Large-Scale Comparative Analyses of Tick Genomes Elucidate Their Genetic Diversity and Vector Capacities.

Among arthropod vectors, ticks transmit the most diverse human and animal pathogens, leading to an increasing number of new challenges worldwide. Here we sequenced and assembled high-quality genomes of six ixodid tick species and further resequenced 678 tick specimens to understand three key aspects of ticks: genetic diversity, population structure, and pathogen distribution. We explored the genetic basis common to ticks, including heme and hemoglobin digestion, iron metabolism, and reactive oxygen species, and unveiled for the first time that genetic structure and pathogen composition in different tick species are mainly shaped by ecological and geographic factors. We further identified species-specific determinants associated with different host ranges, life cycles, and distributions. The findings of this study are an invaluable resource for research and control of ticks and tick-borne diseases.

Identifying SARS-CoV-2-related coronaviruses in Malayan pangolins.

The ongoing outbreak of viral pneumonia in China and across the world is associated with a new coronavirus, SARS-CoV-21. This outbreak has been tentatively associated with a seafood market in Wuhan, China, where the sale of wild animals may be the source of zoonotic infection2. Although bats are probable reservoir hosts for SARS-CoV-2, the identity of any intermediate host that may have facilitated transfer to humans is unknown. Here we report the identification of SARS-CoV-2-related coronaviruses in Malayan pangolins (Manis javanica) seized in anti-smuggling operations in southern China. Metagenomic sequencing identified pangolin-associated coronaviruses that belong to two sub-lineages of SARS-CoV-2-related coronaviruses, including one that exhibits strong similarity in the receptor-binding domain to SARS-CoV-2. The discovery of multiple lineages of pangolin coronavirus and their similarity to SARS-CoV-2 suggests that pangolins should be considered as possible hosts in the emergence of new coronaviruses and should be removed from wet markets to prevent zoonotic transmission.

A Statovirus-like virus from respiratory tracts of patients, China.

The emerging evidence of human infections with emerging viruses suggests their potential public health importance. A novel taxon of viruses named Statoviruses (for stool-associated Tombus-like viruses) was recently identified in the gastrointestinal tracts of multiple mammals. Here we report the discovery of respiratory Statovirus-like viruses (provisionally named Restviruses) from the respiratory tracts of five patients experiencing acute respiratory disease with Human coronavirus OC43 infection through the retrospective analysis of meta-transcriptomic data. Restviruses shared 53.1%-98.8% identities of genomic sequences with each other and 39.9%-44.3% identities with Statoviruses. The phylogenetic analysis revealed that Restviruses together with a Stato-like virus from nasal-throat swabs of Vietnamese patients with acute respiratory disease, formed a well-supported clade distinct from the taxon of Statoviruses. However, the consistent genome characteristics of Restviruses and Statoviruses suggested that they might share similar evolutionary trajectories. These findings warrant further studies to elucidate the etiological and epidemiological significance of the emerging Restviruses.

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.

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.

Antibody and T-cellular response to COVID-19 booster vaccine in SARS-CoV-1 survivors.

The severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) survivors are more likely to produce a potent immune response to SARS-CoV-2 after booster vaccination. We assessed humoral and T cell responses against SARS-CoV-2 in previously vaccinated SARS-CoV-1 survivors and naïve healthy individuals (NHIs) after a booster Ad5-nCoV dose. Boosted SARS-CoV-1 survivors had a high neutralization of SARS-CoV-2 Wuhan-Hu-1 (WA1), Beta, and Delta but is limited to Omicron subvariants (BA.1, BA.2, BA.2.12.1, and BA.4/BA.5). Most boosted SARS-CoV-1 survivors had robust SARS-CoV-2-specific CD4+ and CD8+ T cell responses. While booster vaccination in NHIs elicited less or ineffective neutralization of WA1, Beta, and Delta, and none of them induced neutralizing antibodies against Omicron subvariants. However, they developed comparable SARS-CoV-2-specific T cell responses compared to boosted SARS-CoV-1 survivors. These findings suggest that boosted Ad5-nCoV would not elicit effective neutralizing antibodies against Omicron subvariants in SARS-CoV-1 survivors and NHIs but induced comparable robust T cell responses. Achieving a high antibody titer in SARS-CoV-1 survivors and NHIs is desirable to generate broad neutralization.

Profile and dynamics of infectious diseases: a population-based observational study using multi-source big data.

