Severe fever with thrombocytopenia syndrome virus infection shapes gut microbiome of the tick vector Haemaphysalis longicornis.

BACKGROUND: Ticks serve as vectors for a diverse array of pathogens, including viruses responsible for both human and livestock diseases. Symbiotic bacteria hold significant potential for controlling tick-borne disease. However, the alteration of tick gut bacterial community in response to pathogen infection has not been analyzed for any tick-borne viruses. Here, the impact of severe fever with thrombocytopenia syndrome virus (SFTSV) infection on bacterial diversity in the gut of Haemaphysalis longicornis is investigated. METHODS: Unfed tick females were artificially infected with SFTSV. The gut samples were collected and the genomic DNA was extracted. We then investigated alterations in gut bacterial composition in response to SFTSV infection through 16S rRNA gene sequencing. RESULTS: The study found that a reduction in the number of operational taxonomic units (OTUs) in the tick gut following SFTSV infection. However, there were no significant changes in alpha diversity indices upon infection. Four genera, including Corynebacterium, Arthrobacter, Sphingomonas, and Escherichia, were identified as biomarkers for the tick gut without SFTSV infection. Notably, the predicted correlation network indicated that the biomarkers Sphingomonas and Escherichia exhibited positive correlations within the same subcommunity, which was altered upon viral infection. CONCLUSIONS: These findings revealed that the change in tick gut bacterial composition upon SFTSV infection and could facilitate the discovery new target for tick-borne viral disease control.

Diversity analysis of the endosymbiotic bacterial community in field-collected Haemaphysalis ticks on the tropical Hainan Island, China.

Ticks are important vectors of various pathogens that cause infectious diseases in humans. Endosymbiotic bacteria have been explored as targets for tick and tick-borne disease control. However, the tick bacterial community on Hainan Island, which is the largest tropical island in China and has an environment favourable to ticks, has not yet been studied. In this study, we surveyed the bacterial community of ticks collected from grass in one village in Haikou. A total of 20 ticks were morphologically and molecularly identified as Haemaphysalis spp. The tick bacterial 16S rRNA hypervariable region amplicon libraries were sequenced on an Illumina MiSeq platform. A total of 10 possible bacterial genera were detected, indicating a low-diversity bacterial community profile. The dominant bacterial genus, Massilia, accounted for 97.85% of the population. Some other bacterial genera, including Arsenophonus and Pseudomonas, have been reported to play a role in tick development and tick-borne pathogen transmission in other tick species. Overall, the study highlights the first descriptive understanding of the tick bacterial community on Hainan Island and provides a basis for deciphering the interactions between the tick microbiome and tick-borne pathogens.

Screening core genes and signaling pathways after SFTSV infection by integrated transcriptome profiling analysis.

A newly discovered tick-borne virus called the severe fever with thrombocytopenia syndrome virus (SFTSV) can cause the severe fever with thrombocytopenia syndrome (SFTS). The mortality and incidence rate of SFTS patients remain extremely high due to the fast global dissemination of its arthropod vectors, and the mechanism of viral pathogenesis remains largely unknown. In this study, high-throughput RNA sequencing (RNA-Seq) was used to sequence HEK 293 cells treated with SFTSV at four time points. 115, 191, 259, and 660 differentially expressed genes (DEGs) were identified at 6, 12, 24, and 48 h post-infection, respectively. We found that SFTSV infection induced the expression of genes responsible for numerous cytokine-related pathways, including TNF, CXCL1, CXCL2, CXCL3, CXCL8, CXCL10, and CCL20. With the extension of infection time, the expression of most genes involved in these pathways increased significantly, indicating the host's inflammatory response to SFTSV. Moreover, the expression levels of GNA13, ARHGEF12, RHOA, ROCK1, and MYL12A, elements of the platelet activation signaling pathway, were downregulated during SFTSV infection, suggesting that the SFTSV infection may cause thrombocytopenia by inhibiting platelet activation. Our results contribute to further understanding the interaction between SFTSV and the host.

