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NIRV_HOST_INT Report Summary

Project ID: 682394
Funded under: H2020-EU.1.1.

Periodic Reporting for period 1 - NIRV_HOST_INT (Population genomics of co-evolution between non-retroviral RNA viruses and their hosts)

Reporting period: 2016-05-01 to 2017-10-31

Summary of the context and overall objectives of the project

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. 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.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

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. Characterise the pattern of viral integrations in wild-caught mosquitoes from the native home range and derived populations. Assess the prevalence of viral integrations in natural populations and correlate NIRVs presence with flavivirus infections.
WORK PERFORMED and RESULTS. The Zika epidemic of 2015-2016 intensified vector control throughout the world, reducing the number of mosquitoes and limiting access to sampling. For this reason, we could not sample in California and Thailand, but we established contacts that allowed sampling in alternative places representative of Ae. albopictus derived and native populations such as Mexico and La Reunion, respectively. For the same reason, we delayed sampling in China to fall 2017. Sampling in Guangdong occured in November 2017. Extensive wild sampling was performmed in Mexico, in Italy and in La Reunion . In Mexico, we sampled mosquitoes between Dicember 2016 and Feruary 2017 the region of Tapachula (Chiapas state), which is endemic for dengue and chikungunya viruses. In Italy, where Ae. albopictus picks between August and September, we sampled in the Lombardia region in late August 2016. In La Reunion, we sampled between March and April 2017 in six localities (Saint Pierre, Tampon, Saint Leu, Cirad, Etang Salé and Saint Luis) from the west coast of the island where arboviruses are circulating based on epidemiological data of the number and location of confirmed dengue cases in 2015. We also sampled in five localities (Saint Benoit, Bras-Panon, Saint Rose, Anse des Cascades and Marlongue) from the east coast, from where no dengue cases were reported in 2015. Nucleic acid was extracted from these samples and whole genome sequencing was done. We are performing bioinformatics analyses to assess the patterns of viral integrations. We recently described the pattern of viral integrations in the genome of the Ae. albopictus reference Foshan strain (Palatini et al., 2017, BMC Genomics 18: 512). We are analysing the sequencing data from wild mosquitoes not only in comparison to what detected in Foshan, but we are also exploring novel bioinformatics tools to assess the presence of novel viral integrations.
As anticipated above, we recently completed a bioinformatics analysis that described all the viral integrations of the genome of the Ae. albopictus reference Foshan strain. This analyses was extended to all mosquito species for which there is a genome assembly available to gain insight on the widespread of viral integrations. We showed that viral integrations are not randomly distributed across mosquito species, but are ten-fold more abundant in the arboviral vectors Ae. aegypti and Ae. albopictus than Anophelinae mosquitoes. In Ae. albopcitus we characterised a total of 72 loci with sequences encompassing a complete or partial open reading frame for viral proteins. All these 72 loci had sequences with similarities to either Flaviviruses or Rhabdoviruses sequences, among the 425 viral species tested. Additionally, we observed that viral integrations are statistically enriched in regions of the genome annotated as piRNA clusters and are associated with sequences of transposable elements (TEs). We also looked at RNAseq vs smallRNAseq data and verified that piRNAs, but not polyadenilated mRNAs map to viral integrations, suggesting that viral integrations produce piRNAs, but have limited transcriptional activity. We are using molecular techniques to validate the viral integrations with similarities to Flaviviruses that we identified bioinformatically. While the finding that viral integrations are located primarily in regions of the genome harbouring repetitions, including piRNA clusters and TEs, is significant because it su

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Our results on the pattern of viral integrations in the Foshan strain and our preliminary results on wild mosquiitoes are significant because they support a connection among viral integrations, the piRNA pathway and TEs. It was recently shown that cDNA fragments of different arboviruses, including dengue and chikungunya viruses, are produced following mosquito infection by the action of an endogenous reverse transcriptase and are recruited by the RNAi machinery to favour the persistence of infection. Results from our bioinformatics analysis suggest that these cDNA fragments could be the substrate for integrations and establish an association between the piRNA pathway and viral integrations. Additionally, our analyses detected only sequences from Flaviviruses and Rhabdoviruses integrated into the genome of Ae. albopictus suggesting lineage-specific interactions between nonretroviral RNA viruses and their host may exist for integration to occur. In the wild samples we analysed so far, we did not detect integrations from Alphaviruses, supporting their current use in gene therapy applications.
Our effort on re-sequencing and re-annotating the Ae. albopictus genome combining PacBio SMART and 10X Genomics approaches will provide a high-quality sequence and assembly from which all the scientific community will benefit.
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