Unbiased High-throughput Sequencing Research Articles

Identification and epidemiology of a novel Hepacivirus in domestic ducks in Hunan province, China.

The genus Hepacivirus comprises a diverse range of genetically distinct viruses that infect both mammalian and non-mammalian hosts, with some posing significant risks to human and animal health. Members of the genus Hepacivirus are typically classified into fourteen species (Hepacivirus A-N), with ongoing discoveries of novel hepaciviruses like Hepacivirus P and Hepacivirus Q. In this study, a novel Hepacivirus was identified in duck liver samples collected from live poultry markets in Hunan province, China, using unbiased high-throughput sequencing and meta-transcriptomic analysis. Through sequence comparison and phylogenetic analysis, it was determined that this newly discovered Hepacivirus belongs to a new subspecies of Hepacivirus Q. Moreover, molecular screening revealed the widespread circulation of this novel virus among duck populations in various regions of Hunan province, with an overall prevalence of 13.3%. These findings significantly enhence our understanding of the genetic diversity and evolution of hepaciviruses, emphasizing the presence of genetically diverse hepaciviruses duck populations in China. Given the broad geographical distribution and relatively high positive rate, further investigations are essential to explore any potential associations between Hepacivirus Q and duck-related diseases.

Bulk and Single-Cell RNA Sequencing Elucidate the Etiology of Severe COVID-19.

Coronavirus disease 2019 (COVID-19) is a type of pneumonia caused by the SARS-CoV-2 coronavirus. It can cause acute pulmonary and systemic inflammation, which can lead to death in severely ill patients. This study explores the potential reasons behind severe COVID-19 and its similarities to systemic autoimmune diseases. This study reviewed unbiased high-throughput gene expression datasets, including next-generation and single-cell RNA sequencing. A total of 27 studies and eight meta-analyses were reviewed. The studies indicated that severe COVID-19 is associated with the upregulation of genes involved in pro-inflammatory, interferon, and cytokine/chemokine pathways. Additionally, changes were observed in the proportions of immune cell types in the blood and tissues, along with degenerative alterations in lung epithelial cells. Genomic evidence also supports the association of severe COVID-19 with various inflammatory syndromes, such as neuronal COVID-19, acute respiratory distress syndrome, vascular inflammation, and multisystem inflammatory syndrome. In conclusion, this study suggests that gene expression profiling plays a significant role in elucidating the etiology of severe COVID-19.

Deep RNA sequencing of muscle tissue reveals absence of viral signatures in dermatomyositis.

Objective: To explore a possible connection between active viral infections and manifestation of dermatomyositis (DM). Methods: Skeletal muscle biopsies were analyzed from patients diagnosed with juvenile (n=10) and adult (n=12) DM. Adult DM patients harbored autoantibodies against either TIF-1γ (n=7) or MDA5 (n=5). Additionally, we investigated skeletal muscle biopsies from non-diseased controls (NDC, n=5). We used an unbiased high-throughput RNA sequencing (HTS) approach to detect viral sequences. To further increase sequencing depth, a host depletion approach was applied. Results: In this observational study, no relevant viral sequences were detected either by native sequencing or after host depletion. The absence of detectable viral sequences makes an active viral infection of the muscle tissue unlikely to be the cause of DM in our cohorts. Discussion: Type I interferons (IFN) play a major role in the pathogenesis of both juvenile and adult DM. The IFN response is remarkably conserved between DM subtypes classified by specific autoantibodies. Certain acute viral infections are accompanied by a prominent type I IFN response involving similar downstream mechanisms as in DM. Aiming to elucidate the pathogenesis of DM in skeletal muscle tissue, we used deep RNA sequencing and a host depletion approach to detect possible causative viruses.

Application of VirCapSeq-VERT and BacCapSeq in the diagnosis of presumed and definitive neuroinfectious diseases.

