Great genetic diversity of vector-borne bacteria and protozoan in wild rodents from Guangxi, China.

BACKGROUND: Rodents are recognized as the hosts of many vector-borne bacteria and protozoan parasites and play an important role in their transmission and maintenance. Intensive studies have focused on their infections in vectors, especially in ticks, however, vector-borne bacterial and protozoan infections in rodents are poorly understood although human cases presenting with fever may due to their infection have been found. METHODS: From May to October 2019, 192 wild rodents were trapped in wild environment of Guangxi Province, and the spleen samples were collected to reveal the presence of vector-borne bacterial and protozoan infections in them. The microorganisms in rodents were identified by detecting their DNA using (semi-)nested PCR. All the PCR products of the expected size were subjected to sequencing, and then analyzed by BLASTn. Furthermore, all the recovered sequences were subjected to nucleotide identity and phylogenetic analyses. RESULTS: As a result, 192 rodents representing seven species were captured, and Bandicota indica were the dominant species, followed by Rattus andamanensis. Based on the (semi-)nested PCR, our results suggested that Anaplasma bovis, Anaplasma capra, Anaplasma ovis, Anaplasma phagocytophilum, "Candidatus Neoehrlichia mikurensis", "Candidatus E. hainanensis", "Candidatus E. zunyiensis", three uncultured Ehrlichia spp., Bartonella coopersplainsensis, Bartonella tribocorum, Bartonella rattimassiliensis, Bartonella silvatica, two uncultured Bartonella spp., Babesia microti and diverse Hepatozoon were identified in six rodent species. More importantly, six species (including two Anaplasma, two Bartonella, "Ca. N. mikurensis" and Bab. microti) are zoonotic pathogens except Anaplasma bovis and Anaplasma ovis with zoonotic potential. Furthermore, dual infection was observed between different microorganisms, and the most common type of co-infection is between "Ca. N. mikurensis" and other microorganisms. Additionally, potential novel Bartonella species and Hepatozoon species demonstrated the presence of more diverse rodent-associated Bartonella and Hepatozoon. CONCLUSIONS: The results in this work indicated great genetic diversity of vector-borne infections in wild rodents, and highlighted the potential risk of human pathogens transmitted from rodents to humans through vectors.

Circulation of multiple Rickettsiales bacteria in ticks from Sichuan province, Southwest China.

During 2021, 403 ticks including Haemaphysalis qinghaiensis, Ixodes ovatus, Ixodes acutitarsus, and Rhipicephalus microplus were collected from three sites (590, 310, and 576 km away from each other) in Sichuan Province, China. A total of nine Rickettsiales species were identified in them, including three Rickettsia spp., five Anaplasma spp., and one Ehrlichia sp. Anaplasma ovis and a novel Rickettsia sp. named "Candidatus Rickettsia liangshanensis" were characterized in I. ovatus ticks from Liangshan, with positive rates of 11.11% and 45.56%, respectively. Anaplasma capra (13.33%) and Anaplasma bovis (15.33%) were detected in H. qinghaiensis ticks from Maerkang. Phylogenetic analysis based on 16S rRNA, gltA, and groEL gene sequences indicated that the A. bovis strains were divided into two groups. Additionally, a novel Ehrlichia species named "Candidatus Ehrlichia maerkangensis" was identified. It is closely related to "Candidatus Ehrlichia zunyiensis" which was previously reported in Berylmys bowersi rats from Zunyi City, Southwest China. In R. microplus from Mianyang, "Candidatus Rickettsia jingxinensis" was detected with a high prevalence (92.99%). Notably, a variant of R. raoultii was identified in I. acutitarsus (33.33%). This may be the first Rickettsiales bacterium reported in I. acutitarsus. Our results reveal the remarkable biodiversity of Rickettsiales in this area. Some of these bacteria are human pathogens, indicating the potential exposure risk to local people.

Emergence of ehrlichiosis by a new tick-borne Ehrlichia species in China.

