The three isolates in this study belong to the Asia group. However, the Central African Republic strain (AR_B_2032) and the India strain (DVTD) form a separate branch, provisionally named the CI group. In the VP2 phylogenetic tree, all PALV strains can be grouped into six significant branches, designated as A through F. The three isolates are clustered in Groups A, E, and F and are shown to belong to the D'Aguilar virus (DAGV), Chuzan virus (CHUV), and Bunyip Creek virus (BCV) serotypes, respectively. Seroepidemiological surveys were conducted on local cattle using neutralization tests, which revealed seroprevalence rates of neutralizing antibodies against BVC, DAGV, and CHUV in Yunnan cattle serum of 3.4% (12/350), 6.9% (24/350), and 16.9% (59/350), respectively. This marks the first-ever isolation of CHUV, specifically BCV and DAGV, from vector Culicoides spp. in Southwest China. It was also proved that in this region, at least three serotypes of PALV circulated among Culicoides spp. in nature and infected domestic cattle.

Introduction We verified that Akabane virus (AKAV) is transmitted through biting midges and infects local domestic animals.Methods In 2013, viruses were isolate from biting midges in Yunnan, China, using BHK-21 and C6/36 cells. Two AKAV strains (No. 52 and 55) that induced cytopathogenic effects (CPE) in BHK-21, MDBK, and Vero cells were characterized.Results The complete genomic sequence of both viruses consisted three RNA segments (S, M, and L). The S segment (856 nucleotides) encoded a 233-amino-acid nucleocapsid protein and a 91-amino-acid nonstructural protein, while the M segment (4309 nucleotides) encoded a 1401-amino-acid polyprotein. The L segment (6869 nucleotides) encoded a 2511-amino-acid RNA-dependent RNA polymerase. Phylogenetic analysis revealed that specimen Nos. 52 and 55 clustered with AKAV genotype Ia viruses isolated from Asia. The AKAV strain (55) neutralizing antibody exhibited a total positive rate of 43.55% (202/466) against serum samples from cattle and goats collected in Yunnan Province. Specifically, the positive rates were 48.77% (139/285) for cattle and 34.81% (63/181) for goats. Neutralizing antibody titers in cattle (1:32-1:128) were higher than those in goats (1:4-1:16).Discussion This study represents the first isolation of AKAV from biting midges in China, along with the detection of high neutralizing antibody titers against AKAV in the serum samples of local cattle and goats. These findings suggested that biting midges are involved in AKAV transmission among domestic animals in Yunnan Province, China.

In China, the Akabane virus (AKAVs) has been reported in several host species. However, data regarding goats is still showing a gap. Akabane virus (AKAV) is an insect-borne virus from the Peribunyaviridae family that in ruminant species, particularly affects pregnant animals, resulting in abortions, stillbirths, and premature broth, often with congenital abnormalities. Therefore, there is a dire need to understand the diversity of this virus in ruminants, particularly in goats. The current study aimed to investigate the genotype characteristics of goat-originated AKAVs in Yunnan, China. For this, blood samples from goats were collected for four consecutive years (2019-2023) during routine disease surveillance in Yunnan province. The serum was harvested and evaluated for the seroprevalence of the AKAVs. The seroprevalence analysis revealed that the majority of goats in Yunnan province are infected with AKAVs, with a detected prevalence of 7.69% (92/1,197 tested), even though detected seroprevalence rose as high as 27.8% in some areas. For the evolutionary analysis of AKAVs of goat, five AKAVs strains were isolated from AKAV serum-positive goat blood samples were whole genome sequenced. The phylogenetic analysis (sequenced small and medium segments) classified the AKAVs into Ia and Ib genogroups, with Ia genogroup strains being more common in Yunnan goats. However, genotype II (TJ2016 and CQ-AKAV-1-2023) emerged in China. Overall, this study emphasizes the importance of implementing prevention and control strategies for AKAVs diseases transmitted in Yunnan, China.