Unbiased high-throughput sequencing (HTS) has enabled new insights into the diversity of agents implicated in central nervous system (CNS) infections. The addition of positive selection capture methods to HTS has enhanced the sensitivity while reducing sequencing costs and the complexity of bioinformatic analysis. Here we report the use of virus capture-based sequencing for vertebrate viruses (VirCapSeq-VERT) and bacterial capture sequencing (BacCapSeq) in investigating CNS infections. Thirty-four samples were categorized: (1) patients with definitive CNS infection by routine testing; (2) patients meeting clinically the Brighton criteria (BC) for meningoencephalitis; (3) patients with presumptive infectious etiology highest on the differential. RNA extracts from cerebrospinal fluid (CSF) were used for VirCapSeq-VERT, and DNA extracts were used for BacCapSeq analysis. Among 8 samples from known CNS infections in group 1, VirCapSeq and BacCapSeq confirmed 3 expected diagnoses (42.8%), were negative in 2 (25%), yielded an alternative result in 1 (11.1%), and did not detect 2 expected negative pathogens. The confirmed cases identified HHV-6, HSV-2, and VZV while the negative samples included JCV and HSV-2. In groups 2 and 3, 11/26 samples (42%) were positive for at least one pathogen; however, 27% of the total samples (7/26) were positive for commensal organisms. No microbial nucleic acids were detected in negative control samples. HTS showed limited promise for pathogen identification in presumed CNS infectious diseases in our small sample. Before conducting larger-scale prospective studies to assess the clinical value of this novel technique, clinicians should understand the benefits and limitations of using this modality.

Discovery of an unrecognized nidovirus associated with granulomatous hepatitis in rainbow trout.

Rainbow trout (Oncorhynchus mykiss) is the principal species of inland-farmed fish in the Western hemisphere. Recently, we diagnosed in farmed rainbow trout a disease in which the hallmark is granulomatous-like hepatitis. No biotic agents could be isolated from lesions. Still, unbiased high-throughput sequencing and bioinformatics analyses revealed the presence of a novel piscine nidovirus that we named "Trout Granulomatous Virus" (TGV). TGV genome (28,767 nucleotides long) is predicted to encode non-structural (1a and 1ab) and structural (S, M, and N) proteins that resemble proteins of other known piscine nidoviruses. High loads of TGV transcripts were detected by quantitative RT-PCR in diseased fish and visualized in hepatic granulomatous sites by fluorescence in situ hybridization. Transmission electron microscopy (TEM) revealed coronavirus-like particles in these lesions. Together, these analyses corroborated the association of TGV with the lesions. The identification and detection of TGV provide means to control TGV spread in trout populations.

Isolation of a novel rhabdovirus and detection of multiple novel viral sequences in Culex species mosquitoes in the United States.

To increase our understanding of the diversity of the mosquito virome, 6956 mosquitoes of five species (Culex erraticus, Culex pipiens, Culex restuans, Culex tarsalis, and Culex territans) collected in Iowa in the United States in 2017 and 2020 were assayed for novel viruses by performing polyethylene glycol precipitation, virus isolation in cell culture, and unbiased high-throughput sequencing. A novel virus, provisionally named "Walnut Creek virus", was isolated from Cx. tarsalis, and its genomic sequence and organization are characteristic of viruses in the genus Hapavirus (family Rhabdoviridae). Replication of Walnut Creek virus occurred in avian, mammalian, and mosquito, but not tick, cell lines. A novel virus was also isolated from Cx. restuans, and partial genome sequencing revealed that it is distantly related to an unclassified virus of the genus Phytoreovirus (family Sedoreoviridae). Two recognized viruses were also isolated: Culex Y virus (family Birnaviridae) and Houston virus (family Mesoniviridae). We also identified sequences of eight novel viruses from six families (Amalgaviridae, Birnaviridae, Partitiviridae,Sedoreoviridae, Tombusviridae, and Totiviridae), two viruses that do not belong to any established families, and many previously recognized viruses. In summary, we provide evidence of multiple novel and recognized viruses in Culex spp. mosquitoes in the United States.

Genomic characterization and phylogenetic analysis of a novel Nairobi sheep disease genogroup Orthonairovirus from ticks, Southeastern China.

