Characterization of a pangolin SARS-CoV-2-related virus isolate that uses the human ACE2 receptor.

Various SARS-CoV-2-related coronaviruses have been increasingly identified in pangolins, showing a potential threat to humans. Here we report the infectivity and pathogenicity of the SARS-CoV-2-related virus, PCoV-GX/P2V, which was isolated from a Malayan pangolin (Manis javanica). PCoV-GX/P2V could grow in human hepatoma, colorectal adenocarcinoma cells, and human primary nasal epithelial cells. It replicated more efficiently in cells expressing human angiotensin-converting enzyme 2 (hACE2) as SARS-CoV-2 did. After intranasal inoculation to the hACE2-transgenic mice, PCoV-GX/P2V not only replicated in nasal turbinate and lungs, but also caused interstitial pneumonia, characterized by infiltration of mixed inflammatory cells and multifocal alveolar hemorrhage. Existing population immunity established by SARS-CoV-2 infection and vaccination may not protect people from PCoV-GX/P2V infection. These findings further verify the hACE2 utility of PCoV-GX/P2V by in vivo experiments using authentic viruses and highlight the importance for intensive surveillance to prevent possible cross-species transmission.

The complete mitogenome of Argas vulgaris (Filippova, 1961) and its phylogenetic status in subgenus Argas (Acari: Argasidae).

Soft ticks (Ixodida: Argasidae) are ectoparasites of terrestrial vertebrates with worldwide distributions. As one representative group of Argasidae, the genus Argas has an important vectorial role in transmitting zoonotic diseases. However, our knowledge of the subgenus Argas in China is still limited, as most literature only lists occurrence records or describes specific case reports without providing detailed morphological characteristics and further molecular data. This study aims to characterize Argas vulgaris through complete mitochondrial sequencing and morphological diagnostic techniques based on a batch of adult specimens collected from Ningxia Hui Autonomous Regions (NXHAR), North China. The morphology and microstructures of Ar. vulgaris and other lectotypes of argasid ticks in the subgenus Argas were also observed using a stereomicroscope. Following DNA extraction and sequencing, a complete mitochondrial sequence of Ar. vulgaris was assembled and analyzed within a phylogenetic context. The 14,479 bp mitogenome of Ar. vulgaris consists of 37 genes, including 13 genes for protein coding, two for ribosomal RNA, 22 for transfer RNA, and one for control region (D-loops). Phylogenetic analysis of Ar. vulgaris showed 98.27%-100% nucleotide identity with Ar. japonicus, indicating a close relationship between the two tick species. The morphological diagnostic features to differentiate Ar. vulgaris from other ticks within the subgenus Argas included the location of the anus and setae on the anterior lip of the female genital aperture. This study provided high-resolution scanning electron microscope images of female Ar. vulgaris and corresponding molecular data, representing valuable resources for future accurate species identification.

Isolation and characterization of a pangolin-borne HKU4-related coronavirus that potentially infects human-DPP4-transgenic mice.

We recently detected a HKU4-related coronavirus in subgenus Merbecovirus (named pangolin-CoV-HKU4-P251T) from a Malayan pangolin1. Here we report isolation and characterization of pangolin-CoV-HKU4-P251T, the genome sequence of which is closest to that of a coronavirus from the greater bamboo bat (Tylonycteris robustula) in Yunnan Province, China, with a 94.3% nucleotide identity. Pangolin-CoV-HKU4-P251T is able to infect human cell lines, and replicates more efficiently in cells that express human-dipeptidyl-peptidase-4 (hDPP4)-expressing and pangolin-DPP4-expressing cells than in bat-DPP4-expressing cells. After intranasal inoculation with pangolin-CoV-HKU4-P251, hDPP4-transgenic female mice are likely infected, showing persistent viral RNA copy numbers in the lungs. Progressive interstitial pneumonia developed in the infected mice, characterized by the accumulation of macrophages, and increase of antiviral cytokines, proinflammatory cytokines, and chemokines in lung tissues. These findings suggest that the pangolin-borne HKU4-related coronavirus has a potential for emerging as a human pathogen by using hDPP4.