Background: Arboviruses, transmitted to humans and animals by blood-feeding arthropods, pose significant public health risks. This study investigates the types, distribution, and epidemiology of arboviruses in the central Yuxi region of Yunnan Province, providing crucial data for the prevention and control of mosquito-borne diseases. Methods: In 2015, mosquitoes were collected from Tonghai County, Huaning County, Jiangchuan District, and Chengjiang County in Yuxi using light traps. The mosquitoes were homogenized, and virus isolation was performed by inoculating the homogenates onto BHK-21 (baby hamster kidney) and C6/36 (Aedes albopictus) cell lines. Reverse Transcription Polymerase Chain Reaction (RT-PCR) amplification was performed with universal and virus-specific primers, followed by sequencing of positive products using bioinformatics tools (DNAstar, MEGA X). Results: A total of 13,050 mosquitoes from four counties were collected, with 261 pools processed for virus detection. The mosquitoes belonged to three genera: Culex Anopheles and Aedes, with Culex tritaeniorhynchus as the predominant species. Cytopathic effects were observed in 95 pools. Nine viruses were identified: Japanese Encephalitis Virus (JEV), Bannavirus, Getah Virus, Tibet Orbivirus (TIBOV), Nam Dinh Virus, Akabane Virus (AKAV), Ngewotan Virus (NWTV), Quang Binh Virus (QBV), and Culex pipiens pallens densovirus. The distribution of viruses varied: six viruses with 35 isolates in Tonghai County; six viruses with 88 isolates in Huaning County; two viruses with five isolates in Chengjiang County; and no viruses were found in Jiangchuan District. TIBOV and NWTV were present in all three counties, while JEV, AKAV, and QBV were found only in Tonghai County. Conclusion: Nine arboviruses were isolated across four counties in Yuxi, with varying distributions. This highlights the epidemiological diversity of mosquito-borne viruses in the region, emphasizing the need for targeted control measures.

IntroductionCulicoides plays a crucial role as an insect vector in the field of veterinary medicine. The transmission of significant viruses such as bluetongue virus (BTV) and African horse sickness virus (AHSV) by this insect poses a substantial threat, leading to the development of severe diseases in domestic animals. This study aimed to explore the Culicoides species, identify their blood-meal sources, and assess the presence of BTV and AHSV carried by Culicoides in Yuanyang County, Yunnan Province. The aim was to gain insights into the potential vectors of these two viruses and elucidate their potential roles in the transmission of pathogens.MethodsThe midges were collected from cattle (Bos indicus), pig (Sus scrofa), and goat (Capra hircus) pens in Yuanyang County, Yunnan Province in June 2020. Initial identification of midges was conducted through morphological characteristics, followed by molecular identification using the cytochrome C oxidase subunit I (COI) gene. The determination of Culicoides blood-meal sources was accomplished using specific primers targeting the cytochrome b (Cyt b) gene from potential hosts. BTV and AHSV RNA were identified in Culicoides pools through the application of reverse transcriptase PCR and quantitative real-time PCR. Nucleotide homology and phylogenetic analysis were performed using MegAlign (DNAStar) and Mega 6.0 software.ResultsA total of 6,300 Culicoides, consisting of C. oxystoma, C. arakawai, C. imicola, and C. innoxius, were collected from cattle, pigs, and goat pens. The engorgement rates for these species were 30.2%, 54.6%, 75%, and 66.7%, respectively. In the cattle pen, the prevailing species is C. oxystoma (100%). In the pig pen, C. arakawai dominates (70%), with C. oxystoma following at 30%. In the goat pen, C. imicola holds the majority (45.45%), trailed by C. oxystoma (25%), C. innoxius (20.45%), and C. arakawai (9.09%). These Culicoides species were identified as feeding on cattle, pigs, goats, chickens (Gallus gallus), and humans (Homo sapiens). The positivity rates for BTV were 20.00% and 11.54% in blood-fed specimens of C. imicola and C. oxystoma, respectively. Conversely, the positivity rates for BTV in non-blood-fed specimens were 0.00% and 6.67% for C. imicola and C. oxystoma, respectively. BTV was not detected in C. arakawai and C. innoxius. The specimens (YY86) from C. imicola that tested positive for BTV had the closest genetic relationship to YTS-4 isolated from Mangshi, Yunnan Province in 1996. All test results for the nucleic acid of AHSV were negative.ConclusionThe study reveals variations in the species distribution, community composition, blood sucking rate, and blood-feeding sources of Culicoides across different habitats. Notably, C. imicola and C. oxystoma emerge as potential vectors for the transmission of BTV in local animals. Accordingly, this investigation provides crucial insights that can serve as a valuable reference for the prevention and control of BTV in local animals, particularly from the perspective of vector management.

