Diversity and Distribution of Mites (ACARI) Revealed by Contamination Survey in Public Genomic Databases.

Acari (mites and ticks) are a biodiverse group of microarthropods within the Arachnida. Because of their diminutive size, mites are often overlooked. We hypothesized that mites, like other closely related microorganisms, could also contaminate public genomic database. Here, using a strategy based on DNA barcodes previously reported, we scanned contaminations related to mites (Acari, exclusive of Ixodida) in Genbank WGS/TSA database. In 22,114 assemblies (17,845 animal and 4269 plant projects), 1717 contigs in 681 assemblies (3.1%) were detected as mite contaminations. Additional taxonomic analysis showed the following: (1) most of the contaminants (1445/1717) were from the specimens of Magnoliopsida, Insecta and Pinopsida; (2) the contamination rates were higher in plant or TSA projects; (3) mite distribution among different classes of hosts varied considerably. Additional phylogenetic analysis of these contaminated contigs further revealed complicated mite-host associations. Overall, we conducted a first systemic survey and analysis of mite contaminations in public genomic database, and these DNA barcode related mite contigs will provide a valuable resource of information for understanding the diversity and phylogeny of mites.

A Large-Scale Study into Protist-Animal Interactions Based on Public Genomic Data Using DNA Barcodes.

With the birth of next-generation sequencing (NGS) technology, genomic data in public databases have increased exponentially. Unfortunately, exogenous contamination or intracellular parasite sequences in assemblies could confuse genomic analysis. Meanwhile, they can provide a valuable resource for studies of host-microbe interactions. Here, we used a strategy based on DNA barcodes to scan protistan contamination in the GenBank WGS/TSA database. The results showed a total of 13,952 metazoan/animal assemblies in GenBank, where 17,036 contigs were found to be protistan contaminants in 1507 assemblies (10.8%), with even higher contamination rates in taxa of Cnidaria (150/281), Crustacea (237/480), and Mollusca (107/410). Taxonomic analysis of the protists derived from these contigs showed variations in abundance and evenness of protistan contamination across different metazoan taxa, reflecting host preferences of Apicomplexa, Ciliophora, Oomycota and Symbiodiniaceae for mammals and birds, Crustacea, insects, and Cnidaria, respectively. Finally, mitochondrial proteins COX1 and CYTB were predicted from these contigs, and the phylogenetic analysis corroborated the protistan origination and heterogeneous distribution of the contaminated contigs. Overall, in this study, we conducted a large-scale scan of protistan contaminant in genomic resources, and the protistan sequences detected will help uncover the protist diversity and relationships of these picoeukaryotes with Metazoa.

Positive Selection and Duplication of Bat TRIM Family Proteins.

Bats have received increasing attention because of some unique biological features they possess. TRIM is a large family of proteins that participate in diverse cellular functions, such as antiviral immunity, DNA damage repair, tumor suppression, and aging. These functional areas appear to be highly consistent with the special characteristics of bats, such as tolerance to viruses and DNA damage generated in flight, low cancer incidence, and longevity. However, there is still a lack of systematic study of the TRIM family in bats. Here, we explored the TRIM family of bats using the genomes of 16 representative species. The results showed that the bat TRIM family contains 70 members, with 24 under positive selection and 7 duplicated. Additional transcriptomic analysis revealed the tissue-specific expressions of TRIM9, 46, 54, 55, 63, and 72. Additionally, following interferon or viral stimulation, TRIM orthologs associated with antiviral immunity reported in humans were also upregulated in bat cells. The present study systematically analyzed the composition, evolution, and expression of bat TRIM genes. It may provide a theoretical basis for studies of bat TRIM in the fields of antiviral immunity, longevity, and tolerance to DNA damage.

Dampened STING-Dependent Interferon Activation in Bats.

Compared with terrestrial mammals, bats have a longer lifespan and greater capacity to co-exist with a variety of viruses. In addition to cytosolic DNA generated by these viral infections, the metabolic demands of flight cause DNA damage and the release of self-DNA into the cytoplasm. However, whether bats have an altered DNA sensing/defense system to balance high cytosolic DNA levels remains an open question. We demonstrate that bats have a dampened interferon response due to the replacement of the highly conserved serine residue (S358) in STING, an essential adaptor protein in multiple DNA sensing pathways. Reversing this mutation by introducing S358 restored STING functionality, resulting in interferon activation and virus inhibition. Combined with previous reports on bat-specific changes of other DNA sensors such as TLR9, IFI16, and AIM2, our findings shed light on bat adaptation to flight, their long lifespan, and their unique capacity to serve as a virus reservoir.

