A cell atlas of the adult female Aedes aegypti midgut revealed by single-cell RNA sequencing.

Aedes aegypti is a primary vector for transmitting various arboviruses, including Yellow fever, dengue and Zika virus. The mosquito midgut is the principal organ for blood meal digestion, nutrient absorption and the initial site of arbovirus infection. Although a previous study delineated midgut's transcriptome of Ae. aegypti at the single-nucleus resolution, there still lacks an established protocol for isolating and RNA sequencing of single cells of Ae. aegypti midgut, which is required for investigating arbovirus-midgut interaction at the single-cell level. Here, we established an atlas of the midgut cells for Ae. aegypti by single-cell RNA sequencing. We annotated the cell clusters including intestinal stem cells/enteroblasts (ISC/EB), cardia cells (Cardia), enterocytes (EC, EC-like), enteroendocrine cells (EE), visceral muscle (VM), fat body cells (FBC) and hemocyte cells (HC). This study will provide a foundation for further studies of arbovirus infection in mosquito midgut at the single-cell level.

Development and evaluation of RT-qPCR assays for two neglected orthobunyaviruses: Oya virus and Ebinur Lake virus.

OBJECTIVES: Oya virus (OYAV) and Ebinur lake virus (EBIV) belong to the genus Orthobunyavirus within the Peribunyaviridae family, and both are recognized as the novel virus with potential threat to the animal or public health. Given their potential to cause outbreaks and their detection in diverse samples across different regions, the need for a reliable and efficient molecular detection method for OYAV and EBIV becomes imperative. METHODS: The S-segment of OYAV and EBIV was used for designing specific primer and probe sets, which were employed in a real-time reverse transcription quantitative PCR (RT-qPCR) assay. The analytical performance of these assays, encompassing specificity, sensitivity, reproducibility, and fitness for purpose, was thoroughly evaluated across various sample matrices. RESULTS: The developed RT-qPCR assays were very specific to their respective targets. Both assays were highly reproducible (%CV<3) and sensitive with the 95% limit of detection (LOD) of 0.80 PFU/mL for OYAV primer probe set and 0.37 PFU/mL for EBIV primer probe set. Furthermore, the assays fitness for purpose was good as it could detect the specific viruses in virus-spiked serum samples, virus-inoculated mosquito samples, field caught mosquitoes and biting midge samples. CONCLUSIONS: Our study has successfully developed specific, sensitive, and reliable RT-qPCR assays for the detection of OYAV and EBIV. These assays hold great promise for their potential application in clinical and field samples in the future.

HostNet: improved sequence representation in deep neural networks for virus-host prediction.

BACKGROUND: The escalation of viruses over the past decade has highlighted the need to determine their respective hosts, particularly for emerging ones that pose a potential menace to the welfare of both human and animal life. Yet, the traditional means of ascertaining the host range of viruses, which involves field surveillance and laboratory experiments, is a laborious and demanding undertaking. A computational tool with the capability to reliably predict host ranges for novel viruses can provide timely responses in the prevention and control of emerging infectious diseases. The intricate nature of viral-host prediction involves issues such as data imbalance and deficiency. Therefore, developing highly accurate computational tools capable of predicting virus-host associations is a challenging and pressing demand. RESULTS: To overcome the challenges of virus-host prediction, we present HostNet, a deep learning framework that utilizes a Transformer-CNN-BiGRU architecture and two enhanced sequence representation modules. The first module, k-mer to vector, pre-trains a background vector representation of k-mers from a broad range of virus sequences to address the issue of data deficiency. The second module, an adaptive sliding window, truncates virus sequences of various lengths to create a uniform number of informative and distinct samples for each sequence to address the issue of data imbalance. We assess HostNet's performance on a benchmark dataset of "Rabies lyssavirus" and an in-house dataset of "Flavivirus". Our results show that HostNet surpasses the state-of-the-art deep learning-based method in host-prediction accuracies and F1 score. The enhanced sequence representation modules, significantly improve HostNet's training generalization, performance in challenging classes, and stability. CONCLUSION: HostNet is a promising framework for predicting virus hosts from genomic sequences, addressing challenges posed by sparse and varying-length virus sequence data. Our results demonstrate its potential as a valuable tool for virus-host prediction in various biological contexts. Virus-host prediction based on genomic sequences using deep neural networks is a promising approach to identifying their potential hosts accurately and efficiently, with significant impacts on public health, disease prevention, and vaccine development.

In vivo and in vitro characterization of a new Oya virus isolate from Culicoides spp. and its seroprevalence in domestic animals in Yunnan, China.

Biting midges are one of the most common hematophagous insects. They are capable of transmitting a wide range of arboviruses and have a significant impact on public health and veterinary medicine. Herein, from midge samples collected in 2013 in Yunnan, China, one sample induced a cell cytopathic effect (CPE) in BHK-21, MA104, and PK15 cell lines. Next-generation sequencing data, RACE and PCR determined the genome sequence of the sample and designated as an Oya virus (OYAV) isolate SZC50. Phylogenetic analysis of the sample revealed that it was cluster into viruses from species Orthobunyavirus catqueense. The open reading frames of S, M, and L segment of OYAV SZC50 were closest to those of OYAV SC0806. Moreover, 831 serum samples (736 pigs, 45 cattle, and 50 sheep) were gathered from 13 cities in Yunnan Province to detect neutralizing antibody of OYAV SZC50. A significant proportion of OYAV SZC50 antibody (more than 30%) was found in Yunnan pig populations, with the positive rate of OYAV SZC50 antibody in pigs from Malipo reaching 95%. To determine the pathogenicity of OYAV SZC50, we chose three animal models: specific pathogen-free Kunming mice, C57BL/6 mice lacking the interferon α/ß receptor, and chicken embryos. At 5, 6, and 7 days post-infection, all adult and suckling C57BL/6 mice, and specific pathogen-free suckling Kunming mice were dead. Our finding was expanding the knowledge about the infection and pathogenic risk of the neglected virus in the Orthobunyavirus.

