Hepatitis delta virus (HDV) is the smallest known RNA virus and encodes a single protein. Until recently, HDV had only been identified in humans, where it is strongly associated with co-infection with hepatitis B virus (HBV). However, the recent discovery of HDV-like viruses in metagenomic samples from birds and snakes suggests that this virus has a far longer evolutionary history. Herein, using additional meta-transcriptomic data, we show that highly divergent HDV-like viruses are also present in fish, amphibians and invertebrates. Notably, the novel viruses identified here share HDV-like genomic features such as a small genome size of ~1.7kb in length, circular genomes, and self-complementary, unbranched rod-like structures. Coiled-coil domains, leucine zippers, conserved residues with essential biological functions and isoelectronic points similar to those in the human hepatitis delta virus antigens (HDAgs) were also identified in the putative non-human HDAgs. Notably, none of these novel HDV-like viruses were associated with hepadnavirus infection, supporting the idea that the HDV-HBV association may be specific to humans. Collectively, these data not only broaden our understanding of the diversity and host range of HDV in non-human species, but shed light on its origin and evolutionary history.

Word count abstract: 247 20 21 Word count importance: 132 22 23 Word count main text: 4113 24 25 26 51 species, Culex pipiens and Culex torrentium, sampled from northern Europe. Our analysis 52 revealed extensive viral diversity, including 28 novel viruses, and was comparable to the 53 levels of diversity found in other temperate and tropical regions globally. Importantly, as well 54 as harbouring RNA viruses that are closely related to other mosquito-derived viruses sampled 55 in diverse global locations, we also described a number of viruses that are unique to specific 56 sampling locations in Sweden. Hence, these data showed that geographical factors can play an 57 important role in shaping virome structure even at local scales. 58 59 60 The mosquito (Diptera; Culicidae) genus Culex comprises more than a thousand species, with 61 representatives found globally (1). Culex species are vectors of a number of important 62 pathogens including West Nile virus (WNV) (Flaviviridae), Japanese encephalitis virus (JEV) 63 (Flaviviridae) and Sindbis virus (SINV) (Togaviridae), as well as a variety of nematodes (1-64 3). One of the most widespread Culex species is the Northern House mosquito, Cx. pipiens, 65 that is distributed across the northern hemisphere. In Europe and the Middle East it occurs 66 together with Cx. torrentium, another Culex species with females and larvae that are 67 morphologically identical to Cx. pipiens. These two species have overlapping distributions 68 and share larval habitats. However, Cx. torrentium dominates in northern Europe while Cx. 69 pipiens is more abundant in the south (4). Both species are vectors for a number of bird-70 associated viruses that can cause disease in Europe; for example, WNV, that may cause a 71 febrile disease with encephalitis, and SINV that may result in long lasting arthritis (2, 5). Cx. 72 pipiens is one of the most common WNV vectors in both southern Europe and North 73 America, and Cx. torrentium is the main vector of SINV in northern Europe (2, 6). Infections 74with these pathogenic viruses occur in late summer when the viral prevalence accumulates in 75 passerine birds, the vertebrate hosts of both of these viruses (7, 8). Despite their importance as 76 vectors, little is known about the detailed biology of Cx. pipiens and Cx. torrentium due to the 77 difficulties in species identification, which can only be reliably achieved through molecular 78 means. Much of the biology of these species, such as their larval habitat and feeding 79 preferences, is considered similar. However, one significant difference between the two 80 species is that while Cx. pipiens harbours a high prevalence of the intracellular bacteria 81 Wolbachia pipientis, it is seemingly absent in Cx. torrentium (9). 82 83 In recent years, studies utilizing RNA-sequencing (RNA-Seq, or 'meta-transcriptomics') have 84 revealed an enormous RNA virus diversity in both vertebrates and invertebrates (10, 11). 85 Mosquitoes are of particular interest as many are well-known vectors of...

The four mosquito-borne dengue virus serotypes (DENV1–DENV4) cause a high burden of disease throughout the tropical and sub-tropical regions of the world. Nevertheless, their precise epidemiological history in Africa, including when and where they originated and were distributed during the 20th century, remains unclear stressing the need for One Health focused research. Accordingly, we conducted a time-scaled molecular epidemiological reconstruction using publicly available and newly sequenced dengue virus genomes of African origin representing all four serotypes to deduce the most likely temporal and spatial transmission routes of each DENV serotype from their ancestral regions to, within and from Africa. Our analyses suggest that during the 20th century, serotypes DENV1–DENV3 were introduced to Africa from South East Asia on multiple occasions. The earliest evidence recorded indicates introduction of DENV2 during the early-1940s and of DENV1 during the mid-1940s to Western Africa from South East Asia. The analysis also implies an early introduction of DENV4 during the mid-1940s to Western Africa, alongside DENV1, probably originating in South East Asia. Establishment of DENV3 in Africa appears to have occurred later in the 1960s, apparently originating from South East Asia. However, with the re-establishment of DENV in the Americas, following the cessation of the PAHO mosquito control programme during the mid-20th century, evidence of introductions of DENV1 and DENV2 from the Americas to Western Africa was also observed. The data also identify intra-regional circulation of DENV, but also inter-regional dispersal of all four serotypes within Africa, which has led to a high degree of geographical overlap among serotypes. It is also noteworthy that DENV from both Eastern and Western Africa, have been introduced into Central Africa but there is no support for the converse relationship. For serotypes DENV1–DENV3, we observed probable exports from within established African DENV clusters (≥2 sequences) primarily to Eastern and Southern Asia. Collectively, our findings support the view that all DENV serotypes, apart from DENV4, have been introduced on multiple occasions to Africa, primarily originating from South East Asia, and subsequently to neighbouring regions within Africa.