The commensal microbiota of the mosquito gut plays a complex role in determining the vector competence for arboviruses. In this study, we identified a bacterium from the gut of fieldAedes albopictusmosquitoes namedRosenbergiellasp. YN46 (Rosenbergiella_YN46) that rendered mosquitoes refractory to infection with dengue and Zika viruses. Inoculation of 1.6 × 103colony forming units (CFUs) ofRosenbergiella_YN46 intoA. albopictusmosquitoes effectively prevents viral infection. Mechanistically, this bacterium secretes glucose dehydrogenase (RyGDH), which acidifies the gut lumen of fed mosquitoes, causing irreversible conformational changes in the flavivirus envelope protein that prevent viral entry into cells. In semifield conditions,Rosenbergiella_YN46 exhibits effective transstadial transmission in field mosquitoes, which blocks transmission of dengue virus by newly emerged adult mosquitoes. The prevalence ofRosenbergiella_YN46 is greater in mosquitoes from low-dengue areas (52.9 to ~91.7%) than in those from dengue-endemic regions (0 to ~6.7%).Rosenbergiella_YN46 may offer an effective and safe lead for flavivirus biocontrol.

Two strains of viruses, JC13C644 and JC13C673, were isolated from Culicoides tainanus collected in Jiangcheng County, Yunnan Province, situated along the border area shared by China, Laos, and Vietnam. JC13C644 and JC13C673 viruses can cause cytopathic effect (CPE) in mammalian cells BHK21 and Vero cells, and cause morbidity and mortality in suckling mice 48 h after intracerebral inoculation. Whole-genome sequencing was performed, yielding complete sequences for all 10 segments from Seg-1 (3942nt) to Seg-10 (810nt). Phylogenetic analysis of the sub-core-shell (T2) showed that the JC13C644 and JC13C673 viruses clustered with the Epizootic Hemorrhagic Disease Virus (EHDV) isolated from Japan and Australia, with nucleotide and amino acid homology of 93.1% to 98.3% and 99.2% to 99.6%, respectively, suggesting that they were Eastern group EHDV. The phylogenetic analysis of outer capsid protein (OC1) and outer capsid protein (OC2) showed that the JC13C644 and JC13C673 viruses were clustered with the EHDV-10 isolated from Japan in 1998, with the nucleotide homology of 98.3% and 98.5%, and the amino acid homology of 99.6% and 99.6–99.8%, respectively, indicating that they belong to the EHDV-10. Seroepidemiological survey results demonstrated that JC13C644 virus-neutralizing antibodies were present in 29.02% (177/610) of locally collected cattle serum and 11.32% (89/786) of goat serum, implying the virus’s presence in Jiangcheng, Yunnan Province. This finding suggests that EHDV-10 circulates not only among blood-sucking insects in nature but also infects local domestic animals in China. Notably, this marks the first-ever isolation of the virus in China and its discovery outside of Japan since its initial isolation from Japanese cattle. In light of these results, it is evident that EHDV Serotype 10 exists beyond Japan, notably in the natural vectors of southern Eurasia, with the capacity to infect local cattle and goats. Therefore, it is imperative to intensify the surveillance of EHDV infection in domestic animals, particularly focusing on the detection and monitoring of new virus serotypes that may emerge in the region and pose risks to animal health.