Virome diversity shaped by genetic evolution and ecological landscape of Haemaphysalis longicornis.

BACKGROUND: Haemaphysalis longicornis is drawing attentions for its geographic invasion, extending population, and emerging disease threat. However, there are still substantial gaps in our knowledge of viral composition in relation to genetic diversity of H. longicornis and ecological factors, which are important for us to understand interactions between virus and vector, as well as between vector and ecological elements. RESULTS: We conducted the meta-transcriptomic sequencing of 136 pools of H. longicornis and identified 508 RNA viruses of 48 viral species, 22 of which have never been reported. Phylogenetic analysis of mitochondrion sequences divided the ticks into two genetic clades, each of which was geographically clustered and significantly associated with ecological factors, including altitude, precipitation, and normalized difference vegetation index. The two clades showed significant difference in virome diversity and shared about one fifth number of viral species that might have evolved to "generalists." Notably, Bandavirus dabieense, the pathogen of severe fever with thrombocytopenia syndrome was only detected in ticks of clade 1, and half number of clade 2-specific viruses were aquatic-animal-associated. CONCLUSIONS: These findings highlight that the virome diversity is shaped by internal genetic evolution and external ecological landscape of H. longicornis and provide the new foundation for promoting the studies on virus-vector-ecology interaction and eventually for evaluating the risk of H. longicornis for transmitting the viruses to humans and animals. Video Abstract.

Investigating the pathogens associated with Dermacentor nuttalli and its global distribution: A study integrating metagenomic sequencing, meta-analysis and niche modeling.

Dermacentor nuttalli, a member of family Ixodidae and genus Dermacentor, is predominantly found in North Asia. It transmits various pathogens of human and animal diseases, such as Lymphocytic choriomeningitis mammarenavirus and Brucella ovis, leading to severe symptoms in patients and posing serious hazards to livestock husbandry. To profile pathogen abundances of wild D. nuttalli, metagenomic sequencing was performed of four field-collected tick samples, revealing that Rickettsia, Streptomyces, and Pseudomonas were the most abundant bacterial genera in D. nuttalli. Specifically, four nearly complete Rickettsia genomes were assembled, closely relative to Rickettsia conorii subsp. raoultii. Then, a comprehensive meta-analysis was performed to evaluate its potential threats based on detected pathogens and geographical distribution positions reported in literature, reference books, related websites, and field surveys. At least 48 pathogens were identified, including 20 species of bacteria, seven species of eukaryota, and 21 species of virus. Notably, Rickettsia conorii subsp. raoultii, Coxiella burnetii, and Brucella ovis displayed remarkably high positivity rates, which were known to cause infectious diseases in both humans and livestock. Currently, the primary distribution of D. nuttalli spans China, Mongolia, and Russia. However, an additional 14 countries in Asia and America that may also be affected by D. nuttalli were identified in our niche model, despite no previous reports of its presence in these areas. This study provides comprehensive data and analysis on the pathogens carried by D. nuttalli, along with documented and potential distribution, suggesting an emerging threat to public health and animal husbandry. Therefore, there is a need for heightened surveillance and thorough investigation of D. nuttalli.

Systematic review and integrated data analysis reveal diverse pangolin-associated microbes with infection potential.

There has been increasing global concern about the spillover transmission of pangolin-associated microbes. To assess the risk of these microbes for emergence as human pathogens, we integrated data from multiple sources to describe the distribution and spectrum of microbes harbored by pangolins. Wild and trafficked pangolins have been mainly recorded in Asia and Africa, while captive pangolins have been reported in European and North American countries. A total of 128 microbes, including 92 viruses, 25 bacteria, eight protists, and three uncharacterized microbes, have been identified in five pangolin species. Out of 128 pangolin-associated microbes, 31 (including 13 viruses, 15 bacteria, and three protists) have been reported in humans, and 54 are animal-associated viruses. The phylogenetic analysis of human-associated viruses carried by pangolins reveals that they are genetically close to those naturally circulating among human populations in the world. Pangolins harbor diverse microbes, many of which have been previously reported in humans and animals. Abundant viruses initially detected in pangolins might exhibit risks for spillover transmission.

