Taxonomy of Phleboviruses, Emphasizing Those That Are Sandfly-Borne

Sandfly-borne phleboviruses (phylum Negarnavaricota, realm Riboviria, kingdom Orthornavirae, genus Phlebovirus) comprise three genome segments of ribonucleic acid (RNA) and which encode an RNA-dependent RNA polymerase, which they use to transcribe the viral RNA genome into messenger RNA and to replicate the genome. At least some of these viruses cause mild 3-day fevers in humans but some also have been associated with more severe illnesses in humans. The 67 recognized phleboviruses are listed here in a table composed by the authors from International Committee on Taxonomy of Viruses reports as well as the scientific literature.

Viruses of the genus Phlebovirus (realm Riboviria, kingdom Orthornaviridae, phylum Negarnavaricota, order Bunyavirales and family Phenuiviridae) comprise three genome segments of ribonucleic acid (RNA) which encode an RNA-dependent RNA polymerase, which they use to transcribe the viral RNA genome into messenger RNA and to replicate the genome [1]. Phleboviruses have a large (L) segment (6.4 kb) that codes for the RNA-dependent RNA polymerase (RdRp); a medium (M) segment (3.2 kb), which encodes for several polyproteins, obtained by leaky scanning and cleaved into several proteins (Nsm-GN, Nsm, NSm , Gn and Gc); and a small (S) segment (1.7 kb) that encodes for two proteins (N and NSs) with an ambisense strategy (https://viralzone.expasy.org/252 accessed on 10 April 2021).
The genus name is derived from Phlebotominae, the taxon of vectors of member species sandfly fever Naples phlebovirus, from the Greek phlebos, meaning "vein". Species in the genus were previously defined by serological cross reactivity.  Table 1 with an asterisk. Some of these viruses have other hematophagous arthropods as their main vectors, such as mosquitoes for Rift Valley fever virus, while Mukawa virus has been isolated from ticks but remains in the genus Phlebovirus, despite the observations that most tick-borne viruses formerly included in the genus Phlebovirus are now included in the genus Uukuvirus. Some phleboviruses have been isolated from vertebrates, such as wild or sentinel rodents in the Americas, and in Africa, such as opossums or sloths. Other phleboviruses have been isolated from febrile patients in South America (Table 1). This variety of sources highlights the possible presence of diverse epidemiological cycles of these viruses. A high rate of vertical transmission of Toscana virus has been demonstrated in sandflies by experimental infections [2,3], suggesting that there is an amplifying role for vertebrate hosts but that maintenance in nature is mainly by sandflies. "Sandfly" (or "sand fly") is a colloquial name for members of any species or genus of flying, biting, blood-sucking dipteran encountered in sandy areas. In the United States, "sandfly" may refer to certain horse flies that are also known as "greenheads", or to members of the family Ceratopogonidae. Outside the United States, "sandfly" may refer to members of the subfamily Phlebotominae within the Psychodidae. The three main genera are Lutzomyia (found in the New World) and Phlebotomus and Sergentomyia (both found in the Old World), the former two genera contain the more relevant species able to transmit viral pathogens [4]. Biting midges are sometimes called "sandflies" or "nosee-ums". New Zealand sandflies are in the genus Austrosimulium, a type of black fly (https://en.wikipedia.org/wiki/Sandfly accessed on 10 April 2021).
Infections with many of these viruses cause mild 3-day fevers, also known as pappataci fevers or phlebotomus fevers [5]. These illnesses are influenza-like and are characterized by a rapid onset. The diseases occur commonly in endemic areas in summer months, especially in August during which sandflies are active. Toscana virus has been associated with benign meningitis and, occasionally, more severe meningitis in humans [6]. The most important phlebovirus is Rift Valley fever virus, which has been responsible for widespread epidemics and epizootics in livestock in Africa, most notably in Egypt [7]. However, it is transmitted principally by mosquitoes, and so it is not mentioned further here.
A diagnostically complicating feature of phlebovirus replication may be reassortant generation resulting from multiple simultaneous phlebovirus infections [8]. As with other segmented RNA viruses, the reassortment of RNA segments of phleboviruses is commonly observed. By this means, the RNA segments of different virus strains become mixed during replication, and the progeny viruses contain genome segments of the parental viruses. Thus, the progeny viruses have new combinations of these segments and possess novel properties and may be confused for one another due to the specificity of the testing procedures. Only complete genetic analyses can be used to definitively identify such progeny [9]. It has been argued that perhaps all available viruses in this virus family may be the most recent of long genetic lineages [8]. Undoubtedly, some (or all) of the viruses listed in Table 1 are reassortant phleboviruses, particularly those detected in Italy [10], possibly due to the co-circulation of multiple phleboviruses in arthropod vectors occurring in close proximity [11].
To assess possible reassortant phleboviruses, amino acid sequences of the RdRp and correspondent M segments have been retrieved from GenBank and aligned with MAFFT [12]. The percentage of identity has been evaluated with MegaX software, using p-distance with a pairwise deletion option [13]. A maximum likelihood phylogenetic tree was obtained with the RdRp aligned using IQtree software [14]. In this tree, in Figure 1, sequences with more than 95% identity, then ascribable to a single species, are highlighted in red.
The likely reassortant phleboviruses have different M segments (with the exception of Ponticelli II and Bregalaka, which are very similar). Reassortment events have been described for phleboviruses of the Candiru antigenic complex [15], among Massilia, Granada and Arrabida viruses [16,17], and likely produced Ponticelli I, Ponticelli II and Ponticelli III, which belong to the Adana phlebovirus species, according to the RdRp threshold. The possibility of reassortment involving the M segment would be a relevant phenomenon in the evolution of this group, as similarly reported for orthobunyaviruses [18]. The M segment is likely responsible for modifying the pathogenic potential of a virus, as has been reported for reassortant orthobunyaviruses [18,19]. Viruses 2021, 13, x FOR PEER REVIEW 5 of 6