Background: Kadipiro virus (KDV) is a species of the new 12 segmented RNA virus grouped under the genus Seadornavirus within the Reoviridae family. It has previously been isolated or detected from mosquito, Odonata, and bat feces in Indonesia, China, and Denmark, respectively. Here, we describe the isolation and characterization of a viral strain from mosquitoes in Yunnan Province, China. Methods: Mosquitoes were collected overnight using light traps in Shizong county, on July 17, 2023. Virus was isolated from the mosquito homogenate and grown using baby hamster kidney and Aedes albopictus (C6/36) cells. Preliminary identification of the virus was performed by agarose gel electrophoresis (AGE). The full-genome sequences of the strain were determined by full-length amplification of cDNAs and sequenced using next-generation sequencing. Results: We isolated a viral strain (SZ_M48) from mosquitoes (Culex tritaeniorhynchus Giles) that caused cytopathogenic effects in C6/36 cells. AGE analysis indicated a genome consisting of 12 segments of double-stranded RNA that demonstrated a "6-5-1" pattern, similar to the migrating bands of KDV. Phylogenetic analysis based on the full-genome sequence revealed that SZ_M48 is more clustered with KDV isolates from Hubei and Shangdong in China than with Indonesian and Danish strains. The identity between SZ_M48 and SDKL1625 (Shandong, China) is slightly lower than that of QTM27331 (Hubei, China), and the identity with JKT-7075 (Indonesia) and 21164-6/M.dau/DK (Denmark) is the lowest. Conclusion: The full-genome sequence of the new KDV strain described in this study may be useful for surveillance of the evolutionary characteristics of KDVs. Moreover, these findings extend the knowledge about the genomic diversity, potential vectors, and the distribution of KDVs in China.

2018 年云南省边境地区某猪场猪群出现高热等症状,部分猪只死亡.为确认发病原因,采集死亡猪只肺脏组织样品进行猪繁殖与呼吸综合征病毒(PRRSV)RT-PCR检测,然后将阳性样品接种Marc-145 细胞,连续传代3 次后发现产生稳定的细胞病变效应(CPE).经间接免疫荧光试验(IFA)、毒价测定及病毒全基因组测序,最终确定病原为PRRSV,将其命名为YNML-2018 株,毒价测定为 10-4.88 TCID50/0.1 mL.全基因组序列分析显示,YNML-2018 毒株基因组全长 15 357 bp,包含 8 个开放阅读框(ORF),其中非结构蛋白 2 编码基因(NSP2)缺失 90 个碱基.病毒全基因组遗传进化分析显示,该毒株序列与国内分离的高致病性PRRSV同源性为 95.3%～99.3%;其NSP2 高变区序列与国内分离的高致病性PRRSV同源性为 92.1%～97.8%;结构蛋白GP3、GP5氨基酸序列与国内分离的高致病性PRRSV毒株同源性分别为84.3%～99.6%和83.1%～99.0%.结果表明,YNML-2018 株与近年来我国PRRSV流行株相比存在一定变异,但变异程度较低,仍与首次报道的高致病性毒株JXA1 同属于亚群V.本研究为掌握云南省边境地区PRRSV流行毒株的遗传变异特征提供了技术支撑,并可为疫苗选用提供数据参考.