Discovery of a rich gene pool of bat SARS-related coronaviruses provides new insights into the origin of SARS coronavirus.

A large number of SARS-related coronaviruses (SARSr-CoV) have been detected in horseshoe bats since 2005 in different areas of China. However, these bat SARSr-CoVs show sequence differences from SARS coronavirus (SARS-CoV) in different genes (S, ORF8, ORF3, etc) and are considered unlikely to represent the direct progenitor of SARS-CoV. Herein, we report the findings of our 5-year surveillance of SARSr-CoVs in a cave inhabited by multiple species of horseshoe bats in Yunnan Province, China. The full-length genomes of 11 newly discovered SARSr-CoV strains, together with our previous findings, reveals that the SARSr-CoVs circulating in this single location are highly diverse in the S gene, ORF3 and ORF8. Importantly, strains with high genetic similarity to SARS-CoV in the hypervariable N-terminal domain (NTD) and receptor-binding domain (RBD) of the S1 gene, the ORF3 and ORF8 region, respectively, were all discovered in this cave. In addition, we report the first discovery of bat SARSr-CoVs highly similar to human SARS-CoV in ORF3b and in the split ORF8a and 8b. Moreover, SARSr-CoV strains from this cave were more closely related to SARS-CoV in the non-structural protein genes ORF1a and 1b compared with those detected elsewhere. Recombination analysis shows evidence of frequent recombination events within the S gene and around the ORF8 between these SARSr-CoVs. We hypothesize that the direct progenitor of SARS-CoV may have originated after sequential recombination events between the precursors of these SARSr-CoVs. Cell entry studies demonstrated that three newly identified SARSr-CoVs with different S protein sequences are all able to use human ACE2 as the receptor, further exhibiting the close relationship between strains in this cave and SARS-CoV. This work provides new insights into the origin and evolution of SARS-CoV and highlights the necessity of preparedness for future emergence of SARS-like diseases.

[Effect of Lep d2 from Lepidoglyphus destructor as a vaccine for specific immunotherapy in murine with asthma].

OBJECTIVE: To assess the effect of Lep d2 from Lepidoglyphus destructor as a vaccine for specific immunotherapy on murine model of asthma. METHODS: Thirty BALB/c mice (SPF) were randomly categorized into a PBS group, an asthma group, and a Lep d2 SIT group. The mice in the asthma group and Lep d2 SIT group were sensitized by intraperitoneal injection with extracts of dust mites on Days 0, 7th, and 14th, while those in the PBS group were injected with PBS. From the 21st day, the asthma group and Lep d2 SIT group exposed to the extracts of dust mites were stimulated by aerosol inhalation for 7 successive days. During the period of the 25th-27th Day, the mice in Lep d2 SIT group were injected intraperitoneally with Lep d2 allergen for SIT 30 min before nasal inhalation, whereas the PBS group and asthma group were treated with only PBS. Twenty-four hours after the final inhalation, all the mice were sacrificed, the bronchoalveolar lavage fluids (BALFs) were collected. The levels of IFN-γ, IL-5 and IL-13 in the BALF and the supernatant of splenocyte culture solution (SSCS) as well as the levels of specific IgE (sIgE) and sIgG2a in the sera were detected by ELISA. The lung tissues of the mice in the above 3 groups were stained by haematoxylin and eosin (H&E) and observed by a microscope. RESULTS: The symptoms of acute asthma attack were observed in the mice of the asthma group and Lep d2 group, but not in the PBS group. The allergic inflammation changes in lung in the Lep d2 SIT group were significantly alleviated compared with those in the asthma group. The concentrations of IFN-γ in BALFs and SSCS of the mice in the Lep d2 SIT group were significantly higher than those in the asthma group (both P < 0.01), while the levels of IL-5 and IL-13 in the former group were significantly lower than those in the latter group (all P < 0.01). Meanwhile, the level of sIgE of mice in the Lep d2 SIT group was significantly lower than those in the asthma group (P < 0.01), while the level of sIgG2a of mice in the former group was higher than those in the latter group (P < 0.01). CONCLUSION: Lep d2 allergen as a vaccine can alleviate the allergic symptoms in the lung of mice effectively after allergen specific immunotherapy.