OBJECTIVES: From March to June 2021, the reported number of clinically diagnosed endemic typhus in Anhui and Hubei provinces of China nearly increased four-fold compared with the monthly average numbers in last 5 years. An etiological and epidemiological investigation was initiated. METHODS: The clinical specimens from the reported patients and the potential vector ticks were collected for molecular and serological detection, as well as cell culturing assay to identify the potential pathogen. RESULTS: Polymerase chain reaction and sequence analysis of rrs and groEL showed that the pathogen from these patients was Ehrlichia sp., isolated from Haemaphysalis longicornis attached to these patients. The phylogenetic analysis based on 39 Ehrlichia genomes suggested that it should be taxonomically classified as a novel species, tentatively named "Candidatus Ehrlichia erythraense". A total of 19 of 106 cases were confirmed as Candidatus Ehrlichia erythraense infections by polymerase chain reaction, sequencing, and/or serological tests. The most frequent symptoms were fever (100%), rashes (100%), asthenia (100%), anorexia (100%), and myalgia (79%). CONCLUSION: The occurrence of the disease presenting with fever and rashes in Anhui and Hubei provinces was caused by a novel species of the genus Ehrlichia; physicians need to be aware of this newly-discovered pathogen to ensure appropriate testing, treatment, and regional surveillance.

Prevalence of Spotted Fever Group Rickettsia and Candidatus Lariskella in Multiple Tick Species from Guizhou Province, China.

Rickettsiales (Rickettsia spp., Ehrlichia spp., and Anaplasma spp., etc.) are generally recognized as potentially emerging tick-borne pathogens. However, some bacteria and areas in China remain uninvestigated. In this study, we collected 113 ticks from mammals in Guizhou Province, Southwest China, and screened for the Rickettsiales bacteria. Subsequently, two spotted fever group Rickettsia species and one Candidatus Lariskella sp. were detected and characterized. "Candidatus Rickettsia jingxinensis" was detected in Rhipicephalus microplus (1/1), Haemaphysalis flava (1/3, 33.33%), Haemaphysalis kitaokai (1/3), and Ixodes sinensis (4/101, 3.96%), whereas Rickettsia monacensis was positive in H. flava (1/3), H. kitaokai (2/3), and I. sinensis ticks (74/101, 73.27%). At least two variants/sub-genotypes were identified in the R. monacensis isolates, and the strikingly high prevalence of R. monacensis may suggest a risk of human infection. Unexpectedly, a Candidatus Lariskella sp. belonging to the family Candidatus Midichloriaceae was detected from Ixodes ovatus (1/4) and I. sinensis (10/101, 9.90%). The gltA and groEL gene sequences were successfully obtained, and they show the highest (74.63-74.89% and 73.31%) similarities to "Candidatus Midichloria mitochondrii", respectively. Herein, we name the species "Candidatus Lariskella guizhouensis". These may be the first recovered gltA and groEL sequences of the genus Candidatus Lariskella.

Severe community-acquired pneumonia caused by Chlamydia psittaci genotype E/B strain circulating among geese in Lishui city, Zhejiang province, China.

Between November 2021 and January 2022, four patients of community-acquired pneumonia were admitted to the hospitals in Lishui city, Zhejiang province, China. Their main clinical manifestations were fever and dry cough as well as radiographic infiltrate, but the empiric antimicrobial therapy or traditional Chinese medicine was not effective for their illness. Clinical specimens from the patients as well as environmental and poultry specimens were collected for the determination of the causative pathogen. The ompA gene and seven housekeeping genes of Chlamydia psittaci were successfully amplified from all the patients, and the sequences of each gene were identical to one another, suggesting that they were infected by the same strain of C. psittaci. A novel strain of C. psittaci (LS strain) was isolated from the bronchoalveolar lavage fluid of patient 2 and its whole genome was obtained. Phylogenetic analyses based on the whole-genome sequences showed that the isolate is most closely related to the strain (WS/RT/E30) identified as genotype E/B. In addition, The ompA gene and four housekeeping genes of C. psittaci were also amplified from two of four faeces samples of geese at the home of patient 2, and the sequences from geese were 100% identical to those from the patients. Accordingly, these cases could be attributed to a circulating C. psittaci strain of genotype E/B in the local geese. Therefore, there is an urgent need to strengthen the regional surveillance on C. psittaci among poultry and humans for prevention and control of the outbreak of psittacosis in the city.