The increasing prevalence and transmission of tick-borne diseases, especially those emerging ones, have posed a significant threat to public health. Thus, the discovery of neglected pathogenic agents carried and transmitted by ticks is urgently needed. Using unbiased high-throughput sequencing, a novel Orthonairovirus designated as Meihua Mountain virus (MHMV), was identified in bloodsucking ticks collected from cattle and wild boars in Fujian province, Southeastern China. The full-length genome was determined by RT-PCR and RACE. Genomic architecture of MHMV shares typical features with orthonairoviruses. Phylogenetic analyses suggested that MHMV is clustered into the Nairobi sheep disease (NSD) genogroup of the genus Orthonairovirus and is closely related to the Hazara virus. The RdRp, GPC, and N protein of MHMV shares 62.3%–83.5%, 37.1%–66.1%, and 53.4%–77.3% amino acid identity with other NSD genogroup viruses, respectively, representing a novel species. The overall pooled prevalence of MHMV in ticks was 2.53% (95% CI: 1.62%–3.73%, 22 positives of 134 tick pools), with 7.38% (95% CI: 3.84%–12.59%, 11 positives of 18 pools) in Haemaphysalis hystricis, 6.02% (95% CI: 1.85%–14.22%, four positives of eight pools) in H. formosensis, 25.03% (95% CI: 9.23%–54.59%, six positive of eight pools) in Dermacentor taiwanensis, and 0.16% (95% CI: 0.01%–0.72%, one positive of 100 pools) in Rhipicephalus microplus. This study presented the first report of tick-carried Orthonairovirus in Fujian province and highlighted the broad geographic distribution and high genetic diversity of orthonairoviruses in China.

A Highly Divergent Hepacivirus Identified in Domestic Ducks Further Reveals the Genetic Diversity of Hepaciviruses.

Hepaciviruses represent a group of viruses that pose a significant threat to the health of humans and animals. During the last decade, new members of the genus Hepacivirus have been identified in various host species worldwide, indicating the widespread distribution of genetically diversified hepaciviruses among animals. By applying unbiased high-throughput sequencing, a novel hepacivirus, provisionally designated Hepacivirus Q, was discovered in duck liver samples collected in Guangdong province of China. Genetic analysis revealed that the complete polyprotein of Hepacivirus Q shares 23.9–46.6% amino acid identity with other representatives of the genus Hepacivirus. Considering the species demarcation criteria for hepaciviruses, Hepacivirus Q should be regarded as a novel hepacivirus species of the genus Hepacivirus within the family Flaviviridae. Phylogenetic analyses also indicate the large genetic distance between Hepacivirus Q and other known hepaciviruses. Molecular detection of this novel hepacivirus showed an overall prevalence of 15.9% in duck populations in partial areas of Guangdong province. These results expand knowledge about the genetic diversity and evolution of hepaciviruses and indicate that genetically divergent hepaciviruses are circulating in duck populations in China.

The bacterial biome of ticks and their wildlife hosts at the urban-wildland interface.

Advances in sequencing technologies have revealed the complex and diverse microbial communities present in ticks (Ixodida). As obligate blood-feeding arthropods, ticks are responsible for a number of infectious diseases that can affect humans, livestock, domestic animals and wildlife. While cases of human tick-borne diseases continue to increase in the northern hemisphere, there has been relatively little recognition of zoonotic tick-borne pathogens in Australia. Over the past 5 years, studies using high-throughput sequencing technologies have shown that Australian ticks harbour unique and diverse bacterial communities. In the present study, free-ranging wildlife (n=203), representing ten mammal species, were sampled from urban and peri-urban areas in New South Wales (NSW), Queensland (QLD) and Western Australia (WA). Bacterial metabarcoding targeting the 16S rRNA locus was used to characterize the microbiomes of three sample types collected from wildlife: blood, ticks and tissue samples. Further sequence information was obtained for selected taxa of interest. Six tick species were identified from wildlife: Amblyomma triguttatum, Ixodes antechini, Ixodes australiensis, Ixodes holocyclus, Ixodes tasmani and Ixodes trichosuri. Bacterial 16S rRNA metabarcoding was performed on 536 samples and 65 controls, generating over 100 million sequences. Alpha diversity was significantly different between the three sample types, with tissue samples displaying the highest alpha diversity (P<0.001). Proteobacteria was the most abundant taxon identified across all sample types (37.3 %). Beta diversity analysis and ordination revealed little overlap between the three sample types (P<0.001). Taxa of interest included Anaplasmataceae , Bartonella , Borrelia , Coxiellaceae , Francisella , Midichloria , Mycoplasma and Rickettsia . Anaplasmataceae bacteria were detected in 17.7% (95/536) of samples and included Anaplasma , Ehrlichia and Neoehrlichia species. In samples from NSW, ‘Ca. Neoehrlichia australis’, ‘Ca. Neoehrlichia arcana’, Neoehrlichia sp. and Ehrlichia sp. were identified. A putative novel Ehrlichia sp. was identified from WA and Anaplasma platys was identified from QLD. Nine rodent tissue samples were positive for a novel Borrelia sp. that formed a phylogenetically distinct clade separate from the Lyme Borrelia and relapsing fever groups. This novel clade included recently identified rodent-associated Borrelia genotypes, which were described from Spain and North America. Bartonella was identified in 12.9% (69/536) of samples. Over half of these positive samples were obtained from black rats (Rattus rattus), and the dominant bacterial species identified were Bartonella coopersplainsensis and Bartonella queenslandensis . The results from the present study show the value of using unbiased high-throughput sequencing applied to samples collected from wildlife. In addition to understanding the sylvatic cycle of known vector-associated pathogens, surveillance work is important to ensure preparedness for potential zoonotic spillover events.