Background: Dengue virus (DENV) can be divided into four serotypes-DENV-1, DENV-2, DENV-3, and DENV-4. In humans, infection leads to dengue fever (DF), dengue hemorrhagic fever, and dengue shock syndrome, both widely prevalent in tropical and subtropical regions. In 2019, a severe outbreak of DF occurred in Xishuangbanna, Yunnan province. Objective: To investigate the etiology and genotype of the causative agents of this severe dengue outbreak in Xishuangbanna. Methods: Between October and November 2019, the sera of patients clinically diagnosed with DF were collected in the first People's Hospital of Xishuangbanna. RNA was extracted from the sera and amplified by RT-PCR with flavivirus primers. Flavivirus-positive sera were then used to inoculate Aedes albopictus cells (C6/36); viral RNA was extracted from these cells, amplified, and sequenced with DENV E gene-specific primers. Sequence splicing and nucleotide homology genetic evolution analysis were carried out by biological software (DNAStar). Unique mutations in the E genes of isolated DENV were analyzed by SWISS-MODEL and PyMOL. Results: Of the 60 samples collected from DF patients, 39 tested positively with flavivirus primers. The DENV was isolated from 25 of the 39 positive seras, of which 20 showed cytopathic effects (CPE) and 5 were no CPE. In these 25 isolated nucleic acids, 21 strains of DENV-1, 3 strains of DENV-2, and 1 strain of DENV-3 were identified according to the sequence of E protein. In the four unique mutations (D52, Y149, L312, T386), D52 and Y149 in the E protein of DENV-1 were predicted to be exposed on the surface of the prefusion conformation. Conclusion: The 2019 outbreak of DF in Xishuangbanna area of Yunnan Province consists of at least three serotypes of DENV-1, DENV-2, and DENV-3, and the sources of these virus strains are of mixed and complicated origin.

[目的]了解牛结节性皮肤病病毒(Lumpy skin disease virus,LSDV)在自然感染状况下病牛的带毒状况及其潜在的传播媒介种类,为牛结节性皮肤病防控提供参考依据.[方法]于2021年7月在云南省文山市采集疑似发病牛不同组织类型标本,2020年8月和2021年7月～2021年8月分别在云南省昆明市、文山市和双江县采集疑似LSDV感染的牛圈舍蠓、蚊标本,并进行形态学鉴定.采用实时荧光定量PCR方法检测发病牛不同组织及库蠓、库蚊标本中LSDV核酸;采用LSDV127基因特异引物对皮肤结节标本进行PCR扩增及测序;利用DNAStar和Mega 6.0等软件进行核苷酸序列相似性比对和系统进化分析.[结果]3头发病牛全血、血清、鼻拭子和皮肤结节标本中均检测到LSDV核酸,且皮肤结节标本中LSDV核酸扩增曲线信号最强;LSDV127基因序列分析结果显示,3份皮肤结节标本(17、18和19)的病毒核苷酸序列相似性为100％,与俄罗斯、泰国、越南和南非等LSDV分离株聚为一支,亲缘关系较近,核苷酸序列相似性在99.7％～100％之间.共采集鉴定条带库蠓、连斑库蠓和尖喙库蠓3种,库蠓标本4 822只,库蚊标本150只.所有采集自发病牛圈舍的库蠓和库蚊标本LSDV核酸检测结果均为阴性.[结论]在自然感染LSDV的牛中,血液、鼻分泌物和皮肤结节等不同部位都可能为节肢动物媒介获得并传播LSDV提供很好的感染源;蠓、蚊等节肢动物媒介是否参与了当地LSDV的传播有必要进一步调查.

Biting midges of the genus Culicoides are important in both medicine and veterinary medicine because their blood-feeding regime enable them to transmit a variety of pathogens. In this study, the morphological characteristics of the new species of Culicoides (Sinocoides) jiangchengensis Wang et Liu sp. nov are described and compared with the other species of female Culicoides in the subgenus Sinocoides. Three morphological characteristics of C. jiangchengensis, such as without sensory pit in 3rd palpus segment, sensilla coeloconica on flagellomeres 1,9-13, and m1 and m2 cell of the wings with pale spots, were different from the other nine species of culicoides in subgenus Sinocoides. Genetically, C. jiangchengensis are most closely related to C. malipoensis, but they were located in different branches and the minimum interspecific distance between them was 12.6%. In addition, a checklist of 10 species in the subgenus Sinocoides Chu, 1983 (Diptera: Ceratopogonidae: Culicoides) in China, including the new species C. jiangchengensis Wang et Liu sp. nov., is provided, and an updated key to species of the subgenus Sinocoides Chu, 1983 was presented.