Infection and transovarial transmission of severe fever with thrombocytopenia syndrome virus in Rhipicephalus sanguineus in Hainan Island, China.

Severe fever with thrombocytopenia syndrome virus (SFTSV) RNA level increased in female ticks after injection with SFTSV. Furthermore, SFTSV RNA was detected in the eggs and larvae that originated from the virus-infected female ticks.

Analysis of microbial diversity in the midgut of Haemaphysalis longicornis infected with severe fever with thrombocytopenia syndrome virus / 中国热带医学

@#Abstract: Objective　To investigate the composition and diversity of midgut microbial community of Haemaphysalis longicornis infected with severe fever with thrombocytopenia syndrome virus (SFTSV)． Methods　The midgut DNA of three group Haemaphysalis longicornis infected with SFTSV was extracted, and the 16S rDNA gene of the sample was sequenced by HiSeq platform. The composition and diversity of endosymbiotic microbial community were clarified by OTU cluster analysis and alpha diversity analysis. Results　The midgut microbial clusters of the three groups infected with SFTSV were 143, 113, 163 OTUs respectively; the sparsity curve and abundance grade curve showed that the data had sufficient sequencing depth, and the midgut of Haemaphysalis longicornis infected with SFTSV was rich in microbial composition, but the species distribution was uneven. The analysis of microbial community composition showed that Proteobacteria, Firmicutes and Actinobacteria were the main dominant bacteria at the phyla level. At the class level, Gammaproteobacteria, Bacilli, Betaproteobacteria and Actinomycetia were the main dominant bacteria. At the order level, Legionellales, Bacillales, Burkholderiales and Actinomycetales were the main dominant orders. At the family level, Coxiellaceae, Bacillaceae, Moraxellaceae and Rhodococcaceae were the main dominant families. At the genus level, the relative abundance of Coxiella was the highest, followed by Aeribaillus and Azonexus. Alpha diversity analysis showed that the average Shannon index was 139.67, the average Simpson index was 0.48, the average Chao index was 145.06, and the average ACE index was 147.11. Conclusions　The species diversity of intestinal microorganisms in Haemaphysalis longicornis infected with SFTSV is rich. The results provide a basis for further exploring the interaction between intestinal microbes of Haemaphysalis longicornis and SFTSV and developing new ideas for the prevention and control of ticks and tick-borne diseases.

Dual roles and evolutionary implications of P26/poxin in antagonizing intracellular cGAS-STING and extracellular melanization immunity.

P26, a homolog of the viral-encoded nuclease poxin that neutralizes the cGAS-STING innate immunity, is widely distributed in various invertebrate viruses, lepidopteran insects, and parasitoid wasps. P26/poxin from certain insect viruses also retains protease activity, though its biological role remains unknown. Given that many P26s contain a signal peptide, it is surmised that P26 may possess certain extracellular functions. Here, we report that a secretory baculoviral P26 suppresses melanization, a prominent insect innate immunity against pathogen invasion. P26 targets the cofactor of a prophenoloxidase-activating protease, and its inhibitory function is independent of nuclease activity. The analysis of P26/poxin homologs from different origins suggests that the ability to inhibit the extracellular melanization pathway is limited to P26s with a signal peptide and not shared by the homologs without it. These findings highlight the independent evolution of a single viral suppressor to perform dual roles in modulating immunity during virus-host adaptation.

Proteomics reveals the hemolymph components of partially fed Hyalomma asiaticum ticks.