Ehrlichia, Coxiella and Bartonella infections in rodents from Guizhou Province, Southwest China.

Rodents are generally recognized to be the reservoir hosts of a great many zoonotic pathogens. In some areas of China, rodent-borne pathogens, as well as the role of rodents in the natural cycle of these pathogens, are still poorly investigated. To increase our knowledge on the distribution and epidemiology of rodent-borne bacterial pathogens, 81 rodent liver samples were collected in three locations of Guizhou province located in Southwest China, and screened for the presence of Ehrlichia, Coxiella, and Bartonella in them. A putative novel Ehrlichia species was identified in 5 Berylmys bowersi samples (100%, 5/5). Its 16S rRNA, gltA, and groEL genes have highest 99.84%, 89.11%, and 98.02% identities to those from known Ehrlichia species, and form distinct clades in the phylogenetic trees. Herein we name it "Candidatus Ehrlichia zunyiensis". Bartonella was tested positive in 8 A. agrarius (striped field mouse), 2 A. chevrieri (Chevrier's field mouse), 1 R. norvegicus (Norway rat), 1 N. confucianus, and 1 N. lotipes, with a total positive rate of 16.05% (13/81). Sequence analysis indicated high genetic diversity in these Bartonella strains. Unexpectedly, two Coxiella strains were identified from the rodents (1 Niviventer confucianus and 1 Mus pahari). Genetic and phylogenetic analysis indicated that both of them are closely related to the Coxiella endosymbiont of ticks. This result supported previous conjectures that vertebrate hosts such as rodents may play a role in the preservation and transmission of Coxiella endosymbiont of ticks.

Molecular Survey of Vector-Borne Pathogens in Ticks, Sheep Keds, and Domestic Animals from Ngawa, Southwest China.

Vector-borne pathogens are mainly transmitted by blood-feeding arthropods such as ticks, mosquitoes, fleas, lice, mites, etc. They pose a significant threat to animal and human health due to their worldwide distribution. Although much work has been performed on these pathogens, some neglected areas and undiscovered pathogens are still to be further researched. In this study, ticks (Haemaphysalis qinghaiensis), sheep keds (Melophagus ovinus), and blood samples from yaks and goats were collected in Ngawa Tibetan and Qiang Autonomous Prefecture located on the eastern edge of the Qinghai-Tibet Plateau, Southwest China. Several vector-borne bacterial pathogens were screened and studied. Anaplasma bovis strains representing novel genotypes were detected in ticks (8.83%, 37/419), yak blood samples (45.71%, 64/140), and goat blood samples (58.93%, 33/56). Two spotted fever group (SFG) Rickettsiae, Candidatus Rickettsia jingxinensis, and a novel Rickettsia species named Candidatus Rickettsia hongyuanensis were identified in ticks. Another Rickettsia species closely related to the Rickettsia endosymbiont of Polydesmus complanatus was also detected in ticks. Furthermore, a Coxiella species was detected in ticks (3.34%, 14/419), keds (1.89%, 2/106), and yak blood (0.71%, 1/140). Interestingly, another Coxiella species and a Coxiella-like bacterium were detected in a tick and a goat blood sample, respectively. These results indicate the remarkable diversity of vector-borne pathogens circulating in this area. Further investigations on their pathogenicity to humans and domestic animals are still needed.