Genomic Characteristics of Emerging Intraerythrocytic Anaplasma capra and High Prevalence in Goats, China.

Anaplasma capra is an emerging tickborne human pathogen initially recognized in China in 2015; it has been reported in ticks and in a wide range of domestic and wild animals worldwide. We describe whole-genome sequences of 2 A. capra strains from metagenomic sequencing of purified erythrocytes from infected goats in China. The genome of A. capra was the smallest among members of the genus Anaplasma. The genomes of the 2 A. capra strains contained comparable G+C content and numbers of pseudogenes with intraerythrocytic Anaplasma species. The 2 A. capra strains had 54 unique genes. The prevalence of A. capra was high among goats in the 2 endemic areas. Phylogenetic analyses revealed that the A. capra strains detected in this study were basically classified into 2 subclusters with those previously detected in Asia. Our findings clarify details of the genomic characteristics of A. capra and shed light on its genetic diversity.

Successful treatment with doxycycline monotherapy for human infection with Babesia venatorum (Babesiidae, Sporozoa) in China: a case report and proposal for a clinical regimen.

BACKGROUND: Human babesiosis is a worldwide disease caused by intraerythrocytic protozoa of the genus Babesia. It is transmitted by bites from ixodid ticks, and mechanically transmitted by blood transfusion. It is primarily treated with quinine and/or atovaquone, which are not readily available in China. In this study, we developed a novel treatment regimen involving doxycycline monotherapy in a patient with severe Babesia venatorum infection as an alternative therapeutic medication. The aim of our study is to provide a guidance for clinical practice treatment of human babesiosis. CASE PRESENTATION: A 73-year-old man who had undergone splenectomy and blood transfusion 8 years prior, presented with an unexplained fever, headache, and thrombocytopenia, and was admitted to the Fifth Medical Center of the PLA General Hospital. He was diagnosed with B. venatorum infection by morphological review of thin peripheral blood smears, which was confirmed by multi-gene polymerase chain reaction (PCR), and sequencing of the entire 18s rRNA and partial ß-tubulin encoding genes, as well as isolation by animal inoculation. The doxycycline monotherapy regimen (peros, 0.1 g bisindie) was administered following pharmacological guidance and an effective outcome was observed. The patient recovered rapidly following the doxycycline monotherapy. The protozoan load in peripheral blood samples decreased by 88% in hematocrit counts after 8 days, and negative PCR results were obtained after 90 days of follow-up at the hospital. The treatment lasted for 3 months without any side effects or sequelae. The nine-month follow-up survey of the patient did not reveal any signs of recrudescence or anti-babesial tolerance. CONCLUSIONS: We have reported a clinical case of successful doxycycline monotherapy for human babesiosis caused by B. venatorum, which provides an optional medical intervention for human babesiosis.

Genomic Characterization of Theileria luwenshuni Strain Cheeloo.