Abstract 11844: Viral Gene Signatures in Patients With Cardiomyopathy After Chemotherapy

Introduction: Chemotherapy for hematologic malignancies can cause severe cardiomyopathy. Chemotherapeutic agents also induce immune suppression increasing the risk of opportunistic infections. Hypothesis: We have hypothesized that immunosuppression after chemotherapy increases opportunistic viral infections, myocarditis and cardiomyopathy. Methods: Viral gene sequences were identified using an unbiased high-throughput sequencing approach of patient blood samples following chemotherapy treatment for hematological malignancies (N = 28). Detected viral sequences were correlated with cardiac ejection fraction (LVEF) measured before and after treatment. Results: In a blinded analysis undertaken by two investigators viral sequences were identified in blood samples from patients after chemotherapy. Low levels of Influenza A, orthomyxovirus and Avian Paramyxovirus sequences were detected but identified individual virus sequences did not have a significant correlation to reduced LVEF post chemotherapy (r = 0.177-0.293). Detected sequences for all RNA viruses combined did indicate some correlation with LVEF, r = 0.621, suggesting potential for nonspecific RNA virus detection ( p &lt; 0.0078). In a secondary broad screen, data mining was performed including all detectable viral sequences without the filtering of human related sequences. In this broad analysis a significant correlation was detected for Paramyxovirus with reduced cardiac LVEF (r = 0.592 post, compared to r = 0.464 prior to chemotherapy; P &lt; .0096). For detected Orthomyxovirus sequences correlations were similar for LVEF pre- and post- chemotherapy (R = 0.483, P &lt; 0.0451). Retroviruses reads were increased but suspect due to human genome retrovirus sequences (R= 0.512; P &lt; 0.0351). No correlation was seen for Anellovirus, a DNA virus. Reduced LVEF did not correlate with chemotherapy (p = NS). Conclusions: This is the first report of screening for RNA viruses in the circulating blood of patients post chemotherapy using a high-throughput sequencing approach. Influenza Orthomyxovirus and Avian Paramyxovirus sequences were detectable in patients with reduced LVEF after chemotherapy. Further analysis for RNA virus infections in cardiomyopathy after chemotherapy is warranted.

A Novel Subtype of Bovine Hepacivirus Identified in Ticks Reveals the Genetic Diversity and Evolution of Bovine Hepacivirus

Hepaciviruses represent a group of viruses that pose a significant threat to the health of humans and animals. New members of the genus Hepacivirus in the family Flaviviridae have recently been identified in a wide variety of host species worldwide. Similar to the Hepatitis C virus (HCV), bovine hepacivirus (BovHepV) is hepatotropic and causes acute or persistent infections in cattle. BovHepVs are distributed worldwide and classified into two genotypes with seven subtypes in genotype 1. In this study, three BovHepV strains were identified in the samples of ticks sucking blood on cattle in the Guangdong province of China, through unbiased high-throughput sequencing. Genetic analysis revealed the polyprotein-coding gene of these viral sequences herein shared 67.7–84.8% nt identity and 76.1–95.6% aa identity with other BovHepVs identified worldwide. As per the demarcation criteria adopted for the genotyping and subtyping of HCV, these three BovHepV strains belonged to a novel subtype within the genotype 1. Additionally, purifying selection was the dominant evolutionary pressure acting on the genomes of BovHepV, and genetic recombination was not common among BovHepVs. These results expand the knowledge about the genetic diversity and evolution of BovHepV distributed globally, and also indicate genetically divergent BovHepV strains were co-circulating in cattle populations in China.