Anaplasma bovis Infection in Fever and Thrombocytopenia Patients - Anhui Province, China, 2021.

What is already known about this topic?: The genusAnaplasma contains seven recognized bacterial species, mainly transmitted by tick bites. The two species, A. phagocytophilum and A. capra, are known commonly to cause diseases in humans. What is added by this report?: Anaplasma bovis was initially thought to be only an animal agent until the first patient case was reported in 2019. This study investigated another two patients who became sick within one month in the same township and were infected with A. bovis in Anhui Province. What are the implications for public health practice?: This study suggested that more A. bovis-infected patients may exist in this area and that patients with anaplasmosis require an early and specific diagnosis.

High Prevalence of Rickettsia bellii in Mosquitoes From Eastern China.

Mosquitoes are the most important vectors carrying significant numbers of human pathogens. Recent studies implicated that mosquitoes play an important role in circulation and transmission of multiple Rickettsia species. In this study, Rickettsia bellii was identified in four mosquito species (Culex pipiens, C. tritaeniorhynchus, Aedes albopictus, and Anopheles sinensis) collected from three Eastern China provinces during 2019-2020. Rickettsia bellii was detected in 37.50 and 26.32% of the C. pipiens pools from Beijing and Jiangsu province, respectively. In C. tritaeniorhynchus and An. sinensis from Shandong, the infection rate is 20.00 and 6.25%, respectively. Additionally, three Ae. albopictus pools (3/42, 7.14%) from Beijing were also detected positive for R. bellii. Genetic and phylogenetic analysis on 16S, gltA, and groEL genes indicates that sequences from all these strains are highly homologous and closely related to other R. bellii strains. This is the first report that Ae. albopictus and C. tritaeniorhynchus harbor R. bellii. The wide host range and high infection rate in certain areas may dramatically increase the exposure of R. bellii to human and other vertebrates. The role of mosquitoes in transmission of rickettsiosis and its potential risk to public health should be further considered.

Identification of Rickettsia spp., Anaplasma spp., and an Ehrlichia canis-like agent in Rhipicephalus microplus from Southwest and South-Central China.

Rhipicephalus microplus is considered to be the most important tick infesting cattle, buffalo, horse, goats as well as other animals. They transmit diseases between domestic animals and act as vectors of a variety of zoonotic pathogens. Although pathogens harbored by R. microplus have been extensively studied, the Rickettsiales pathogens vectored by R. microplus in some areas of China remained largely unexplored. From August to October 2020, a total of 291 R. microplus ticks were collected from goats and cattle in three Southern China provinces, Guangxi (n = 138), Sichuan (n = 120) and Hubei (n = 33) provinces. Phylogenetic analysis based on COI gene sequences shows that these ticks are divided into three distinct clades, indicating the remarkable genetic diversity of R. microplus ticks in China. These samples were subsequently screened for the presence of Rickettsia, Anaplasma and Ehrlichia using conventional PCR and sequencing. Subsequently, five bacterial species were identified. Out of the 120 tick DNA samples from Sichuan province, 35.83% (43/120) were positive for Rickettsia sp. belonging to spotted fever group (SFG), 12.50% (15/120) were positive for Anaplasma marginale and 0.83% (1/120) was identified as A. platys. From the 138 DNA samples from Guangxi province, an Ehrlichia canis-like and Rickettsia sp. were detected, with a positive rate of 11.59% (16/138) and 2.17% (3/138), respectively. A. capra DNA was detected in 4 out of 33 (12.12%) samples from Hubei province. Notably, the 16S, gltA and groEL sequences of the E. canis-like are closely related to the E. canis strain previously identified from China, and form a distinct cluster in the phylogenetic trees. Collectively, our results expand the knowledge on tick-borne Rickettsiales pathogens in China. Because the state of engorgement of ticks was not recorded, it is not clear at this stage whether these pathogens are infecting the ticks or are simply present in the blood meal. Given the public health significance of SFG Rickettsia, A. capra, A. platys and E. canis, a thorough investigation of the diversity and presence of pathogens in R. microplus in areas with tick-associated diseases are needed.

