Bats are natural reservoirs for a wide range of microorganisms, including many notable zoonotic pathogens. However, the infectome of bat kidneys remains poorly understood. To address this gap, we performed meta-transcriptomic sequencing on kidney tissues from 142 bats, spanning ten species sampled at five locations in Yunnan province, China. This analysis identified 22 viral species, including 20 novel viruses, two of which represented newly discovered henipaviruses closely related to the highly pathogenic Hendra and Nipah viruses. These henipaviruses were found in the kidneys of bats inhabiting an orchard near villages, raising concerns about potential fruit contamination via bat urine and transmission risks to livestock or humans. Additionally, we identified a novel protozoan parasite, tentatively named Klossiella yunnanensis, along with two highly abundant bacterial species, one of which is a newly discovered species, Flavobacterium yunnanensis. These findings broaden our understanding of the bat kidney infectome, underscore critical zoonotic threats, and highlight the need for comprehensive, full-spectrum microbial analyses of previously understudied organs to better assess spillover risks from bat populations. ### Competing Interest Statement The authors have declared no competing interest.

Understanding wildlife-pathogen interactions is crucial for mitigating zoonotic risk. Through meta-transcriptomic sequencing we profiled the infectomes of 1,922 samples from 67 mammalian species across China, uncovering a remarkable diversity of viral, bacterial, fungal, and parasitic pathogens. Of the 195 pathogens identified, 62 were novel, including a bi-segmented coronavirus in diseased lesser pandas, which we propose represents a new genus - Zetacoronavirus. The orders Carnivora and Rodentia exhibited the highest pathogen diversity and were implicated in numerous host-jumping events. Comparative analysis of diseased versus healthy animals revealed a trend of higher pathogen loads in the former, with possible differences in tissue tropisms. In total, 48 zoonotic and 17 epizootic pathogens were identified, with frequent cross-species transmission, emphasizing the potential for emerging public health threats. This study highlights the urgent need for wildlife pathogen surveillance to inform proactive disease management strategies. ### Competing Interest Statement The authors have declared no competing interest.

Yunnan province in China shares its borders with three neighboring countries: Myanmar, Vietnam, and Laos. The region is characterized by a diverse climate and is known to be a suitable habitat for various arthropods, including midges which are notorious for transmitting diseases which pose significant health burdens affecting both human and animal health. A total of 431,100 midges were collected from 15 different locations in the border region of Yunnan province from 2015 to 2020. These midges were divided into 37 groups according to the collection year and sampling site. These 37 groups of midges were then homogenized to extract nucleic acid. Metatranscriptomics were used to analyze their viromes. Based on the obtained cytochrome C oxidase I gene (COI) sequences, three genera were identified, including one species of Forcipomyia, one species of Dasyhelea, and twenty-five species of Culicoides. We identified a total of 3199 viruses in five orders and 12 families, including 1305 single-stranded positive-stranded RNA viruses (+ssRNA) in two orders and seven families, 175 single-stranded negative-stranded RNA viruses (−ssRNA) in two orders and one family, and 1719 double-stranded RNA viruses in five families. Six arboviruses of economic importance were identified, namely Banna virus (BAV), Japanese encephalitis virus (JEV), Akabane virus (AKV), Bluetongue virus (BTV), Tibetan circovirus (TIBOV), and Epizootic hemorrhagic disease virus (EHDV), all of which are capable, to varying extents, of causing disease in humans and/or animals. The survey sites in this study basically covered the current distribution area of midges in Yunnan province, which helps to predict the geographic expansion of midge species. The complexity and diversity of the viral spectrum carried by midges identified in the study calls for more in-depth research, which can be utilized to monitor arthropod vectors and to predict the emergence and spread of zoonoses and animal epidemics, which is of great significance for the control of vector-borne diseases.

Seoul orthohantavirus (SEOV) has been identified as one of the main causative agents of hemorrhagic fever with renal syndrome (HFRS) in China. The virus was found circulating in rodent populations in almost all provinces of the country, reflecting the wide distribution of HFRS. Here, using the direct immunofluorescence assay (DFA) and real-time quantitative reverse transcription PCR (qRT-PCR) approach, we performed screening in 1784 small mammals belonging to 14 species of three orders captured in the main areas of HFRS endemicity in Yunnan province (southwestern China) and identified 37 SEOV-positive rats (36 Rattus norvegicus and 1 Rattus tanezumi). A 3-year surveillance of HFRS epidemics and dynamics of rodent reservoir density and virus prevalence implied a potential correlation between them. The subsequent meta-transcriptomic sequencing and phylogenetic analyses revealed three SEOV variants, among which two are completely novel. The ancestral character state reconstruction (ACSR) analysis based on both novel variants and documented strains from 5 continents demonstrated that SEOV appeared to originate near the southwestern area (Yunnan-Kweichow Plateau) of China, then could spread to other regions and countries by their rodent carriers, resulting in a global distribution today. In summary, these data furthered the understanding regards genetic diversity and the potential origin for SEOV. However, the expanding endemic foci in the province suggest that the virus is spreading over a wider region and is much more diverse than previous depicted, which means that increased sampling is necessary.