目前世界范围内仅分别从日本西南的与那国岛(2015年)及中国云南省景洪市勐罕镇(2019年)的哨兵动物牛上分离到过新型环状病毒Yonaguni orbivirus(YONOV),但尚缺乏该病毒核酸的特异性检测方法.该研究对比我国分离的YONOV毒株JH2019C603和日本毒株(ON-7/E/15)的基因序列,选择编码非结构蛋白3(Non-structural protein 3,NS3)的Segment-10(Seg-10)节段的保守区设计引物和探针,并分别进行了特异性、灵敏度、重复性和临床血液样品的测试,建立了牛YONOV的RT-qPCR和RT-PCR检测方法.结果显示,两种方法能特异性扩增YONOV核酸片段,对其他环状病毒无有效扩增;RT-qPCR和RT-PCR检出YONOV核酸浓度的下限分别为11.5 copies/μL和115.0 copies/μL;两种方法均能检测出毒株或动物临床血样中的YONOV核酸,并且能在动物感染并产生中和抗体前1周检测出病毒核酸,因此更适用于病毒感染动物的早期诊断.RT-qPCR因其更高的灵敏度和实效性,能用于大量临床样品的快速检测,而RT-PCR的扩增产物可以进行测序,获取待测毒株的基因信息,两种检测方法相互辅助,可以为YONOV的核酸检测及动物感染该病毒的诊断及流行病学研究提供技术支撑.

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.

IntroductionBanna virus (BAV), a potential pathogen that may cause human encephalitis, is the prototype species of genus Seadornaviru within the family Reoviridae, and has been isolated from a variety of blood-sucking insects and mammals in Asia.MethodsCulicoides, Mosquitoes, and Ticks were collected overnight in Yunnan, China, during 2016-2023 using light traps. Virus was isolated from these collected blood-sucking insects and grown using Aedes albopictus (C6/36) cells. Preliminary identification of the virus was performed by agarose gel electrophoresis (AGE). The full genome sequences of the BAVs were determined by full-length amplification of cDNAs (FLAC) and sequenced using next-generation sequencing.ResultsIn this study, 13 strains BAV were isolated from Culicoides, Mosquitoes and Ticks. Their viral genome consisted of 12 segments of double-stranded RNA (dsRNA), and with three distinct distribution patterns. Sequence analysis showed that Seg-5 of four strains (SJ_M46, SJ_M49, JC_M19-13 and JC_C24-13) has 435 bases nucleotide sequence insertions in their ORF compared to other BAVs, resulting in the length of Seg-5 up to 2128 nt. There are 34 bases sequence deletion in Seg-9 of 3 strains (WS_T06, MS_M166 and MS_M140). Comparison of the coding sequences of VP1, VP2, VP5, VP9 and VP12 of the 13 BAV strains, the results show that VP1, VP2 and VP12 are characterised by high levels of sequence conservation, while VP9 is highly variable, under great pressure to adapt and may be correlated with serotype. While also variable, VP5 appears to be under less adaptive pressure than VP9. Additionally, phylogenetic analysis indicates that the 13 BAV strains locate in the same evolutionary cluster as BAVs isolated from various blood-sucking insects, and are clustered according to geographical distribution.ConclusionThe data obtained herein would be beneficial for the surveillance of evolutionary characteristics of BAV in China and neighboring countries as well as extend the knowledge about its genomic diversity and geographic distribution.