Diverse and abundant resistome in terrestrial and aquatic vertebrates revealed by transcriptional analysis.

Despite increasing evidence that antibiotic resistant pathogens are shared among humans and animals, the diversity, abundance and patterns of spread of antibiotic resistance genes (ARGs) in wildlife remains unclear. We identified 194 ARGs associated with phenotypic resistance to 13 types of antibiotic in meta-transcriptomic data generated from a broad range of lower vertebrates residing in both terrestrial and aquatic habitats. These ARGs, confirmed by PCR, included those that shared high sequence similarity to clinical isolates of public health concern. Notably, the lower vertebrate resistome varied by ecological niche of the host sampled. The resistomes in marine fish shared high similarity and were characterized by very high abundance, distinct from that observed in other habitats. An assessment of ARG mobility found that ARGs in marine fish were frequently co-localized with mobile elements, indicating that they were likely spread by horizontal gene transfer. Together, these data reveal the remarkable diversity and transcriptional levels of ARGs in lower vertebrates, and suggest that these wildlife species might play an important role in the global spread of ARGs.

Identification and characterization of Jingmen tick virus in rodents from Xinjiang, China.

Jingmen tick virus (JMTV) is a recently identified virus which provides an unexpected connection between segmented and unsegmented RNA viruses. Recent investigations reveal that JMTV including JMTV-like virus (Alongshan virus) could be associated with human disease, suggesting the significance of JMTV in public health. To better understand the genetic diversity and host range of JMTV, a total of 164 rodents representing 8 species were collected in Qapqal Xibe county of Xinjiang Uygur Autonomous Region, China, and were screened for JMTVs using RT- PCR. Consequently, JMTV was identified in 42 rodents including 23 Microtus arvalis voles (24.5%), 9 Apodemus uralensis mice (29.0%), 5 Mus musculus mice, 1 Rhombomys opimus gerbil, 1 Meriones tamariscinus gerbil, 1 Meriones libycus gerbil, 1 Cricetulus migratorius hamster and 1 Microtus gregalis vole. Interestingly, nearly complete genome sequences were successfully recovered from 7 JMTV positive samples. Although the newly identified rodent JMTVs were closely related to those previously identified in ticks from China, based on the phylogenetic analysis of the S1, S2 and S3 segments, the newly identified rodent viruses clustered into two genetic groups. One group comprised of viruses only found in M. arvalis, while another group included viruses from A. uralensis, C. migratorius and M. gregalis. However, all rodent viruses clustered together in the S4 tree. Considering rodents live in close proximity to humans, more efforts are needed to investigate the role of rodents in the evolution and transmission of JMTV in nature.

Metatranscriptomics reveals that the death of a Mongolian wild ass was caused by Clostridium botulinum in Inner Mongolia, China.

Clostridium botulinum is an important pathogen that causes botulism in humans and animals worldwide. C. botulinum group III strains, which produce a single toxin of type C or D or a chimeric toxin of type C/D or D/C, are responsible for botulism in a wide range of animal species including cattle and birds. We used unbiased high-throughput RNA sequencing (i.e., metatranscriptomics) to identify a strain of group III C. botulinum from a deceased Mongolian wild ass (Equus hemionus). The strain was closely related to some European strains. Genetic analysis of the recovered bacterial sequences showed that the C. botulinum strain identified might represent a type C/D strain of group III. Infection by C. botulinum producing the mosaic toxin of type C/D is the most likely cause of the death of the wild ass.

Diversity and circulation of Jingmen tick virus in ticks and mammals.