Sequence recombination and conservation of Varroa destructor virus-1 and deformed wing virus in field collected honey bees (Apis mellifera).

We sequenced small (s) RNAs from field collected honeybees (Apis mellifera) and bumblebees (Bombuspascuorum) using the Illumina technology. The sRNA reads were assembled and resulting contigs were used to search for virus homologues in GenBank. Matches with Varroadestructor virus-1 (VDV1) and Deformed wing virus (DWV) genomic sequences were obtained for A. mellifera but not B. pascuorum. Further analyses suggested that the prevalent virus population was composed of VDV-1 and a chimera of 5'-DWV-VDV1-DWV-3'. The recombination junctions in the chimera genomes were confirmed by using RT-PCR, cDNA cloning and Sanger sequencing. We then focused on conserved short fragments (CSF, size > 25 nt) in the virus genomes by using GenBank sequences and the deep sequencing data obtained in this study. The majority of CSF sites confirmed conservation at both between-species (GenBank sequences) and within-population (dataset of this study) levels. However, conserved nucleotide positions in the GenBank sequences might be variable at the within-population level. High mutation rates (Pi>10%) were observed at a number of sites using the deep sequencing data, suggesting that sequence conservation might not always be maintained at the population level. Virus-host interactions and strategies for developing RNAi treatments against VDV1/DWV infections are discussed.

Targeting of dicer-2 and RNA by a viral RNA silencing suppressor in Drosophila cells.

RNA interference (RNAi) is a eukaryotic gene-silencing mechanism that functions in antiviral immunity in diverse organisms. To combat RNAi-mediated immunity, viruses encode viral suppressors of RNA silencing (VSRs) that target RNA and protein components in the RNAi machinery. Although the endonuclease Dicer plays key roles in RNAi immunity, little is known about how VSRs target Dicer. Here, we show that the B2 protein from Wuhan nodavirus (WhNV), the counterpart of Flock House virus (FHV), suppresses Drosophila melanogaster RNAi by directly interacting with Dicer-2 (Dcr-2) and sequestering double-stranded RNA (dsRNA) and small interfering RNA (siRNA). Further investigations reveal that WhNV B2 binds to the RNase III and Piwi-Argonaut-Zwille (PAZ) domains of Dcr-2 via its C-terminal region, thereby blocking the activities of Dcr-2 in processing dsRNA and incorporating siRNA into the RNA-induced silencing complex (RISC). Moreover, we uncover an interrelationship among diverse activities of WhNV B2, showing that RNA binding enhances the B2-Dcr-2 interaction by promoting B2 homodimerization. Taken together, our findings establish a model of suppression of Drosophila RNAi by WhNV B2 targeting both Dcr-2 and RNA and provide evidence that an interrelationship exists among diverse activities of VSRs to antagonize RNAi.

RNA binding by a novel helical fold of b2 protein from wuhan nodavirus mediates the suppression of RNA interference and promotes b2 dimerization.

Wuhan nodavirus (WhNV) is a newly identified member of the Nodaviridae family with a bipartite genome of positive-sense RNAs. A nonstructural protein encoded by subgenomic RNA3 of nodaviruses, B2, serves as a potent RNA silencing suppressor (RSS) by sequestering RNA duplexes. We have previously demonstrated that WhNV B2 blocks RNA silencing in cultured Drosophila cells. However, the molecular mechanism by which WhNV B2 functions remains unknown. Here, we successfully established an RNA silencing system in cells derived from Pieris rapae, a natural host of WhNV, by introducing into these cells double-stranded RNA (dsRNA)-expressing plasmids or chemically synthesized small interfering RNAs (siRNAs). Using this system, we revealed that the WhNV B2 protein inhibited Dicer-mediated dsRNA cleavage and the incorporation of siRNA into the RNA-induced silencing complex (RISC) by sequestering dsRNA and siRNA. Based on the modeled B2 3-dimensional structure, serial single alanine replacement mutations and N-terminal deletion analyses showed that the RNA-binding domain of B2 is formed by its helices α2 and α3, while helix α1 mediates B2 dimerization. Furthermore, positive feedback between RNA binding and B2 dimerization was uncovered by gel shift assay and far-Western blotting, revealing that B2 dimerization is required for its binding to RNA, whereas RNA binding to B2 in turn promotes its dimerization. All together, our findings uncovered a novel RNA-binding mode of WhNV B2 and provided evidence that the promotion effect of RNA binding on dimerization exists in a viral RSS protein.