Theileria, a tick-borne intracellular protozoan, can cause infections of various livestock and wildlife around the world, posing a threat to veterinary health. Although more and more Theileria species have been identified, genomes have been available only from four Theileria species to date. Here, we assembled a whole genome of Theileria luwenshuni, an emerging Theileria, through next-generation sequencing of purified erythrocytes from the blood of a naturally infected goat. We designated it T. luwenshuni str. Cheeloo because its genome was assembled by the researchers at Cheeloo College of Medicine, Shandong University, China. The genome of T. lunwenshuni str. Cheeloo was the smallest in comparison with the other four Theileria species. T. luwenshuni str. Cheeloo possessed the fewest gene gains and gene family expansion. The protein count of each category was always comparable between T. luwenshuni str. Cheeloo and T. orientalis str. Shintoku in the Eukaryote Orthologs annotation, though there were remarkable differences in genome size. T. luwenshuni str. Cheeloo had lower counts than the other four Theileria species in most categories at level 3 of Gene Ontology annotation. Kyoto Encyclopedia of Genes and Genomes annotation revealed a loss of the c-Myb in T. luwenshuni str. Cheeloo. The infection rate of T. luwenshuni str. Cheeloo was up to 81.5% in a total of 54 goats from three flocks. The phylogenetic analyses based on both 18S rRNA and cox1 genes indicated that T. luwenshuni had relatively low diversity. The first characterization of the T. luwenshuni genome will promote better understanding of the emerging Theileria. IMPORTANCE Theileria has led to substantial economic losses in animal husbandry. Whole-genome sequencing data of the genus Theileria are currently limited, which has prohibited us from further understanding their molecular features. This work depicted whole-genome sequences of T. luwenshuni str. Cheeloo, an emerging Theileria species, and reported a high prevalence of T. luwenshuni str. Cheeloo infection in goats. The first assembly and characterization of T. luwenshuni genome will benefit exploring the infective and pathogenic mechanisms of the emerging Theileria to provide scientific basis for future control strategies of theileriosis.

Continuous evolution and emerging lineage of seasonal human coronaviruses: A multicenter surveillance study.

The seasonal human coronaviruses (HCoVs) have zoonotic origins, repeated infections, and global transmission. The objectives of this study are to elaborate the epidemiological and evolutionary characteristics of HCoVs from patients with acute respiratory illness. We conducted a multicenter surveillance at 36 sentinel hospitals of Beijing Metropolis, China, during 2016-2019. Patients with influenza-like illness (ILI) and severe acute respiratory infection (SARI) were included, and submitted respiratory samples for screening HCoVs by multiplex real-time reverse transcription-polymerase chain reaction assays. All the positive samples were used for metatranscriptomic sequencing to get whole genomes of HCoVs for genetical and evolutionary analyses. Totally, 321 of 15 677 patients with ILI or SARI were found to be positive for HCoVs, with an infection rate of 2.0% (95% confidence interval, 1.8%-2.3%). HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1 infections accounted for 18.7%, 38.3%, 40.5%, and 2.5%, respectively. In comparison to ILI cases, SARI cases were significantly older, more likely caused by HCoV-229E and HCoV-OC43, and more often co-infected with other respiratory pathogens. A total of 179 full genome sequences of HCoVs were obtained from 321 positive patients. The phylogenetical analyses revealed that HCoV-229E, HCoV-NL63 and HCoV-OC43 continuously yielded novel lineages, respectively. The nonsynonymous to synonymous ratio of all key genes in each HCoV was less than one, indicating that all four HCoVs were under negative selection pressure. Multiple substitution modes were observed in spike glycoprotein among the four HCoVs. Our findings highlight the importance of enhancing surveillance on HCoVs, and imply that more variants might occur in the future.

Genomic representation predicts an asymptotic host adaptation of bat coronaviruses using deep learning.

Introduction: Coronaviruses (CoVs) are naturally found in bats and can occasionally cause infection and transmission in humans and other mammals. Our study aimed to build a deep learning (DL) method to predict the adaptation of bat CoVs to other mammals. Methods: The CoV genome was represented with a method of dinucleotide composition representation (DCR) for the two main viral genes, ORF1ab and Spike. DCR features were first analyzed for their distribution among adaptive hosts and then trained with a DL classifier of convolutional neural networks (CNN) to predict the adaptation of bat CoVs. Results and discussion: The results demonstrated inter-host separation and intra-host clustering of DCR-represented CoVs for six host types: Artiodactyla, Carnivora, Chiroptera, Primates, Rodentia/Lagomorpha, and Suiformes. The DCR-based CNN with five host labels (without Chiroptera) predicted a dominant adaptation of bat CoVs to Artiodactyla hosts, then to Carnivora and Rodentia/Lagomorpha mammals, and later to primates. Moreover, a linear asymptotic adaptation of all CoVs (except Suiformes) from Artiodactyla to Carnivora and Rodentia/Lagomorpha and then to Primates indicates an asymptotic bats-other mammals-human adaptation. Conclusion: Genomic dinucleotides represented as DCR indicate a host-specific separation, and clustering predicts a linear asymptotic adaptation shift of bat CoVs from other mammals to humans via deep learning.