Since its initial identification in ticks in 2010, Jingmen tick virus (JMTV) has been described in cattle, rodents, and primates. To better understand the diversity, evolution, and transmission of JMTV, we sampled 215 ticks, 104 cattle bloods, 216 bats, and 119 rodents in Wenzhou city, Zhejiang Province, China as well as 240 bats from Guizhou and Henan Provinces. JMTV was identified in 107 ticks (from two species), 54 bats (eleven species), 8 rodents (three species), and 10 cattle, with prevalence levels of 49.8, 11.8, 6.7, and 9.6 per cent, respectively, suggesting that bats may be a natural reservoir of JMTV. Phylogenetic analyses revealed that all the newly identified JMTVs were closely related to each other and to previously described viruses. Additionally, all tick and mammalian JMTV sampled in Wenzhou shared a consistent genomic structure, suggesting that the virus can cocirculate between ticks and mammals without observable variation in genome organization. All JMTVs sampled globally could be divided into two phylogenetic groups, Mantel tests suggested that geographic isolation, rather than host species, may be the main driver of JMTV diversity. However, the exact geographical origin of JMTV was difficult to determine, suggesting that this virus has a complex evolutionary history.

Redefining the invertebrate RNA virosphere.

Current knowledge of RNA virus biodiversity is both biased and fragmentary, reflecting a focus on culturable or disease-causing agents. Here we profile the transcriptomes of over 220 invertebrate species sampled across nine animal phyla and report the discovery of 1,445 RNA viruses, including some that are sufficiently divergent to comprise new families. The identified viruses fill major gaps in the RNA virus phylogeny and reveal an evolutionary history that is characterized by both host switching and co-divergence. The invertebrate virome also reveals remarkable genomic flexibility that includes frequent recombination, lateral gene transfer among viruses and hosts, gene gain and loss, and complex genomic rearrangements. Together, these data present a view of the RNA virosphere that is more phylogenetically and genomically diverse than that depicted in current classification schemes and provide a more solid foundation for studies in virus ecology and evolution.

Divergent Viruses Discovered in Arthropods and Vertebrates Revise the Evolutionary History of the Flaviviridae and Related Viruses.

UNLABELLED: Viruses of the family Flaviviridae are important pathogens of humans and other animals and are currently classified into four genera. To better understand their diversity, evolutionary history, and genomic flexibility, we used transcriptome sequencing (RNA-seq) to search for the viruses related to the Flaviviridae in a range of potential invertebrate and vertebrate hosts. Accordingly, we recovered the full genomes of five segmented jingmenviruses and 12 distant relatives of the known Flaviviridae ("flavi-like" viruses) from a range of arthropod species. Although these viruses are highly divergent, they share a similar genomic plan and common ancestry with the Flaviviridae in the NS3 and NS5 regions. Remarkably, although these viruses fill in major gaps in the phylogenetic diversity of the Flaviviridae, genomic comparisons reveal important changes in genome structure, genome size, and replication/gene regulation strategy during evolutionary history. In addition, the wide diversity of flavi-like viruses found in invertebrates, as well as their deep phylogenetic positions, suggests that they may represent the ancestral forms from which the vertebrate-infecting viruses evolved. For the vertebrate viruses, we expanded the previously mammal-only pegivirus-hepacivirus group to include a virus from the graceful catshark (Proscyllium habereri), which in turn implies that these viruses possess a larger host range than is currently known. In sum, our data show that the Flaviviridae infect a far wider range of hosts and exhibit greater diversity in genome structure than previously anticipated. IMPORTANCE: The family Flaviviridae of RNA viruses contains several notorious human pathogens, including dengue virus, West Nile virus, and hepatitis C virus. To date, however, our understanding of the biodiversity and evolution of the Flaviviridae has largely been directed toward vertebrate hosts and their blood-feeding arthropod vectors. Therefore, we investigated an expanded group of potential arthropod and vertebrate host species that have generally been ignored by surveillance programs. Remarkably, these species contained diverse flaviviruses and related viruses that are characterized by major changes in genome size and genome structure, such that these traits are more flexible than previously thought. More generally, these data suggest that arthropods may be the ultimate reservoir of the Flaviviridae and related viruses, harboring considerable genetic and phenotypic diversity. In sum, this study revises the traditional view on the evolutionary history, host range, and genomic structures of a major group of RNA viruses.