Metagenomic sequencing has transformed our understanding of viral diversity in wildlife and its threat to human health. Despite this progress, many studies have lacked systematic and ecologically informed sampling, which has left numerous potentially emergent viruses undiscovered, and the drivers of their ecology and evolution poorly understood. We conducted an extensive analysis of viruses in the lung, spleen, and gut of 1,688 animals from 38 mammalian species across 428 sites in Yunnan, China—a hotspot for zoonotic diseases. We identified 162 mammalian viral species, including 102 novel species and 24 posing potential risks to humans due to their relationships with known pathogens associated with serious diseases and their ability to cross major host species barriers. Our findings offer an in-depth view of virus organotropism, cross-species transmission, host sharing patterns, and the ecological factors influencing viral evolution, all of which are critical for anticipating and mitigating future zoonotic outbreaks. ### Competing Interest Statement The authors have declared no competing interest.

Metagenomic sequencing has transformed the understanding of viral diversity in wildlife and the potential threats these viruses pose to human health.Despite this progress,such sequencing studies often have lacked systematic and ecologically informed sampling,thereby likely missing many potential human pathogens and the drivers behind their ecology,evolution and emergence.We conducted an extensive search for viruses in the lungs,spleens and guts of 1688 mammals from 38 species across 428 sites in Yunnan Province,China-a hotspot for zoonoses emergence.We identified 162 mammalian viruses,including 102 new ones and 24 posing potential risks to humans due to their relationships with known human pathogens associated with serious diseases or their ability to cross major host species barriers.Our findings offer an in-depth view of virus organotropism,cross-host associations,host sharing patterns,and the ecological factors influencing viral evolution,all of which are critical for anticipating and mitigating future zoonotic outbreaks.

Ticks, an arthropod known for transmitting various pathogens such as viruses, bacteria, and fungi, pose a perpetual public health concern. A total of 2,570 ticks collected from Nujiang Prefecture in Yunnan Province between 2017 and 2022 were included in the study. Through the meta-transcriptomic sequencing of four locally distributed tick species, we identified 13 RNA viruses belonging to eight viral families, namely, Phenuiviridae, Nairoviridae, Peribunyaviridae, Flaviviridae, Chuviridae, Rhabdoviridae, Orthomyxoviridae, and Totiviridae. The most prevalent viruses were members of the order Bunyavirales, including three of Phenuiviridae, two were classified as Peribunyaviridae, and one was associated with Nairoviridae. However, whether they pose a threat to human health still remains unclear. Indeed, this study revealed the genetic diversity of tick species and tick-borne viruses in Nujiang Prefecture based on COI gene and tick-borne virus research. These data clarified the genetic evolution of some RNA viruses and furthered our understanding of the distribution pattern of tick-borne pathogens, highlighting the importance and necessity of monitoring tick-borne pathogens.

Bats are reservoir hosts for many zoonotic viruses. Despite this, relatively little is known about the diversity and abundance of viruses within individual bats, and hence the frequency of virus co-infection and spillover among them. We characterize the mammal-associated viruses in 149 individual bats sampled from Yunnan province, China, using an unbiased meta-transcriptomics approach. This reveals a high frequency of virus co-infection (simultaneous infection of bat individuals by multiple viral species) and spillover among the animals studied, which may in turn facilitate virus recombination and reassortment. Of note, we identify five viral species that are likely to be pathogenic to humans or livestock, based on phylogenetic relatedness to known pathogens or in vitro receptor binding assays. This includes a novel recombinant SARS-like coronavirus that is closely related to both SARS-CoV and SARS-CoV-2. In vitro assays indicate that this recombinant virus can utilize the human ACE2 receptor such that it is likely to be of increased emergence risk. Our study highlights the common occurrence of co-infection and spillover of bat viruses and their implications for virus emergence.

