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. The detection of new phleboviruses, not often available for serological assays, prompted the change of classification rules. Now, viral species are defined by 95% or greater identity in the amino acid sequences of their RdRp [TaxoProp 2019.026M.A.v1.Phenuiviridae_4gen79sp]. The genus currently comprises 67 species, listed in Table 1. Phleboviruses mentioned in this Special Issue have not all been detected in sandflies. Those that have are noted in 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.

Table 1

Viruses of the genus Phlebovirus, modified from [1].

Species 1	Virus 2	Abbreviation
Adana phlebovirus	Adana virus *	ADAV
	Ponticelli I virus *
	Ponticelli II virus *
	Ponticelli III virus *
	Bregalaka virus *
Aguacate phlebovirus	Aguacate virus *	AGUV
Alcube phlebovirus	Alcube virus *	ACBV
Alenquer phlebovirus	Alenquer virus	ALEV
Ambe phlebovirus	Ambe virus *	ABEV
Anhanga phlebovirus	Anhangá virus	ANHV
Arumowot phlebovirus	Arumowot virus	AMTV
Bogoria phlebovirus	Bogoria virus	BGRV
Buenaventura phlebovirus	Buenaventura virus *	BUEV
Bujaru phlebovirus	Bujaru virus	BUJV
Cacao phlebovirus	Cacao virus *	CACV
Campana phlebovirus	Campana virus *	CMAV
Candiru phlebovirus 3
	Ariquemes virus	ARQV
	Candirú virus	CDUV
	Jacundá virus	JCNV
	Morumbi virus	MRBV
	Mucura virus	MCRV
	Serra Norte virus	SRNV
Chagres phlebovirus	Chagres virus *	CHGV
Cocle phlebovirus	Coclé virus	CCLV
Corfou phlebovirus	Corfou virus *	CFUV
Dashli phlebovirus	Dāshlī virus *	DASV
Durania phlebovirus	Durania virus *	DRNV
Echarate phlebovirus	Echarate virus	ECHV
Embossos phlebovirus	Embossos virus *	EMBV
Gabek phlebovirus	Gabek forest virus	GFV
Gordil phlebovirus	Gordil virus	GORV
Icoaraci phlebovirus	Icoaraci virus	ICOV
Itaituba phlebovirus	Itaituba virus	ITAV
Itaporanga phlebovirus	Itaporanga virus	ITPV
Ixcanal phlebovirus	Ixcanal virus *	IXCV
Karimabad phlebovirus	Karimabad virus *	KARV
Kiborgoch phlebovirus	Kiborgoch virus *	KBGV
La Gloria phlebovirus	La Gloria virus *	LAGV
Lara phlebovirus	Rio Claro virus	RICV
Leticia phlebovirus	Leticia virus *	LTCV
Maldonado phlebovirus	Maldonado virus	MLOV
Mariquita phlebovirus	Mariquita virus *	MRQV
Massilia phlebovirus	Massilia virus *	MASV
Medjerda phlebovirus	Medjerda Valley virus *	MVV
Mona Grita phlebovirus	Mona Grita virus *	MOGV
Mukawa phlebovirus	Mukawa virus	MKWV
Munguba phlebovirus	Munguba virus *	MUNV
Naples phlebovirus 3
	Arrabida virus *	ARRV
	Balkan virus *	BALKV
	Fermo virus *	FERV
	Granada virus *	GRAV
	Saddaguia virus *	SADV
	sandfy fever Naples virus *	SFNV
Nique phlebovirus	Nique virus *	NIQV
Ntepes phlebovirus	Ntepes virus *	NTPV
Odrenisrou phlebovirus	Odrénisrou virus	ODRV
Oriximina phlebovirus	Oriximiná virus *	ORXV
Pena Blanca phlebovirus	Peña Blanca virus *	PEBV
Penshurt phlebovirus	Penshurt virus	PEHV
Perkerra phlebovirus	Perkerra virus	PKEV
Punique phlebovirus	Punique virus*	PUNV
Punta Toro phlebovirus 3
	Buenaventura virus *	BUEV
	Capira virus *	CAPIV
	Punta Toro virus *	PTV
Rift Valley fever phlebovirus 4	Rift Valley fever virus	RVFV
	Hedi virus * [20]	HEDV
Rio Grande phlebovirus	Rio Grande virus	RGV
Saint Floris phlebovirus	Saint-Floris virus	SAFV
Salanga phlebovirus	Salanga virus	SLGV
Salehabad phlebovirus 3
	Adria virus *	ADRV
	Arbia virus *	ARBV
	Olbia virus *	OLBV
	Salehabad virus *	SALV
	Zaba virus *	ZABAV
Salobo phlebovirus	Salobo virus *	SLBOV
Sicilian phlebovirus	sandfy fever Sicilian virus *	SFSV
Tapara phlebovirus	Tapará virus *	TPRV
Tehran phlebovirus	Tehran virus *	THEV
Tico phlebovirus	Tico virus *	TICV
Toros phlebovirus	Toros virus *	TORV
Toscana phlebovirus	Toscana virus *	TOSV
Tres Almendras phlebovirus	Tres Almendras virus *	TRAV
Turuna phlebovirus	Turuna virus *	TUAV
Uriurana phlebovirus	Uriurana virus *	URIV
Urucuri phlebovirus	Urucuri virus	URUV
Viola phlebovirus	Viola virus *	VIOV
Zerdali phlebovirus	Zerdali virus *	ZERV

1 Taxon names are always italicized and always begin with a capital letter. Note that viruses are real objects that are assigned to concepts that are called taxa. Species, genera, subfamilies, families and orders are taxa; 2 virus names are not italicized and are not capitalized, except if the name or a name component is a proper noun. This column lists the virus names with their correct (including lack of) capitalization; 3 lists of viruses within a given species are provisional at this point and will likely be amended in the near future; 4 type of species; * detected in 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 “no-see-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 wide-spread 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.

Figure 1

Midpoint rooted maximum likelihood tree obtained with amino acid sequences of the RdRp of available phleboviruses. Sequences in red: ascribable to the same species according the 95% identity threshold. Bootstrap values >85% are shown over the branch.

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].