BACKGROUND: The current surveillance system only focuses on notifiable infectious diseases in China. The arrival of the big-data era provides us a chance to elaborate on the full spectrum of infectious diseases. METHODS: In this population-based observational study, we used multiple health-related data extracted from the Shandong Multi-Center Healthcare Big Data Platform from January 2013 to June 2017 to estimate the incidence density and describe the epidemiological characteristics and dynamics of various infectious diseases in a population of 3,987,573 individuals in Shandong province, China. RESULTS: In total, 106,289 cases of 130 infectious diseases were diagnosed among the population, with an incidence density (ID) of 694.86 per 100,000 person-years. Besides 73,801 cases of 35 notifiable infectious diseases, 32,488 cases of 95 non-notifiable infectious diseases were identified. The overall ID continuously increased from 364.81 per 100,000 person-years in 2013 to 1071.80 per 100,000 person-years in 2017 (χ2 test for trend, P < 0.0001). Urban areas had a significantly higher ID than rural areas, with a relative risk of 1.25 (95% CI 1.23-1.27). Adolescents aged 10-19 years had the highest ID of varicella, women aged 20-39 years had significantly higher IDs of syphilis and trichomoniasis, and people aged ≥ 60 years had significantly higher IDs of zoster and viral conjunctivitis (all P < 0.05). CONCLUSIONS: Infectious diseases remain a substantial public health problem, and non-notifiable diseases should not be neglected. Multi-source-based big data are beneficial to better understand the profile and dynamics of infectious diseases.

LncRNA LBX2-AS1 promotes colorectal cancer progression and 5-fluorouracil resistance.

BACKGROUND: Recent reports suggest that the long non-coding RNA LBX2 antisense RNA 1 (LBX2-AS1) acts as an important regulator in cancer progression, but its significance in colorectal cancer (CRC) remains undetermined. METHODS: LBX2-AS1 expression levels in CRC were determined from the GEPIA database and CRC tissues to investigate clinical relevance. meRIP-PCR assays investigated the molecular mechanisms underlying the function of m6A in LBX2-AS1. Loss of function experiments was used to define the role of LBX2-AS1 in the progression of CRC. The ceRNA function of LBX2-AS1 was evaluated by RNA immunoprecipitation. In vitro and PDX models were used to determine if LBX2-AS1 promotes 5-fluorouracil resistance. RESULTS: Data from the TCGA and our institutional patient cohorts established that LBX2-AS1 levels were significantly upregulated in most CRC tissues relative to normal adjacent colon tissues. Moreover, LBX2-AS1 levels were positively correlated with aggressive disease characteristics, constituting an independent prognostic indicator of overall patient survival. Mechanistic investigations suggested that the increased LBX2-AS1 in CRC was mediated by METTL3-dependent m6A methylation. In vitro experiments indicated that knockdown of LBX2-AS1 inhibited CRC proliferation, migration and invasion with this phenotype linked to LBX2-AS1-mediated regulation of AKT1, acting as a ceRNA to sponge miR-422a. Ex vivo analysis of patient-derived CRC xenografts showed that low LBX2-AS1 expression cases exhibited 5-FU responsiveness and clinical investigations confirmed that low LBX2-AS1 expression was associated with improved clinical benefits from 5-FU therapy. CONCLUSIONS: Together these results suggest that LBX2-AS1 may serve as a therapeutic target and predictor of 5-FU benefit in CRC patients.

Isolation and Identification of Rickettsia raoultii in Human Cases: A Surveillance Study in 3 Medical Centers in China.

Background: Rickettsia raoultii is frequently detected in multiple tick species, whereas human infection remains scarcely studied. Methods: A surveillance study was performed at 3 sentinel hospitals in China, to recruit participants with suspected tick exposure. Rickettsia raoultii infection was identified through polymerase chain reaction, followed by sequencing, and confirmed serologically. Isolation by cell culture was performed and the isolates were genome sequenced. Results: Twenty-six subjects were determined to have R. raoultii infection, including 7 with asymptomatic infection, 15 with mild to moderate illness, and 4 with severe illness. Common nonspecific manifestations in the 19 patients with mild to moderate or severe illness included fever (100%), malaise (95%), myalgia (58%), lymphadenopathy (53%), and nausea (42%). Only 5% of them had rash, and 16% had eschar. Scalp eschar and neck lymphadenopathy after a tick bite syndrome was only seen in 2 patients. Of the 4 patients with severe complications, 3 developed pulmonary edema, and 1 developed clouding of consciousness and lethargy. Frequent abnormalities of laboratory testing included leukopenia, thrombocytopenia, lymphopenia, neutropenia, hypoproteinemia, and elevated levels of total bilirubin, hepatic aminotransferases, lactate dehydrogenase, and creatine kinase. All the 19 patients recovered without sequelae after receiving doxycycline treatment. Two R. raoultii strains were isolated, and a significantly less degraded genome was observed than other more virulent Rickettsia strains, indicating a low pathogenicity of the current strain. Conclusions: Human infection with R. raoultii has a wide clinical spectrum that ranged from subclinical infection to severe complications. Physicians need to be aware of the high potential and clinical complexity of R. raoultii infection, to ensure appropriate testing and treatment in endemic regions.