Skin infectome of patients with a tick bite history.

Introduction: Ticks are the most important obligate blood-feeding vectors of human pathogens. With the advance of high-throughput sequencing, more and more bacterial community and virome in tick has been reported, which seems to pose a great threat to people. Methods: A total of 14 skin specimens collected from tick-bite patients with mild to severe symptoms were analyzed through meta-transcriptomic sequencings. Results: Four bacteria genera were both detected in the skins and ticks, including Pseudomonas, Acinetobacter, Corynebacterium and Propionibacterium, and three tick-associated viruses, Jingmen tick virus (JMTV), Bole tick virus 4 (BLTV4) and Deer tick mononegavirales-like virus (DTMV) were identified in the skin samples. Except of known pathogens such as pathogenic rickettsia, Coxiella burnetii and JMTV, we suggest Roseomonas cervicalis and BLTV4 as potential new agents amplified in the skins and then disseminated into the blood. As early as 1 day after a tick-bite, these pathogens can transmit to skins and at most four ones can co-infect in skins. Discussion: Advances in sequencing technologies have revealed that the diversity of tick microbiome and virome goes far beyond our previous understanding. This report not only identifies three new potential pathogens in humans but also shows that the skin barrier is vital in preventing horizontal transmissions of tick-associated bacteria or virus communities to the host. It is the first research on patients' skin infectome after a tick bite and demonstrates that more attention should be paid to the cutaneous response to prevent tick-borne illness.

Geographical distribution of Ixodes persulcatus and associated pathogens: Analysis of integrated data from a China field survey and global published data.

The increasing incidence and range expansion of tick-borne diseases have caused global threats to human and animal health under the background of climate and socioeconomic changes. As an efficient vector in transmission of tick-borne diseases, a growing burden caused by Ixodes persulcatus and associated pathogens could not be underestimated. This study summarized the distribution, hosts, and pathogens of I. persulcatus, and predicted the suitable habitats of this tick species worldwide. An integrated database involving a field survey, reference book, literature review, and related website was constructed. Location records of I. persulcatus and associated pathogens were incorporated into distribution maps using ArcGIS software. Positive rates for I. persulcatus-associated agents were estimated by meta-analysis. The global distribution of the tick species was predicted using Maxent model. I. persulcatus was distributed in 14 countries across the Eurasian continent, involving Russia, China, Japan, and several Baltic Sea states, which ranged between 21°N to 66°N. The tick species fed on 46 species of hosts, and 51 tick-borne agents could be harbored by I. persulcatus. The predictive model showed that I. persulcatus could be predominantly distributed in northern Europe, western Russia, and northern China. Our study fully clarified the potential public health risks posed by I. persulcatus and I. persulcatus-borne pathogens. Surveillance and control measures of tick-borne diseases should be enhanced to promote the health of humans, animals, and ecosystems.

Morphological and genomic characteristics of two novel actinomycetes, Ornithinimicrobium sufpigmenti sp. nov. and Ornithinimicrobium faecis sp. nov. isolated from bat faeces (Rousettus leschenaultia and Taphozous perforates).

