Insect-Specific Viruses Regulate Vector Competence in Aedes Aegypti 2 Mosquitoes Via Expression of Histone H4 3 4 Roenick P
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bioRxiv preprint doi: https://doi.org/10.1101/2021.06.05.447047; this version posted June 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Insect-specific viruses regulate vector competence in Aedes aegypti 2 mosquitoes via expression of histone H4 3 4 Roenick P. Olmo1,2*, Yaovi M. H. Todjro1*, Eric R. G. R. Aguiar1,3*, João Paulo P. de 5 Almeida1, Juliana N. Armache1, Isaque J. S. de Faria1, Flávia V. Ferreira1, Ana 6 Teresa S. Silva1, Kátia P. R. de Souza1, Ana Paula P. Vilela1, Cheong H. Tan4, 7 Mawlouth Diallo5, Alioune Gaye5, Christophe Paupy6, Judicaël Obame-Nkoghe7,8, 8 Tessa M. Visser9, Constantianus J. M. Koenraadt9, Merril A. Wongsokarijo10, Ana 9 Luiza C. Cruz11, Mariliza T. Prieto12, Maisa C. P. Parra13, Maurício L. Nogueira13, 10 Vivian Avelino-Silva14, Renato N. Mota15, Magno A. Z. Borges16, Betânia P. 11 Drumond11, Erna G. Kroon11, Luigi Sedda17, Eric Marois2, Jean-Luc Imler2 & João T. 12 Marques1,2, # 13 14 1 Department of Biochemistry and Immunology, Instituto de Ciências Biológicas, 15 Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, Brazil. 16 2 Université de Strasbourg, CNRS UPR9022, INSERM U1257, 67084 Strasbourg, 17 France. 18 3 Department of Biological Science (DCB), Center of Biotechnology and Genetics 19 (CBG), State University of Santa Cruz (UESC), 45662-900, Ilhéus, Brazil. 20 4 Environmental Health Institute, Vector Biology & Control Division, National 21 Environment Agency, 138667, Singapore. 22 5 Pôle de Zoologie Médicale, Institut Pasteur de Dakar, Dakar, Senegal 23 6 Maladies Infectieuses et Vecteurs: Écologie, Génétique, Évolution et Contrôle 24 (MIVEGEC); Université de Montpellier, Institut de Recherche pour le Développement, 25 CNRS, 34394 Montpellier, France. 26 7 Laboratoire de Biologie Moléculaire et Cellulaire, Département de Biologie, 27 Université des Sciences et Techniques de Masuku, 901 Franceville, Gabon. 28 8 Écologie des Systèmes Vectoriels, Centre Interdisciplinaire de Recherches 29 Médicales de Franceville, 769 Franceville, Gabon. 30 9 Laboratory of Entomology, Wageningen University & Research, 6708, Wageningen, 31 The Netherlands. 32 10 Central Laboratory of the Bureau of Public Health, Paramaribo, Suriname. 33 11 Department of Microbiology, Instituto de Ciências Biológicas, Universidade Federal 34 de Minas Gerais (UFMG), 31270-901, Belo Horizonte, Brazil. 35 12 Secretaria Municipal de Saúde, Seção de Controle de Vetores, Santos City Hall, 36 11013-151, Santos, Brazil. 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.05.447047; this version posted June 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 37 13 Laboratory of Research in Virology, Faculdade de Medicina de São José do Rio 38 Preto (FAMERP), 15090-000, São José do Rio Preto, Brazil. 39 14 Department of Infectious and Parasitic Diseases, Faculdade de Medicina da 40 Universidade de São Paulo (FMUSP), 01246-903, Cerqueira Cesar, Brazil. 41 15 Health Surveillance (Zoonosis Control), Brumadinho City Hall, 35460-000, 42 Brumadinho, Brazil. 43 16 Center for Biological and Health Sciences, Universidade Estadual de Montes 44 Claros, 39401-089, Montes Claros, Brazil. 45 17 Lancaster Medical School, Lancaster University, Lancaster, LA1 4YG, United 46 Kingdom. 47 * These authors contributed equally to this work. 48 # Corresponding author 49 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.05.447047; this version posted June 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 50 Abstract 51 52 Aedes aegypti and Aedes albopictus are major mosquito vectors for arthropod-borne 53 viruses (arboviruses) such as dengue (DENV) and Zika (ZIKV) viruses. Mosquitoes 54 also carry insect-specific viruses (ISVs) that may affect the transmission of 55 arboviruses. Here, we analyzed the global virome in urban Aedes mosquitoes and 56 observed that two insect-specific viruses, Phasi Charoen-like virus (PCLV) and 57 Humaita Tubiacanga virus (HTV), were the most prevalent in A. aegypti worldwide 58 except for African cities, where transmission of arboviruses is low. Spatiotemporal 59 analysis revealed that presence of HTV and PCLV led to a 200% increase in the 60 chances of having DENV in wild mosquitoes. In the laboratory, we showed that HTV 61 and PCLV prevented downregulation of histone H4, a previously unrecognized 62 proviral host factor, and rendered mosquitoes more susceptible to DENV and ZIKV. 63 Altogether, our data reveals a molecular basis for the regulation of A. aegypti vector 64 competence by highly prevalent ISVs that may impact how we analyze the risk of 65 arbovirus outbreaks. 