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Population genomics of co-evolution between non-retroviral RNA viruses and their hosts

Periodic Reporting for period 4 - NIRV_HOST_INT (Population genomics of co-evolution between non-retroviral RNA viruses and their hosts)

Reporting period: 2020-11-01 to 2022-04-30

The project NIRV_HOST_INT aims at studying integrations of sequences from nonretroviral RNA viruses in the genome of their eukaryotic hosts. Nonretroviral RNA viruses are the most common viruses that infect eukaryotes and include arboviruses (i.e. viruses transmitted by an arthropod host) with high public health relevance, such as Dengue, Zika and West Nile viruses (Flavivirus genus). Despite having different genome structures and replication strategies, all nonretroviral RNA viruses do not encode for reverse transcriptase and integrase. As a consequence, nonretroviral RNA viruses should be present in host cells transiently, a property that favoured their application in medicine as delivery vectors for vaccine and drugs. However, the application of next-generation sequencing technologies and metagenomic analyses led to the discovery of sequences from nonretroviral RNA viruses integrated into the genome of many eukaryotes. The safe application of nonretroviral RNA viruses in medicine requires the understanding of which viral species integrate into host genomes and under which circumstances this phenomenon occurs. Sequences from nonretroviral RNA viruses have been found integrated also into the genomes of arboviral vectors, including the Asian tiger mosquito Aedes albopictus, which is the main arboviral vector in temperate regions of the world, including Europe. It is still unclear whether viral integrations are transcribed, facilitate the establishment and the progression of viral infection or, on the contrary, they prevent further infections with cognate viruses. Understanding the impact of viral integrations on mosquito vectors is important because virus-vector interaction processes could be manipulated to develop novel genetic-based strategies of mosquito control.
This project uses the model system “Aedes albopictus-Flavivirus” and combines molecular work in a laboratory setting with sampling of mosquitoes in the wild to investigate whether integration of sequences from nonretroviral RNA viruses is a common phenomenon in nature, which viruses integrate into the genome of Ae. albopictus, whether there are hot spot of integrations in the genome which could lead to hypothesis on mechanisms of integrations and whether the presence of viral integrations affect the outcome of subsequent viral infections.
An atlas of viral integrations in the genome of Ae. albopictus was produced, showing that viral integrations are not distributed homogeneously across the genome, but they are enriched next to transposable elements (TEs) in piRNA clusters and produce piRNAs, suggesting they are antiviral. Some viral integrations are also part of coding sequences, which are expressed at the mRNA level, indicating that there is not a universal role for viral integrations. Analyses of the genome of wild caught mosquitoes showed variations in the pattern of viral integrations, including the novel acquisition of viral integrations. The number of novel viral integrations and their similarity to currently circulating viruses seem to indicate that most integrations occurred millions of years ago, but the acquisition of viral sequences is an ongoing process, with few integrations having built in only recently. These results emphasise the importance of studying viral integrations in the context of the genetic-make up and population history of their hosts. The acquisition of viral sequences from the environment and the role of these sequences in subsequent response to the same stimulus (challenge with cognate virus), are hallmarks of adaptive immunity, a new concept for invertebrates.
During the course of the project it was realised that the available bioinformatic tools to annotate viral integrations and identify new integration sites, which had been mostly produced in the context of human cancer genetics, do not perform when applied to the genome of mosquitoes due to the richness in repetitive elements. Thus a new pipeline to account for intrasample variability was developed and included in a a step-to-step protocol to identify viral integrations and study their polymorphism (https://github.com/BonizzoniLab). A new assembly for the Ae. albopictus genome based on long-read sequencing and Hi-C was also made available for the scientific community.
This project studies nonretroviral integrated RNA virus sequences (NIRVs) through two aims, each with different goals. Work performed and main achievements are reported below, under each aim.

Aim 1) Population genomics analyses of NIRVs in Ae. albopictus
TASK determine the patterns of viral integrations and their prevalence in the genome of wild-collected mosquitoes.
WORK PERFORMED and RESULTS. We generated a new genome assembly of Ae. albopicuts based on long read sequencing approaches (published in Genome Biology in August 2020, 21:215) and a bioinformatic program called ViR (published in BMC Bioinformatics in 2021, 10.1186/s12859-021-03980-5) and annotated viral integrations in the genome of Ae. albopictus. Using these resources we looked for viral integrations in wild-collected mosquitoes and were able to identify a total of 7. We demonstrated that viral integrations occur in repetitive regions of the genome in close association with trasposable elements, mostly in piRNA clusters and produce piRNAs.
We also generated a public database (http://www.nreves.com) for the viral integrations of Aedes spp. mosquitoes

Aim 2) Biological Impact of NIRVs in Ae. albopictus.
TASK1 potential effects of NIRVs on mosquito fitness and heritability of NIRVs
WORK PERFORMED and RESULTS. Life-table parameters of different Ae. albopictus strains were built and data analysed. We studied the landscape of viral integrations in the reference Foshan strain in details (Pischedda et al., 2019, 10.3389/fgene.2019.0009). We generated mosquito families by doing single matings, we are analysing whole genome sequencing data of these samples collected at G0 and G12.
TAKS2 potential effects of NIRVS in presence of subsequent infections
We tried to generate an Ae. albopictus transgenic line by inserting into a viral integration within a piRNA cluster a synthetic viral integration made of fragments from the genome of different viruses. Because this approach did not work, we reverted to using a cell-line based approach. We identified three promising viral integrations which seem to reduce subsequent infection with cognate viruses and we are testing knock down of these NIRVS.
Data from infection experiments with different Ae. albopictus strains and the arboviruses Dengue and Chukungunya viruses, which were done at the Institut Pasteur were analysed and published in 2021 (Viruses,10.3390/v13040553). We also opted for a cell-line approach to further understand the origin of viral integrations and characterise integration frequencies.

Overall this project led to a new research avenue that intersects insect genomics with insect immunity.
The new genome assembly of Ae. albopictus (AalbF2) represents the most up-to-date collective knowledge of the Ae. albopictus genome and is expected to aid in the understanding of the adaptation potential and the epidemiological relevance of this species and foster the development of innovative control measures. The database of viral integrations we produced (http://www.nreves.com) will foster new investigations on the role and widespread distribution of viral integrations.
home page of the database for viral integration