Four Gram-staining-positive, aerobic, non-motile, circle-shaped bacteria were isolated from the faeces of bats (Rousettus leschenaultia and Taphozous perforates) collected from Guangxi autonomous region (E106°49'20″, N22°20'54″) and Yunnan province (E102°04'39″, N25°09'10″) of South China. Strains HY006T and HY008 shared highly 16S rRNA gene sequence similarity to those of Ornithinimicrobium pratense W204T (99.3%) and O. flavum CPCC 203535T (97.3%), while the strains HY1745 and HY1793T were closest to the type strains O. ciconiae H23M54T (98.7%), O. cavernae CFH 30183T (98.3%), and O. murale 01-Gi-040T (98.1%). Furthermore, when compared to the other members of the genus Ornithinimicrobium, the digital DNA-DNA hybridization and average nucleotide identity values of the four novel strains were within the ranges of 19.6-33.7% and 70.6-87.4%, respectively, both of which were below the respective recommended cutoff values of 70.0% and 95-96%. Significantly, strain HY006T was resistant to chloramphenicol and linezolid whereas strain HY1793T was resistant to erythromycin, clindamycin (intermediately), and levofloxacin (intermediately). The main cellular fatty acids (>20.0%) of our isolates were iso-C15:0 and iso-C16:0. Strains HY006T and HY1793T contained ornithine as the diagnostic diamino acid, also along with the alanine, glycine and glutamic acid in their cell wall. Based on phylogenetic, chemotaxonomic and phenotypic analyses, these four strains could be classified as two novel species of the genus Ornithinimicrobium, for which the names Ornithinimicrobium sufpigmenti sp. nov. and Ornithinimicrobium faecis sp. nov. are proposed. The type strains are HY006T (=CGMCC 1.16565T =JCM 33397T) and HY1793T (=CGMCC 1.19143T =JCM 34881T), respectively.

Metavirome of 31 tick species provides a compendium of 1,801 RNA virus genomes.

The increasing prevalence and expanding distribution of tick-borne viruses globally have raised health concerns, but the full repertoire of the tick virome has not been assessed. We sequenced the meta-transcriptomes of 31 different tick species in the Ixodidae and Argasidae families from across mainland China, and identified 724 RNA viruses with distinctive virome compositions among genera. A total of 1,801 assembled and complete or nearly complete viral genomes revealed an extensive diversity of genome architectures of tick-associated viruses, highlighting ticks as a reservoir of RNA viruses. We examined the phylogenies of different virus families to investigate virome evolution and found that the most diverse tick-associated viruses are positive-strand RNA virus families that demonstrate more ancient divergence than other arboviruses. Tick-specific viruses are often associated with only a few tick species, whereas virus clades that can infect vertebrates are found in a wider range of tick species. We hypothesize that tick viruses can exhibit both 'specialist' and 'generalist' evolutionary trends. We hope that our virome dataset will enable much-needed research on vertebrate-pathogenic tick-associated viruses.

Interference with LTßR signaling by tick saliva facilitates transmission of Lyme disease spirochetes.

Lyme spirochetes have coevolved with ticks to optimize transmission to hosts using tick salivary molecules (TSMs) to counteract host defenses. TSMs modulate various molecular events at the tick-host interface. Lymphotoxin-beta receptor (LTßR) is a vital immune receptor and plays protective roles in host immunity against microbial infections. We found that Ltbr knockout mice were more susceptible to Lyme disease spirochetes, suggesting the involvement of LTßR signaling in tick-borne Borrelia infection. Further investigation showed that a 15-kDa TSM protein from Ixodes persulcatus (I. persulcatus salivary protein; IpSAP) functioned as an immunosuppressant to facilitate the transmission and infection of Lyme disease spirochetes. IpSAP directly interacts with LTßR to block its activation, thus inhibiting the downstream signaling and consequently suppressing immunity. IpSAP immunization provided mice with significant protection against I. persulcatus-mediated Borrelia garinii infection. Notably, the immunization showed considerable cross-protection against other Borrelia infections mediated by other ixodid ticks. One of the IpSAP homologs from other ixodid ticks showed similar effects on Lyme spirochete transmission. Together, our findings suggest that LTßR signaling plays an important role in blocking the transmission and pathogenesis of tick-borne Lyme disease spirochetes, and that IpSAP and its homologs are promising candidates for broad-spectrum vaccine development.