Molecular and Cellular Insights into the Development of Uterine Fibroids.

Uterine leiomyomas represent the most common benign gynecologic tumor. These hormone-dependent smooth-muscle formations occur with an estimated prevalence of ~70% among women of reproductive age and cause symptoms including pain, abnormal uterine bleeding, infertility, and recurrent abortion. Despite the prevalence and public health impact of uterine leiomyomas, available treatments remain limited. Among the potential causes of leiomyomas, early hormonal exposure during periods of development may result in developmental reprogramming via epigenetic changes that persist in adulthood, leading to disease onset or progression. Recent developments in unbiased high-throughput sequencing technology enable powerful approaches to detect driver mutations, yielding new insights into the genomic instability of leiomyomas. Current data also suggest that each leiomyoma originates from the clonal expansion of a single transformed somatic stem cell of the myometrium. In this review, we propose an integrated cellular and molecular view of the origins of leiomyomas, as well as paradigm-shifting studies that will lead to better understanding and the future development of non-surgical treatments for these highly frequent tumors.

Transcriptome of DNA Repair Genes in CMML: Rationale for Targeting ERCC1 and CDKN1A As a Synthetic Lethality Approach

Background: Chronic myelomonocytic leukemia (CMML) is characterized by features of both myeloproliferative neoplasm and myelodysplastic syndrome (MDS). Despite the main role of DNA repair pathways in cancer, in myelodysplastic disorders, somatic mutations are not found in these pathways. The novel discovery of non-classical leukaemogenesis mediated by splicing defects, profound influence of epigenetic anomalies, deeper knowledge of the DNA repair network, and development of unbiased high-throughput sequencing approaches, prompted us to revisit whether these processes might be disrupted by non-mutational mechanisms in CMML and related myeloid disorders. Study aims: i) to screen the transcriptome of DNA repair genes in a CMML discovery cohort; ii) to validate their misregulation in an independent large CMML cohort; and iii) determining how those candidates behave through the spectrum of myeloid malignancies. Methods: RNA-seq was performed in the discovery cohort: 27 CMML bone marrow samples (BM) at diagnosis and 9 healthy BM samples, and in 7 patients treated with Azacitidine in BM collected before and after 4 cycles. RT-qPCR was used to validate 9 DEGs through the myeloid spectrum and in a CMML validation cohort: 74 additional CMML, 70 MDS, 66 AML , and 25 CML patients. Candidates were chosen based on clinical considerations: i) druggable oncogenes highly overexpressed ii) oncogenes infra-expressed with inhibitory molecules already being tested in myeloid neoplasms. Global pattern of DNA repair gene expression was compared with MDS and AML extracted from MILE study data. The DEGs methylation status was studied using available public data and we confirmed by pyrosequencing the methylation status of our main candidates. The CMML validation cohort was mutationally characterized by targeted sequencing of 18 CMML recurrently mutated genes. Alternative splice sites, skipped exons, mutually exclusive exons and retained introns were quantified using rMATS.Results: Of 27 CMML patients and 9 healthy donors, the expression of 30 genes was significantly different between the two groups (8 genes up-regulated and 22 down-regulated). Defects on genes predominantly unique to a single strand breaks repair pathway included: NEIL1 and OGG1 in base excision repair, XPA and MSS19 in nucleotide excision repair and RPA4 in mismatch repair. XRCC4 and MSH4 overexpression and PRKDC down-regulation were found as significant changes among the genes associated exclusively to double strand breaks repair. We next extracted the DNA repair transcriptional components from MILE study: 206 MDS, 47 ckAML and 73 healthy donors. MDS misregulation was characterized by upregulated genes while ckAML showed a global disruption, mostly downregulation of Homologous Recombination genes. Some genes showed opposite sense of misregulation depending on the myeloid entity: TDP1 was upregulated in CMML cases and downregulated in ckAML, and viceversa for BAP1 . CDK1 and EXO1 were upregulated in MDS cases but downregulated in ckAML cases. Statistically significant and direction-concordant dysregulation of CDKN1A, ERCC1, XRCC4, NEIL1, MSH4, PARP1 and BAP1 were confirmed in our independent CMML validation cohort. The methylation study showed a global demethylated status of our misregulated DNA repair genes in CMML. Either responder s (n=3) or non-responders (n=2) to azacitidine failed to achieve a substantial expression change in their DNA Repair DEGs. PARP1 infraexpresion was associated with TET2 mut cases. Lower CDKN1A and BAP1 expression was related to chromatin regulator mutations. SRSF2 mutations induced widespread aberrant splicing events in DNA repair genes in CMMLConclusions: We have identified by means of an unbiased high-throughput approach, and validated in an independent cohort, a subset of DNA repair genes consistently misregulated in CMML. The genes identified warrant further study as potential novel therapeutic targets, both directly and through modulation of associated compensatory pathways. CDKN1A and ERCC1 in particular emerge as realistic candidates for a synthetic lethality approach. Our findings suggest that the promise of PARP1 inhibition appears less pertinent to CMML than for other myeloid diseases. These differences might also partially explain the different genomic and phenotypic manifestations of different myeloid neoplasms, and shed insights into their distinctive biology. DisclosuresMaciejewski:Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Speaker Fees; Apellis Pharmaceuticals: Consultancy; Ra Pharma: Consultancy.