66 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.05.447047; this version posted June 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 67 Main Text 68 69 Arthropod-borne viruses (arboviruses) transmitted by mosquitoes, such as DENV, 70 ZIKV and chikungunya (CHIKV) viruses, are a great threat to human health 71 worldwide (1). DENV alone is estimated to cause 400 million infections per year, 72 leading to approximately 20,000 deaths (2). DENV transmission has increased 73 several folds over the past decades and newly emerged mosquito borne viruses such 74 as CHIKV and ZIKV have become important (1, 3). Increased transmission of 75 arboviruses has been enabled by the global spread of Aedes aegypti and Aedes 76 albopictus that are their major urban mosquito vectors. Globalization, urbanization 77 and climate change have positively impacted the distribution of these Aedes 78 mosquitoes worldwide (4–6). Importantly, we still lack vaccines or treatments for 79 most arboviral diseases (1). 80 81 Arboviruses circulate primarily in enzootic cycles involving sylvatic mosquitoes and 82 vertebrate animals causing rare accidental human infections (6, 7). However, 83 arboviruses may become able to infect humans efficiently and be maintained by 84 human-mosquito-human transmission in nature that will no longer require animal 85 reservoirs (1). In this scenario, virologic surveillance of urban Aedes mosquitoes can 86 lead to early identification of circulating viruses and help raise preparedness to 87 prevent outbreaks (8). However, studies monitoring the collection of viruses, the 88 virome, in Aedes mosquitoes have mostly identified a large diversity of insect-specific 89 viruses (ISVs) (9–13). ISVs do not infect vertebrates but can affect the capacity of the 90 mosquito to be infected, maintain and transmit arboviruses, usually referred to as 91 vector competence (8, 14, 15). However, most data on ISV-arbovirus interactions 92 have been obtained in cell lines using closely related viruses that may compete for 93 common resources (16–26). In addition, effects were often reported for ISVs that 94 belong to the same family as arboviruses and are likely to compete for common 95 resources, a phenomenon referred to as superinfection exclusion (17, 19, 23). 96 Mechanistic understanding of how ISVs affect arboviruses beyond the competition 97 between related viruses remains scarce, especially in vivo, and we still lack evidence 98 for interactions in natural conditions. In this regard, the presence of ISVs could help 99 explain differences in vector competence between mosquito populations thus 100 contributing to outbreaks. 101 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.05.447047; this version posted June 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 102 Here, we characterize the virome of urban Aedes mosquitoes worldwide and provide 103 novel mechanistic understanding of the interactions between ISVs and arboviruses in 104 natural conditions. 105 5 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.05.447047; this version posted June 5, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 106 Results 107 108 A. aegypti and A. albopictus mosquitoes are the most prolific and widespread vectors 109 for arboviruses (5, 8). To assess the collection of viruses found in these mosquitoes, 110 we took advantage of a network of field researchers wordlwide. Adult A. aegypti and 111 A. albopictus mosquitoes were collected directly from the field in 12 different sites 112 from 6 countries on 4 continents (Figure 1a). In total, 815 adult mosquitoes were 113 pooled according to species, location and date of collection resulting in 91 samples 114 derived from 69 A. aegypti and 22 A. albopictus pools or individual insects 115 (Supplementary Table 1). These 91 samples were used to construct small RNA 116 libraries that were sequenced and analyzed using a metagenomic strategy, 117 previously described by our group (9), optimized to detect viruses (Figure 1b). In 118 total, we were able to assemble 7260 contiguous sequences (contigs) larger than 119 200 nt from the 91 individual libraries (Figure 1b). A summary of the assembly 120 results is shown in Supplementary Table 2. Out of these 7260 contigs assembled, 121 1448 contigs were identified as putative viral sequences by sequence similarity 122 searches against non-redundant nucleotide and protein databases (NT and NR, 123 respectively) at GenBank (Figure 1b, Supplementary Table 3). Although the 124 number of contigs assembled per library varied greatly, we observed high abundance 125 and diversity of viral contigs in most samples (Figure 1c). Comparing results from 126 the two mosquito species, the percentage of viral contigs was strikingly smaller in A. 127 albopictus libraries compared to A. aegypti (Figure 1c). In addition, we noted more 128 variation in the number of assembled contigs and larger proportions of unknown 129 contigs in libraries from A.