Leptospirosis has been identified as a zoonotic disease caused by pathogenic spirochetes of the bacterial genus Leptospira. Rodents are considered the primary hosts of these bacteria, whereas many recent studies suggest that bats may serve as potential natural reservoirs. However, studies on pathogenic spirochetes hosted by bat populations still need to be completed in China. In this study, a total of 276 bats belonging to five genera collected in Yunnan Province (Southwest China) from 2017 to 2021 were included in the screening. Pathogenic spirochetes were detected by PCR amplification and sequencing targeting four genes (rrs, secY, flaB, and LipL32), resulting in 17 positive samples. Phylogenetic analysis based on multi-loci concatenated sequences, inferred by MLST approach, identified the strains as two novel Leptospira species within the pathogenic group. Of note, only Rousettus leschenaultii was found to harbor these spirochetes, suggesting it may be one of the potential natural reservoirs in circulating leptospires in this region. Nevertheless, the pathogenesis and transmission dynamics still need to be fully understood, requiring in-depth studies on other animals and the surrounding population.

Hantaviruses are zoonotic pathogens that are widely distributed worldwide. Hantaan virus (HTNV) and Seoul virus (SEOV) are two most common hantaviruses that infect humans and cause hemorrhagic fever with renal syndrome (HFRS). Rapid and sensitive detection of HTNV and SEOV are crucial for surveillance, clinical treatment and management of HFRS. This study aimed to develop a rapid HFman probe-based mulstiplex reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to simultaneously detect HTNV and SEOV. A novel multiplex RT-LAMP assay was developed, and 46 serum samples obtained from clinically suspected patients were used for evaluation. The novel RT-LAMP assay can detect as low as 3 copies/reaction of hantaviruses with a detection limit of 41 and 73 copies per reaction for HTNV and SEOV, respectively. A clinical evaluation showed that the consistencies of the multiplex RT-LAMP with RT-qPCR assay were 100% and 97.8% for HTNV and SEOV, respectively. In view of the high prevalence of HTNV and SEOV in rural areas with high rodent density, a colorimetric visual determination method was also developed for point-of-care testing (POCT) for the diagnosis of the two viruses. The novel multiplex RT-LAMP assay is a sensitive, specific, and efficient method for simultaneously detecting HTNV and SEOV.

Bartonella species has been validated as blood-borne bacteria in mammals and has a substantial opportunity to be harbored by a variety of hematophagous arthropod vectors. Bats, along with their ectoparasites, are recognized worldwide as one of the natural reservoir hosts for these bacteria. However, there have been few investigations of Bartonella bacteria toward a broad range of obligated bat ectoparasites in China. Here, molecular detection of Bartonella species was performed to survey the infection among bat ectoparasites and follow-up phylogenetic analyses to further characterize the evolutionary relationships of the genus. A total of 434 bat ectoparasites involving four types of arthropods, namely, bat mites, bat tick, bat fleas, and bat flies (further divided into traditionally fly-like bat flies and wingless bat flies) were collected in 10 trapping sites in Yunnan Province, southwestern China. Bartonella was detected by PCR amplification and sequencing through four gene target fragments (gltA, ftsZ, rpoB, and ITS). Accordingly, diverse Bartonella species were discovered, including both the validated species and the novel genotypes, which were characterized into several geographical regions with high prevalence. Phylogenetic analyses based on gltA and multi-locus concatenated sequences both demonstrated strong phylogeny–trait associations of Bartonella species from bats and their parasitic arthropods, suggesting the occurrence of host switches and emphasizing the potential connecting vector role of these ectoparasites. Nevertheless, the maintenance and transmission of Bartonella in both bat and hemoparasite populations have not been fully understood, as well as the risk of spillage to humans, which warrants in-depth experimental studies focusing on these mammals and their ectoparasites.

目的 了解云南省怒江州吸血蠓类携带流行性乙型脑炎病毒(Japanese encephalitis virus,JEV)情况,为流行性乙型脑炎(乙脑)的预防控制提供科学依据.方法 2017年7月在怒江州用诱蚊灯采集蠓类标本,研磨后将上清液接种BHK-21细胞进行病毒分离,并用逆转录聚合酶链式反应检测JEV核酸,测序获得JEVC/PrM基因序列后进行系统进化和核苷酸、氨基酸同源性分析.结果 在怒江州泸水、福贡和贡山县共捕获蠓类约15 000只,每300只蠓为1组,共52组,从泸水市捕获的一批蠓(NJC1718)中检测到JEV核酸,测序获得C/PrM基因核酸序列,进化分析显示,NJC1718株与基因Ⅰ型JEV处于同一进化分支,与2014年及2015年柬埔寨基因Ⅰ型分离株(639A37Cx-tri和C081)亲缘关系较近;与10株JEV基因Ⅰ型参考株(云南省5株、泰国1株和柬埔寨4株)的核苷酸和氨基酸同源性为90.8％～98.8％和96.2％～100.0％.结论 怒江州吸血蠓携带基因Ⅰ型JEV,表明当地蠓类在JEV传播中起一定作用,需加强该地区乙脑病例及其媒介蚊和蠓的监测和防控.