Unprecedented genomic diversity of RNA viruses in arthropods reveals the ancestry of negative-sense RNA viruses.

Although arthropods are important viral vectors, the biodiversity of arthropod viruses, as well as the role that arthropods have played in viral origins and evolution, is unclear. Through RNA sequencing of 70 arthropod species we discovered 112 novel viruses that appear to be ancestral to much of the documented genetic diversity of negative-sense RNA viruses, a number of which are also present as endogenous genomic copies. With this greatly enriched diversity we revealed that arthropods contain viruses that fall basal to major virus groups, including the vertebrate-specific arenaviruses, filoviruses, hantaviruses, influenza viruses, lyssaviruses, and paramyxoviruses. We similarly documented a remarkable diversity of genome structures in arthropod viruses, including a putative circular form, that sheds new light on the evolution of genome organization. Hence, arthropods are a major reservoir of viral genetic diversity and have likely been central to viral evolution.

A tick-borne segmented RNA virus contains genome segments derived from unsegmented viral ancestors.

Although segmented and unsegmented RNA viruses are commonplace, the evolutionary links between these two very different forms of genome organization are unclear. We report the discovery and characterization of a tick-borne virus--Jingmen tick virus (JMTV)--that reveals an unexpected connection between segmented and unsegmented RNA viruses. The JMTV genome comprises four segments, two of which are related to the nonstructural protein genes of the genus Flavivirus (family Flaviviridae), whereas the remaining segments are unique to this virus, have no known homologs, and contain a number of features indicative of structural protein genes. Remarkably, homology searching revealed that sequences related to JMTV were present in the cDNA library from Toxocara canis (dog roundworm; Nematoda), and that shared strong sequence and structural resemblances. Epidemiological studies showed that JMTV is distributed in tick populations across China, especially Rhipicephalus and Haemaphysalis spp., and experiences frequent host-switching and genomic reassortment. To our knowledge, JMTV is the first example of a segmented RNA virus with a genome derived in part from unsegmented viral ancestors.

Co-circulation of multiple hemorrhagic fever diseases with distinct clinical characteristics in Dandong, China.

Hemorrhagic fevers (HF) caused by viruses and bacteria are a major public health problem in China and characterized by variable clinical manifestations, such that it is often difficult to achieve accurate diagnosis and treatment. The causes of HF in 85 patients admitted to Dandong hospital, China, between 2011-2012 were determined by serological and PCR tests. Of these, 34 patients were diagnosed with Huaiyangshan hemorrhagic fever (HYSHF), 34 with Hemorrhagic Fever with Renal Syndrome (HFRS), one with murine typhus, and one with scrub typhus. Etiologic agents could not be determined in the 15 remaining patients. Phylogenetic analyses of recovered bacterial and viral sequences revealed that the causative infectious agents were closely related to those described in other geographical regions. As these diseases have no distinctive clinical features in their early stage, only 13 patients were initially accurately diagnosed. The distinctive clinical features of HFRS and HYSHF developed during disease progression. Enlarged lymph nodes, cough, sputum, and diarrhea were more common in HYSHF patients, while more HFRS cases presented with headache, sore throat, oliguria, percussion pain kidney area, and petechiae. Additionally, HYSHF patients displayed significantly lower levels of white blood cells (WBC), higher levels of creations kinase (CK) and alanine aminotransferase (ALT), while HFRS patients presented with an elevation of blood urea nitrogen (BUN) and creatinine (CREA). These clinical features will assist in the accurate diagnosis of both HYSHF and HFRS. Overall, our data reveal the complexity of pathogens causing HFs in a single Chinese hospital, and highlight the need for accurate early diagnosis and a better understanding of their distinctive clinical features.