Molecular detection of cosaviruses in a patient with acute flaccid paralysis and in sewage samples in Germany

Cosaviruses (CoSV) were first identified in stool samples collected from non-polio acute flaccid paralysis (AFP) cases and their healthy contacts in Pakistan in 2003. The clinical importance of CoSV remains unclear as data on epidemiology are scarce and no routine diagnostic testing is done. In this study, we characterized human CoSV (HCoSV) in a child with non-polio AFP and in sewage samples collected in Berlin, Germany.Using unbiased high-throughput sequencing and specific PCR, we characterized a HCoSV-D in stool samples of a three-year-old child hospitalized in Germany with non-polio AFP and travel history to Pakistan. The shedding pattern and absence of other relevant pathogens suggests that HCoSV-D may have been involved in the genesis of AFP. The HCoSV-RNA concentration was high, with 2.57 × 106 copies per mL fecal/suspension, decreasing in follow-up samples. To investigate the possibility of local circulation of HCoSV, we screened Berlin sewage samples collected between 2013 and 2018. Molecular testing of sewage samples has shown the presence of CoSV in several parts of the world, but until now not in Germany. Of our sewage samples, 54.3 % were positive for CoSV, with up to three viral species identified in samples. Phylogenetically, the German sequences clustered intermixed with sequences obtained globally.Together, these findings emphasize the need for further clinical, epidemiological, environmental, pathogenicity and phylogenetic studies of HCoSV.

Discovery and Surveillance of Tick-Borne Pathogens.

Within the past 30 yr molecular assays have largely supplanted classical methods for detection of tick-borne agents. Enhancements provided by molecular assays, including speed, throughput, sensitivity, and specificity, have resulted in a rapid increase in the number of newly characterized tick-borne agents. The use of unbiased high throughput sequencing has enabled the prompt identification of new pathogens and the examination of tick microbiomes. These efforts have led to the identification of hundreds of new tick-borne agents in the last decade alone. However, little is currently known about the majority of these agents beyond their phylogenetic classification. Our article outlines the primary methods involved in tick-borne agent discovery and the current status of our understanding of tick-borne agent diversity.

Distinct T-cell receptor profiles associated with hepatitis B e antigen seroconversion during entecavir treatment.