为了解云南省汉坦病毒的分子流行病学特征,本研究在云南省祥云县、泸西县、禄车县和楚雄市4个地区布点,采集鼠类动物标本,运用实时荧光RT-PCR检测鼠肺标本中的汉坦病毒中的汉滩病毒和首尔病毒核酸,通过PCR扩增S节段的全基因序列,并进行核苷酸同源性分析和遗传进化分析.结果 共捕获鼠类动物70份,包含10个鼠种,其中褐家鼠30只,占42.9％,其次是鼩鼱12只,占17.1％;核酸检测阳性标本19份,均为首尔病毒,检出率为27.1％,源自祥云县的标本检出率在四个地区中最高(32.0％).从鼠肺标本中扩增得到8条汉坦病毒S节段全基因序列,相似性为87.0％～99.7％,遗传进化分析显示基因序列聚集形成2个分支,一个分支属于S1亚型,另一个分支在遗传进化分析和blast比对分析中与其他亚型同源性均小于90％,属首尔病毒中独立的新的遗传进化分支,提示了云南省鼠类动物间多基因亚型首尔汉坦病毒流行,应进一步关注由宿主动物溢出感染人的潜力.

Bats are reservoir hosts for many zoonotic viruses. Despite this, relatively little is known about the diversity and abundance of viruses within bats at the level of individual animals, and hence the frequency of virus co-infection and inter-species transmission. Using an unbiased meta-transcriptomics approach we characterised the mammalian associated viruses present in 149 individual bats sampled from Yunnan province, China. This revealed a high frequency of virus co-infection and species spillover among the animals studied, with 12 viruses shared among different bat species, which in turn facilitates virus recombination and reassortment. Of note, we identified five viral species that are likely to be pathogenic to humans or livestock, including a novel recombinant SARS-like coronavirus that is closely related to both SARS-CoV-2 and SARS-CoV, with only five amino acid differences between its receptor-binding domain sequence and that of the earliest sequences of SARS-CoV-2. Functional analysis predicts that this recombinant coronavirus can utilize the human ACE2 receptor such that it is likely to be of high zoonotic risk. Our study highlights the common occurrence of inter-species transmission and co-infection of bat viruses, as well as their implications for virus emergence.

目的 进一步了解云南省肾综合征出血热(HFRS)宿主动物的种类和汉坦病毒自然感染情况,为云南省HFRS的防控提供科学依据.方法 收集2020年全省HFRS疫情资料;在固定监测点(泸西县、楚雄市和祥云县)和流动监测点临沧市(1区7县)进行宿主动物调查,捕获样本进行分类鉴定后,用直接免疫荧光法对肺组织标本进行HFRS抗原检测.结果 2020年云南省共报告HFRS病例213例,发病率为0.44/10万,病例主要集中在大理白族自治州和楚雄彝族自治州,占全省发病数的89.20％.此次调查共捕获宿主动物10属15种1 451只,其中固定监测点宿主动物优势种居民区为褐家鼠,野外为高山姬鼠和大绒鼠;流动监测点居民区优势种为黄胸鼠.固定监测点泸西县、楚雄市和祥云县宿主动物汉坦病毒阳性率分别为14.18％(37/261)、13.60％(17/125)和9.78％(22/225),流动监测点临沧市汉坦病毒阳性率为13.21％(111/840).阳性宿主动物为褐家鼠、黄胸鼠、斯氏家鼠、高山姬鼠、灰麝鼩、大绒鼠、臭鼩和四川短尾鼩.结论 云南省啮齿动物和食虫动物中汉坦病毒自然感染普遍存在,近年云南省HFRS发病处于较高的流行水平,因此需在全省加强防鼠灭鼠、病例监测、个人防护和疫苗接种为主的综合防控措施,进一步做好HFRS的监测防控工作.