A global dataset of sequence, diversity and biosafety recommendation of arbovirus and arthropod-specific virus.

Arthropod-borne virus (arbovirus) and arthropod-specific virus (ASV) are viruses circulating amongst hematophagous arthropods that are broadly transmitted in ecological systems. Arbovirus may replicate in both vertebrates and invertebrates and some are known to be pathogenic to animals or humans. ASV only replicate in invertebrate arthropods yet they are basal to many types of arboviruses. We built a comprehensive dataset of arbovirus and ASV by curating globally available data from the Arbovirus Catalog, the arbovirus list in Section VIII-F of the Biosafety in Microbiological and Biomedical Laboratories 6th edition, Virus Metadata Resource of International Committee on Taxonomy of Viruses, and GenBank. Revealing the diversity, distribution and biosafety recommendation of arbovirus and ASV at a global scale is essential to the understanding of potential interactions, evolution, and risks associated with these viruses. Moreover, the genomic sequences associated with the dataset will enable the investigation of genetic patterns distinguishing the two groups, as well as aid in predicting the vector/host relationships of the newly discovered viruses.

Efficient rescue of a newly classified Ebinur lake orthobunyavirus with GFP reporter and its application in rapid antiviral screening.

Orthobunyaviruses have been reported to cause severe diseases in humans or animals, posing a potential threat to human health and socio-economy. Ebinur lake virus (EBIV) is a newly classified orthobunyavirus, which can induce the histopathogenic change and even the high mortality of infected BALB/c mice. Therefore, it is needed to further study the viral replication and pathogenesis, and develop the therapies to cope with its potential infection to human or animals. Here, through the reverse genetics system, the recombinant EBIV of wild type (rEBIV/WT) and NP-conjugated-eGFP (rEBIV/eGFP/S) were rescued for the application of the high-content screening (HCS) of antiviral drug. The eGFP fluorescence signal of the rEBIV/eGFP/S was stable in the process of successive passage in BHK-21 cells (over 10 passages) and this recombinant virus could replicate in various cell lines. Compared to the wild type EBIV, the rEBIV/eGFP/S caused the smaller plaques (diameter around 1 mm on 3 dpi) and lower peak titers (105 PFU/mL), suggesting attenuation due to the eGFP insertion. Through the high-content screening (HCS) system, two antiviral compounds, ribavirin and favipiravir, which previously reported to have effect to some bunyavirus were tested firstly. Ribavirin showed an inhibitory effect on the rEBIV/eGFP/S (EC50 = 14.38 µM) as our expect, while favipiravir with no inhibitory effect even using high doses. Furthermore, Tyrphostin A9 (EC50 = 0.72 µM for rEBIV/eGFP/S, EC50 = 0.05 µM for EBIV-WT) and UNC0638 (EC50 = 1.26 µM for rEBIV/eGFP/S, EC50 = 1.10 µM for rEBIV/eGFP/S) were identified with strong antiviral effect against EBIV in vitro from 150 antiviral compounds. In addition, the time-of-addition assay indicated that Tyrphostin A9 worked in the stage of viral post-infection, and the UNC0638 in all pre-, co-, and post-infection stages. This robust reverse genetics system will facilitate the investigation into the studying of viral replication and assembly mechanisms, and the development of drug and vaccine for EBIV in the future.

Experimental Viral Infection in Adult Mosquitoes by Oral Feeding and Microinjection.

Mosquito-borne viruses (MBVs), which are infectious pathogens to vertebrates, are spread by many mosquito species, posing a severe threat to public health. Once ingested, the viruses must overcome the mosquito midgut barrier to reach the hemolymph, from where they might potentially spread to the salivary glands. When a mosquito bites, these viruses are spread to new vertebrate hosts. Similarly, the mosquito may pick up different viruses. In general, only a tiny portion of viruses may enter the salivary glands via the gut. The transmission efficiency of these viruses to the glands is be affected by the two physical barriers found in different mosquito species: midgut barriers and salivary glands barriers. This protocol presents a method for virus detection in salivary glands of Aedes aegypti's following oral feeding and intrathoracic injection infection. Furthermore, determining whether the guts and/or salivary glands hinder viral spread can aid in the risk assessments of MBVs transmitted by Aedes aegypti.

In Vitro and In Vivo Characterization of a New Strain of Mosquito Flavivirus Derived from Culicoides.

Mosquito-specific flaviviruses comprise a group of insect-specific viruses with a single positive RNA, which can affect the duplication of mosquito-borne viruses and the life growth of mosquitoes, and which have the potential to be developed as a vaccine platform for mosquito-borne viruses. In this study, a strain of mosquito flavivirus (MFV) YN15-283-02 was detected in Culicoides collected from Yunnan, China. The isolation of the purified MFV YN15-283-02 from cell culture failed, and the virus was then rescued by an infectious clone. To study the biological features of MFV YN15-283-02 in vitro and in vivo, electron microscopy, phylogenetic tree, and viral growth kinetic analyses were performed in both cell lines and mosquitoes. The rescued MFV (rMFV) YN15-283-02 duplicated and reached a peak in C6/36 cells at 6 d.p.i. with approximately 2 × 106 RNA copies/µL (RNA to cell ratio of 0.1), but without displaying a cytopathic effect. In addition, the infection rate for the rMFV in Ae.aegypti show a low level in both larvae (≤15%) and adult mosquitoes (≤12%).