Transmission of Severe Fever with Thrombocytopenia Syndrome Virus by Haemaphysalis longicornis Ticks, China.

We demonstrate maintenance and transmission of severe fever with thrombocytopenia syndrome virus by Haemaphysalis longicornis ticks in the larva, nymph, and adult stages with dissemination in salivary gland, midgut, and ovarian tissues. The H. longicornis tick is a competent vector to transmit this virus in both transovarial and transstadial modes.

Severe Fever with Thrombocytopenia Syndrome Complicated by Co-infection with Spotted Fever Group Rickettsiae, China.

During 2013-2015 in central China, co-infection with spotted fever group rickettsiae was identified in 77 of 823 patients infected with severe fever with thrombocytopenia syndrome virus. Co-infection resulted in delayed recovery and increased risk for death, prompting clinical practices in the region to consider co-infection in patients with severe fever with thrombocytopenia syndrome.

Human Infection with Novel Spotted Fever Group Rickettsia Genotype, China, 2015.

Only 4 species of spotted fever group rickettsiae have been detected in humans in China. However, phylogenetic analysis of samples from 5 ill patients in China indicated infection with a novel spotted fever group Rickettsia, designated Rickettsia sp. XY99. Clinical signs resembled those of severe fever with thrombocytopenia syndrome.

Genomic characters of Anaplasma bovis and genetic diversity in China.

The emergence of Anaplasma bovis or A. bovis-like infection in humans from China and the United States of America has raised concern about the public health importance of this pathogen. Although A. bovis has been detected in a wide range of ticks and mammals in the world, no genome of the pathogen is available up to now, which has prohibited us from better understanding the genetic basis for its pathogenicity. Here we describe an A. bovis genome from metagenomic sequencing of an infected goat in China. Anaplasma bovis had the smallest genome of the genus Anaplasma, and relatively lower GC content. Phylogenetic analysis of single-copy orthologue sequence showed that A. bovis was closely related to A. platys and A. phagocytophilum, but relatively far from intraerythrocytic Anaplasma species. Anaplasma bovis had 116 unique orthogroups and lacked 51 orthogroups in comparison to other Anaplasma species. The virulence factors of A. bovis were significantly less than those of A. phagocytophilum, suggesting less pathogenicity of A. bovis. When tested by specific PCR assays, A. bovis was detected in 23 of 29 goats, with an infection rate up to 79.3% (95% CI: 64.6% ∼94.1%). The phylogenetic analyses based on partial 16S rRNA, gltA and groEL genes indicated that A. bovis had high genetic diversity. The findings of this study lay a foundation for further understanding of the biological characteristics and genetic diversity of A. bovis, and will facilitate the formulation of prevention and control strategies.

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.

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.

Ecoclimate drivers shape virome diversity in a globally invasive tick species.

Spillovers of viruses from animals to humans occur more frequently under warmer conditions, particularly arboviruses. The invasive tick species Haemaphysalis longicornis, the Asian longhorned tick, poses a significant public health threat due to its global expansion and its potential to carry a wide range of pathogens. We analyzed meta-transcriptomic data from 3595 adult H. longicornis ticks collected between 2016 and 2019 in 22 provinces across China encompassing diverse ecological conditions. Generalized additive modeling revealed that climate factors exerted a stronger influence on the virome of H. longicornis than other ecological factors, such as ecotypes, distance to coastline, animal host, tick gender, and antiviral immunity. To understand how climate changes drive the tick virome, we performed a mechanistic investigation using causality inference with emphasis on the significance of this process for public health. Our findings demonstrated that higher temperatures and lower relative humidity/precipitation contribute to variations in animal host diversity, leading to increased diversity of the tick virome, particularly the evenness of vertebrate-associated viruses. These findings may explain the evolution of tick-borne viruses into generalists across multiple hosts, thereby increasing the probability of spillover events involving tick-borne pathogens. Deep learning projections have indicated that the diversity of the H. longicornis virome is expected to increase in 81.9% of regions under the SSP8.5 scenario from 2019 to 2030. Extension of surveillance should be implemented to avert the spread of tick-borne diseases.

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.

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.