目的 分析2013-2018年云南省肾综合征出血热(hemorrhagic fever with renal syndrome,HFRS)监测点宿主动物分布特点及其自然感染汉坦病毒(Hantavirus,HTV)的状况,为防控HFRS提供科学依据.方法 在云南省泸西、祥云、禄丰和大姚县的居住区和野外采用笼夜法和夹夜法捕鼠.用直接免疫荧光法检测鼠肺HTV抗原.使用Excel 2007与SPSS 20.0软件处理数据和系统分析.结果 2013-2018年在上述4个监测县共布放鼠夹66 716夹次,捕获鼠型动物2506只,平均鼠密度为3.76％.其中居住区鼠密度3.34％,野外鼠密度4.35％,居住区和野外鼠密度差异有统计学意义(x2=45.943,P＜0.01).居住区共捕鼠8种,优势鼠种为褐家鼠,HTV带病毒率为9.22％;野外捕鼠26种,优势鼠种为大绒鼠,HTV带病毒率14.83％.结论 云南省HFRS监测点主要宿主动物密度和带病毒率均较高,仍需加强以防鼠灭鼠为主的综合防控,并在重点地区落实开展国家HFRS扩大免疫规划项目工作.

Objective:To study the whole genome and genetic evolution characteristics of the Banna virus carried by Culicoides in Cangyuan County. Methods:Culicoides were collected in Cangyuan county in 2018. After grinding, the supernatant was inoculated onto BHK-21 cells for virus isolation. At the same time, the virus carrying status of Culicoides was detected by metatranscriptomics technology. Perl, Mafft and other bioinformatics softwares were used for sequence alignment, homology and molecular genetic evolution analysis. Results:Through metatranscriptomics method, the whole genome of Banna virus CYC1801 strain was obtained. Analysis of the whole genome showed that segment 4 and segment 7 are closely related to the Vietnamese strains, other segments are closely related to China strains. According to the phylogenetic analysis of the segment 12 gene, CYC1801 and the A2 gene subtype are in the same evolutionary branch and belong to the A2 gene subtype of Banna virus.Conclusions:Banna virus was carried by Culicoides collected in Cangyuan county, which may cause disease epidemic as a vector, so monitoring and prevention should be strengthened.

目的 了解云南省蛛蝇和蝠蝇中沃尔巴克氏体(Wolbachia)的感染状况及其基因型别.方法 2012-2017年,在云南省芒市、腾冲、畹町、孟连、勐腊、祥云、墨江、永德县和保山市隆阳区共9个采样点采集蝙蝠体表蛛蝇和蝠蝇,PCR扩增沃尔巴克氏体的核糖体小亚基16S(16S rRNA)和细菌表皮蛋白(wsp)基因并测序,对所有16S rRNA和wsp基因进行系统进化分析.结果 2012-2017年,在云南省9个采集点采集蝙蝠体表蛛蝇747只(48批)和蝠蝇19只(6批),检测54批标本获得沃尔巴克氏体16S rRNA和wsp基因序列各24条.云南省蝙蝠体表蛛蝇和蝠蝇中,蛛虱蝇属(Phthiridium)6批和Eucampsipoda属37批中检出沃尔巴克氏体阳性分别为2批(2/6)和19批(19/37),蛛虱蝇属(Nycteribia)(0/4)和笔虱蝇属(Penicillidia)(0/1)未检出;蝠蝇均属于蝠虱蝇属(Brachytarsina),6批中检出沃尔巴克氏体阳性的有3批(3/6).16S rRNA基因系统进化结果提示,24株沃尔巴克氏体均位于一个大的分支中,与1株来源于白蚁(DQ837204)的同源性最高达98.80％～100％.wsp基因的系统进化分析提示,24株沃尔巴克氏体位于B组和F组基因型.结论 沃尔巴克氏体在云南省蛛蝇和蝠蝇中广泛而长期存在,wsp基因系统进化分析属于B组和F组基因型.

目的 评估“攻击性”作为犬类狂犬病典型症状在监测和临床诊断中的作用与意义.方法 2011-2017年,从云南省收集到可疑狂犬病犬脑组织标本239份,使用DFA检验.将攻击性定义为咬人和动物总数≥2,并按可疑犬咬人和动物数,将可疑病犬分为“0”,“1”,“2”,“3”和“≥4”共5类.结果 239只可疑犬,总阳性率为64.02％;当咬人和动物数=0,1,2,3和≥4时,可疑犬阳性率分别为21.57％、61.76％、73.34％、77.50％和88.37％.当咬人和动物数≥2时,阳性率为78.57％.统计学分析显示:“1”类可疑犬阳性率与“2”和“3”类无统计学差异.结论 本研究结果支持WHO关于“攻击性”定义示例说明(咬两人或更多人或者动物和/或无生命物体),并为犬伤风险评估提供了参考数据.本研究建议未来犬类狂犬病监测项目的策划者和实施者在采用WHO目前关于“攻击性”的定义时,并关注咬人或动物数=1的可疑犬,如肇事犬死亡或逃逸,应深入调查其是否有吞食或咬或攻击异物(如石头、木块、移动车辆等)等异常行为.