Hemolymph infection facilitates pathogen invasion of internal tick tissues. However, the overall protein composition of the hemolymph has not been analyzed for any tick species. Here, a gel based liquid chromatography tandem mass spectrometry method was used to characterize the hemolymph proteome of Hyalomma asiaticum females during blood feeding. A total of 311 proteins were identified. Hemelipoglyco-carrier proteins, apolipophorin-like proteins, and intracellular proteins were the most abundant proteins. Thirteen immunity-related proteins were identified, including peptidoglycan recognition protein (PGRP), Thioester-containing proteins (TEPs), clip­serine proteinases, serpins and Dome. The presence of hemocytin, proclotting enzyme homologs, serpins, TEPs, factor D-like protein and the lack of coagulin, hemocyanin, and prophenoloxidase suggest ticks may possess a unique coagulation system, which is largely different from that of insects. Taken together, the study revealed the constitution, level, and possible functions of global hemolymph proteins in H. asiaticum and could facilitate the discovery of new targets for control of tick-borne pathogens.

The Micrococcus luteus infection activates a novel melanization pathway of cSP10, cSP4, and cSP8 in Helicoverpa armigera.

Melanization is a key immune response mediated by serine protease (SP) cascade in insects. Multiple SP pathways exist in different species and it is unclear how conserved these cascades are. The cotton bollworm Helicoverpa armigera is a major worldwide agricultural pest. We reported a conserved melanization pathway in this species, which consists of SP41, cSP1, and cSP6. In this study, we attempted to identify an insect pathogen that elicits the cascade and test whether or not there are other SP cascades in H. armigera. After Micrococcus luteus, Enterobacter cloacae, Beauveria bassiana, or Helicoverpa armigera nucleopolyhedrovirus were injected into larvae, pathogen-induced hemolymph samples were collected for in vitro biochemical assays, which failed to detect proSP41 or procSP1 activation. In contrast, we found that procSP4, a protein proposed to participate in H. armigera melanization, was activated in M. luteus infected hemolymph. We further revealed that cSP8 was a prophenoloxidase (PPO) activating protease downstream of cSP4, and cSP4 was activated by cSP10. The pathway of cSP10-cSP4-cSP8 activated PPO in vitro. Efficiently cleaved procSPH11 and procSPH50 by cSP8 substantially enhanced phenoloxidase activity, suggesting they work together as a cofactor for cSP8 mediated PPO activation. Hemolymph from larvae challenged with M. luteus or its peptidoglycan effectively activated procSP10. Collectively, these results revealed a new PPO activation cascade specifically triggered by the bacterium. In addition, we found that the PPO activation cascades in H. armigera and Manduca sexta are conserved.

Microbiota modulates gut immunity and promotes baculovirus infection in Helicoverpa armigera.

Baculoviruses are natural enemies of agricultural and forest insect pests and play an important role in biological pest control. Oral infection by baculovirus in the insect midgut is necessary for establishing systemic infection and eventually killing the insect. Since the insect midgut continuously encounters microbiota, the gut microbiota could affect baculovirus infection. Here, we demonstrated that gut microbiota modulates immune responses and promotes baculovirus infection in the cotton bollworm, Helicoverpa armigera. After oral infection, numerous host immunity-related genes including genes encoding Toll and immune deficiency (IMD) pathway components were upregulated in the midgut. Elimination of the gut microbiota significantly increased the resistance to viral infection in H. armigera. Quantitative real-time reverse transcription polymerase chain reaction and proteomic analysis showed that downregulation of the antiviral factor prophenoloxidase (PPO) could be mediated by microbiota during infection. It implied that midgut microbiota diminishes the expression of PPO to facilitate viral infection in H. armigera. Our findings revealed that the microbiota plays an important role in modulating the resistance of H. armigera to baculovirus infection, providing new insights in applying biopesticide.

Transcriptome analysis of the innate immune system of Hyalomma asiaticum.