Hedgehogs as Amplifying Hosts of Severe Fever with Thrombocytopenia Syndrome Virus, China.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tickborne bandavirus mainly transmitted by Haemaphysalis longicornis ticks in East Asia, mostly in rural areas. As of April 2022, the amplifying host involved in the natural transmission of SFTSV remained unidentified. Our epidemiologic field survey conducted in endemic areas in China showed that hedgehogs were widely distributed, had heavy tick infestations, and had high SFTSV seroprevalence and RNA prevalence. After experimental infection of Erinaceus amurensis and Atelerix albiventris hedgehogs with SFTSV, we detected robust but transitory viremias that lasted for 9-11 days. We completed the SFTSV transmission cycle between hedgehogs and nymph and adult H. longicornis ticks under laboratory conditions with 100% efficiency. Furthermore, naive H. longicornis ticks could be infected by SFTSV-positive ticks co-feeding on naive hedgehogs; we confirmed transstadial transmission of SFTSV. Our study suggests that the hedgehogs are a notable wildlife amplifying host of SFTSV in China.

Coinfection of Two Rickettsia Species in a Single Tick Species Provides New Insight into Rickettsia-Rickettsia and Rickettsia-Vector Interactions.

Rickettsiae are obligate intracellular bacteria that can cause life-threatening illnesses. There is an ongoing debate as to whether established infections by one Rickettsia species preclude the maintenance of the second species in ticks. Here, we identified two Rickettsia species in inoculum from Haemaphysalis montgomeryi ticks and subsequently obtained pure isolates of each species by plaque selection. The two isolates were classified as a transitional group and spotted fever group rickettsiae and named Rickettsia hoogstraalii str CS and Rickettsia rhipicephalii str EH, respectively. The coinfection of these two Rickettsia species was detected in 25.6% of individual field-collected H. montgomeryi. In cell culture infection models, R. hoogstraalii str CS overwhelmed R. rhipicephalii str EH with more obvious cytopathic effects, faster plaque formation, and increased cellular growth when cocultured, and R. hoogstraalii str CS seemed to polymerize actin tails differently from R. rhipicephalii str EH in vitro. This work provides a model to investigate the mechanisms of both Rickettsia-Rickettsia and Rickettsia-vector interactions. IMPORTANCE The rickettsiae are a group of obligate intracellular Gram-negative bacteria that include human pathogens causing an array of clinical symptoms and even death. There is an important question in the field, that is whether one infection can block the superinfection of other rickettsiae. This work demonstrated the coinfection of two Rickettsia species in individual ticks and further highlighted that testing the rickettsial competitive exclusion hypothesis will undoubtedly be a promising area as methods for bioengineering and pathogen biocontrol become amenable for rickettsiae.

Trafficked Malayan pangolins contain viral pathogens of humans.

Pangolins are the most trafficked wild animal in the world according to the World Wildlife Fund. The discovery of SARS-CoV-2-related coronaviruses in Malayan pangolins has piqued interest in the viromes of these wild, scaly-skinned mammals. We sequenced the viromes of 161 pangolins that were smuggled into China and assembled 28 vertebrate-associated viruses, 21 of which have not been previously reported in vertebrates. We named 16 members of Hunnivirus, Pestivirus and Copiparvovirus pangolin-associated viruses. We report that the L-protein has been lost from all hunniviruses identified in pangolins. Sequences of four human-associated viruses were detected in pangolin viromes, including respiratory syncytial virus, Orthopneumovirus, Rotavirus A and Mammalian orthoreovirus. The genomic sequences of five mammal-associated and three tick-associated viruses were also present. Notably, a coronavirus related to HKU4-CoV, which was originally found in bats, was identified. The presence of these viruses in smuggled pangolins identifies these mammals as a potential source of emergent pathogenic viruses.