T-cell receptor (TCR) repertoire is ambiguously changed in chronic hepatitis B (CHB) patients during antivirus therapy. We tried to assess TCR repertoire dynamics and its clinical significance upon HBeAg seroconversion in CHB patients. Twenty CHB patients undergoing 1-year entecavir (ETV) treatment were enrolled, including 10 complete response (CR) vs 10 non-complete response (NCR) patients based on HBeAg seroconversion at week 48. The TCRβ complementarity-determining region 3 (CDR3) of peripheral CD4+ and CD8+ T cells at weeks 0, 12 and 48 was analyzed by unbiased high-throughput sequencing. The TCR repertoire profiles and their correlations with serological parameters were analyzed. The diversity of TCRβ repertoires was decreasing in CR patients but increasing in NCR patients. The distribution pattern of TCR repertoires stratified according to clonotype frequencies changed in the opposite direction between CR and NCR patients. Narrow amounts of newly appearing clonotypes in CR patients experienced a more intensive and robust expansion and this phenomenon could occur as early as week 12 for the CD4+ subset but later at week 48 for the CD8+ subset. There existed some CR-exclusive clonotypes with a relatively low but increasing frequency at week 48. The number of unique TCRβ clonotypes was positively correlated with the ALT or HBV DNA level in CR patients but showed no or negative correlation in NCR patients. Distinct TCR profiles contribute to predicting HBeAg seroconversion in CHB patients during ETV treatment and certain TCRβ CDR3 motif may be utilized for CHB immunotherapy in the future.

The Diversity and Distribution of Viruses Associated with Culex annulirostris Mosquitoes from the Kimberley Region of Western Australia.

Metagenomics revealed an impressive breadth of previously unrecognized viruses. Here, we report the virome of the Culex annulirostris Skuse mosquito, an important vector of pathogenic arboviruses in Australia. Mosquitoes were collected from three sites in the Kimberley region of Western Australia. Unbiased high-throughput sequencing (HTS) revealed the presence of 16 novel viral sequences that share less than 90% identity with known viruses. None were closely related to pathogenic arboviruses. Viruses were distributed unevenly across sites, indicating a heterogeneous Cx. annulirostris virome. Polymerase chain reaction assays confirmed HTS data and identified marked variation between the virus prevalence identified at each site.

Detection of Recombinant Rousettus Bat Coronavirus GCCDC1 in Lesser Dawn Bats (Eonycteris spelaea) in Singapore.

Rousettus bat coronavirus GCCDC1 (RoBat-CoV GCCDC1) is a cross-family recombinant coronavirus that has previously only been reported in wild-caught bats in Yúnnan, China. We report the persistence of a related strain in a captive colony of lesser dawn bats captured in Singapore. Genomic evidence of the virus was detected using targeted enrichment sequencing, and further investigated using deeper, unbiased high throughput sequencing. RoBat-CoV GCCDC1 Singapore shared 96.52% similarity with RoBat-CoV GCCDC1 356 (NC_030886) at the nucleotide level, and had a high prevalence in the captive bat colony. It was detected at five out of six sampling time points across the course of 18 months. A partial segment 1 from an ancestral Pteropine orthoreovirus, p10, makes up the recombinant portion of the virus, which shares high similarity with previously reported RoBat-CoV GCCDC1 strains that were detected in Yúnnan, China. RoBat-CoV GCCDC1 is an intriguing, cross-family recombinant virus, with a geographical range that expands farther than was previously known. The discovery of RoBat-CoV GCCDC1 in Singapore indicates that this recombinant coronavirus exists in a broad geographical range, and can persist in bat colonies long-term.

Retrieval of the Complete Coding Sequence of the UK-Endemic Tatenale Orthohantavirus Reveals Extensive Strain Variation and Supports Its Classification as a Novel Species.

Orthohantaviruses are globally distributed viruses, associated with rodents and other small mammals. However, data on the circulation of orthohantaviruses within the UK, particularly the UK-endemic Tatenale virus, is sparse. In this study, 531 animals from five rodent species were collected from two locations in northern and central England and screened using a degenerate, pan- orthohantavirus RT-PCR assay. Tatenale virus was detected in a single field vole (Microtus agrestis) from central England and twelve field voles from northern England. Unbiased high-throughput sequencing of the central English strain resulted in the recovery of the complete coding sequence of a novel strain of Tatenale virus, whilst PCR-primer walking of the northern English strain recovered almost complete coding sequence of a previously identified strain. These findings represented the detection of a third lineage of Tatenale virus in the United Kingdom and extended the known geographic distribution of these viruses from northern to central England. Furthermore, the recovery of the complete coding sequence revealed that Tatenale virus was sufficiently related to the recently identified Traemersee virus, to meet the accepted criteria for classification as a single species of orthohantavirus.

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.