Ticks are considered to be the second most important vectors of human infectious diseases. The innate immune system is the key factor that affects its vector competence. Hyalomma asiaticum is the primary vector of Crimean-Congo hemorrhagic fever virus (CCHFV). However, the immune system of H. asiaticum remains virtually unknown. Here, a high throughput full-length mRNA sequencing method was adopted to define the immunotranscriptome of H. asiaticum infected with the fungal pathogen Beauveria bassiana and gram-negative bacterium Enterobacter cloacae. The analysis yielded 22,300 isoforms with an average length of 3233 bps. In total, 68 potential immunity-related genes were identified based on similarity to the homologs known to be involved in immunity. These included most members of the Toll and JAK/STAT signaling pathways, but not the IMD signaling pathway. Moreover, two copies of Dicer-2 and five copies of Argonaute-2 were detected. These genes are postulated to be involved in the RNA interference (RNAi) pathway, which is an important defense against RNA viruses. Overall, this study provides the foundation for understanding the immune response of H. asiaticum to CCHFV.

Identification of a Conserved Prophenoloxidase Activation Pathway in Cotton Bollworm Helicoverpa armigera.

Melanization is a prominent insect humoral response for encapsulation of and killing invading pathogens. It is mediated by a protease cascade composed of a modular serine protease (SP), and clip domain SPs (cSPs), which converts prophenoloxidase (PPO) into active phenoloxidase (PO). To date, melanization pathway in cotton bollworm Helicoverpa armigera, an important agricultural pest, remains largely unclear. To biochemically reconstitute the pathway in vitro, the putative proteases along with modified proteases containing the factor Xa cleavage site were expressed by Drosophila S2 cell expression system. Purified recombinant proteins were used to examine their role in activating PPO. It is revealed that cascade is initiated by a modular SP-SP41, followed by cSP1 and cSP6. The three-step SP41/cSP1/cSP6 cascade could further activate PPO, and the PO activity was significantly enhanced in the presence of two cSP homologs (cSPHs), cSPH11 and cSPH50, suggesting the latter are cofactors for PPO activation. Moreover, baculovirus infection was efficiently blocked by the reconstituted PPO activation cascade, and the effect was boosted by cSPH11 and cSPH50. Taken together, we unraveled a conserved PPO activation cascade in H. armigera, which is similar to that exists in lepidopteran biochemical model Manduca sexta and highlighted its role in antagonizing viral infection.

Prophenoloxidase-Mediated Ex Vivo Immunity to Delay Fungal Infection after Insect Ecdysis.

Skin immunity protects animals from airborne pathogen infection. Unlike mammals, arthropods, including insects, undergo periodic ecdysis to grow and develop. Newly molted insects emerge with unsclerotized thin cuticles but successfully escape pathogenic infections during the post-molt period. Here we show that prophenoloxidases (PPOs) in molting fluids remain bioactive on the integument and impede fungal infection after ecdysis. We found that the purified plasma PPOs or recombinant PPOs could effectively bind to fungal spores (conidia) by targeting the cell wall components chitin and ß-1,3-glucan. Pretreatment of the spores of the fungal pathogen Beauveria bassiana with PPOs increased spore hydrophilicity and reduced spore adhesion activity, resulting in a significant decrease in virulence as compared with mock infection. We also identified a spore-secreted protease BPS8, a member of peptidase S8 family of protease that degrade PPOs at high levels to benefit fungal infection, but which at lower doses activate PPOs to inhibit spore germination after melanization. These data indicate that insects have evolved a distinct strategy of ex vivo immunity to survive pathogen infections after ecdysis using PPOs in molting fluids retained on the underdeveloped and tender integument of newly molted insects for protection against airborne fungal infection.

Inhibition of melanization by serpin-5 and serpin-9 promotes baculovirus infection in cotton bollworm Helicoverpa armigera.

Melanization, an important insect defense mechanism, is mediated by clip-domain serine protease (cSP) cascades and is regulated by serpins. Here we show that proteolytic activation of prophenoloxidase (PPO) and PO-catalyzed melanization kill the baculovirus in vitro. Our quantitative proteomics and biochemical experiments revealed that baculovirus infection of the cotton bollworm, Helicoverpa armigera, reduced levels of most cascade members in the host hemolymph and PO activity. By contrast, serpin-9 and serpin-5 were sequentially upregulated after the viral infection. The H. armigera serpin-5 and serpin-9 regulate melanization by directly inhibiting their target proteases cSP4 and cSP6, respectively and cSP6 activates PPO purified from hemolymph. Furthermore, serpin-5/9-depleted insects exhibited high PO activities and showed resistance to baculovirus infection. Together, our results characterize a part of the melanization cascade in H. armigera, and suggest that natural insect virus baculovirus has evolved a distinct strategy to suppress the host immune system.

Dynamics of the Interaction between Cotton Bollworm Helicoverpa armigera and Nucleopolyhedrovirus as Revealed by Integrated Transcriptomic and Proteomic Analyses.

Over the past decades, Helicoverpa armigera nucleopolyhedrovirus (HearNPV) has been widely used for biocontrol of cotton bollworm, which is one of the most destructive pest insects in agriculture worldwide. However, the molecular mechanism underlying the interaction between HearNPV and host insects remains poorly understood. In this study, high-throughput RNA-sequencing was integrated with label-free quantitative proteomics analysis to examine the dynamics of gene expression in the fat body of H. armigera larvae in response to challenge with HearNPV. RNA sequencing-based transcriptomic analysis indicated that host gene expression was substantially altered, yielding 3,850 differentially expressed genes (DEGs), whereas no global transcriptional shut-off effects were observed in the fat body. Among the DEGs, 60 immunity-related genes were down-regulated after baculovirus infection, a finding that was consistent with the results of quantitative real-time RT-PCR. Gene ontology and functional classification demonstrated that the majority of down-regulated genes were enriched in gene cohorts involved in energy, carbohydrate, and amino acid metabolic pathways. Proteomics analysis identified differentially expressed proteins in the fat body, among which 76 were up-regulated, whereas 373 were significantly down-regulated upon infection. The down-regulated proteins are involved in metabolic pathways such as energy metabolism, carbohydrate metabolism (CM), and amino acid metabolism, in agreement with the RNA-sequence data. Furthermore, correlation analysis suggested a strong association between the mRNA level and protein abundance in the H. armigera fat body. More importantly, the predicted gene interaction network indicated that a large subset of metabolic networks was significantly negatively regulated by viral infection, including CM-related enzymes such as aldolase, enolase, malate dehydrogenase, and triose-phosphate isomerase. Taken together, transcriptomic data combined with proteomic data elucidated that baculovirus established systemic infection of host larvae and manipulated the host mainly by suppressing the host immune response and down-regulating metabolism to allow viral self-replication and proliferation. Therefore, this study provided important insights into the mechanism of host-baculovirus interaction.

The outer membrane protein, LamB (maltoporin), is a versatile vaccine candidate among the Vibrio species.

Maltoporin (LamB) is a family of outer membrane proteins. There has been no report of immunological characteristics of LamB in the Vibrio species so far. In this study, lamB genes from eight Vibrio strains were cloned and sequenced. The bioinformatics analysis indicated that sequence similarities of LamB proteins were ranged from 46.7% to 81.1%. Further, the result showed that their antigenic epitopes were highly conserved implying that LamB might be a shared antigen among Vibrios. The Western blot of rabbit sera against recombinant LamB from V. alginolyticus ATCC 33787 with cell lysate of 18 Vibrio strains showed cross-recognition. Bands observed on cell lysate of Vibrio strains immunoblotted with the anti-LamB sera ranged between 40 and 49 kDa. The Whole-cell ELISA assay further confirmed that the antisera of recombinant LamB recognized the tested Vibrio strains indicating the surface-exposed of LamB. Finally, the cross-protective property of recombinant LamB was evaluated through vaccination and subsequent challenge with heterogeneous virulent Vibrio strains in zebrafish. Recorded relative percent survival (RPS) of the vaccinated group varied from 54.1% to 77.8%, showing that zebrafish were protected from Vibrio infection after immunization with LamB protein. The cumulative evidences in this study suggested that LamB was a conserved antigen among tested Vibrio species and might be a potentially versatile vaccine candidate for the prevention of Vibriosis.