is an order of negative-sense single-stranded RNA (-ssRNA) viruses [1
]. They typically have three genomic segments (large, L; medium, M; small, S) encoding a viral RNA-dependent RNA polymerase L (RDRP L), a surface glycoprotein precursor, and a nucleoprotein, respectively [2
]. Additional ORFs, usually involved in counteracting the host antiviral response, may be present in S or M segments [3
]. Each viral segment has terminal complementary sequences governing its interaction with the polymerase. Furthermore, multiple molecules of a nucleoprotein wrap around genomic RNA following helical symmetry [4
]. Together, an RNA molecule, a polymerase, and the nucleoproteins form a functional viral ribonucleoprotein (vRNP) capable of transcription and replication [5
]. Virions are usually 90–100 nm in diameter and consist of vRNPs of each genomic segment enclosed by a lipid membrane with incorporated viral glycoproteins [3
]. Many bunyaviruses (a generic term for Bunyavirales
) are causative agents of arthropod-borne diseases of vertebrates and plants [6
Recent metatranscriptomic studies revealed a plethora of deep branching bunyaviruses from vertebrates and invertebrates, suggesting a long-term coevolution of these viruses with their hosts and vectors [7
]. Of note, some bunyaviruses are capable of infecting distantly related eukaryotic cells. For example, Orthotospovirus
(the tomato spotted wilt virus (Bunyavirales
)) can replicate in both plant and insect cells [10
The kinetoplastid flagellates of the family Trypanosomatidae are a eukaryotic group, whose viruses recently started attracting attention [12
]. Trypanosomatids are obligate parasites of invertebrates, vertebrates, and plants [13
]. They either have one or two hosts in their life cycle (monoxenous and dixenous species, respectively) [14
]. Dixenous trypanosomatids originate from their monoxenous relatives and many of them are of medical or economic importance [17
Members of the genus Leishmania
infect vertebrates; they are transmitted by phlebotomine sand flies or, possibly, biting midges and cause a variety of diseases collectively named leishmaniases [20
]. These diseases manifest with a wide spectrum of clinical symptoms from relatively harmless skin lesions to fatal cases involving failure of visceral organs. Currently, the genus Leishmania
is subdivided into four subgenera: Leishmania
), and L.
]. These groups are phylogenetically distinct and differ in host specificity or clinical symptoms. The recently established subgenus Mundinia
is the most understudied one [23
Thus far, only the representatives of the subgenera Viannia
were extensively screened for viral presence, resulting in the discovery of Leishmania
RNA viruses (LRVs). The first virus of this group was documented in L.
more than 30 years ago [25
]. This double-stranded RNA (dsRNA) virus is classified as Leishmaniavirus
within the family Totiviridae
based on sequence similarity to the yeast L-A totivirus [26
]. The genus Leishmaniavirus
is subdivided into LRV1, infecting New World Leishmania
], and LRV2 described from Old World Leishmania
]. Recently, new representatives of this viral genus were unexpectedly found in unrelated trypanosomatids, members of the monoxenous genus Blechomonas
parasitizing fleas [32
An increased interest in leishmaniaviruses was stimulated by the discovery that LRV1 presence may augment pathogenicity of some New World Leishmania
species. It was shown that viral dsRNA interacts with Toll-like receptor 3 (TLR3) in the parasitophorous vacuole of a macrophage, initiating production of pro-inflammatory cytokines, including interferon-β [33
] and subverts innate immunity via TLR3-mediated NLRP3 (NACHT, LRR and PYD domains-containing protein 3) inhibition of inflammasomes. This, in turn, leads to chronic inflammation that counteracts anti-leishmanial immune response and contributes to the metastatic potential of Leishmania
]. It is argued that in this way the virus confers a selective advantage to Leishmania
, resulting in its retention [36
]. Only two strains of L.
were tested for LRV presence by PCR and both documented as negative [23
No viruses other than LRVs were found in Leishmania
]. At the same time, recent studies reveal numerous bunyaviruses infecting other trypanosomatids, including monoxenous relatives of Leishmania
]. They all have a typical tripartite genome arrangement, although their M segment is markedly reduced in size and amino acid sequences of the M-encoded putative glycoprotein are extremely divergent. Sequences of their RDRPs and terminal complementary repeats are closest to those of Phenuiviridae
. Leishbunyaviruses (LBVs, proposed family Leishbunyaviridae) form a single and well separated clade on a Bunyavirales
tree, suggesting that they acquired the ability to infect trypanosomatids only once. Comparison of LBV and trypanosomatid phylogenies revealed cases of both co-evolution and horizontal viral transmissions [32
In this work, we describe the first Leishmania-infecting leishbunyavirus as the first non-LRV virus in trypanosomatids of this genus.
In this study, we describe the first leishbunyavirus of Leishmania
. Previously, these viruses were discovered in monoxenous trypanosomatids of the subfamily Leishmaniinae (mainly in Crithidia
spp.), genus Blechomonas
(subfamily Blechomonadinae), as well as in one plant-infecting dixenous Phytomonas
sp. (Figure 5
]. Leishbunyaviral sequences are also found in metatranscriptomes of insects infected by flagellates of other trypanosomatid genera, such as Strigomonas
, light grey) [38
]. This is the most widespread and species-rich group of RNA viruses in trypanosomatids known to date. This fact, along with the discordance of viral and trypanosomatid phylogenies documented in the previous studies [32
], strongly suggests that host-to-host transition is significantly facilitated in this group of viruses. It is explained when taking into account two facts: (i) LBVs are able to form membrane-bound viral particles [68
] and (ii) the flagellar pocket of trypanosomatids is an organelle-governing intensive exchange with the milieu by endo- and exocytosis [69
]. The documented particles of LBVs measure about 100 nm [38
] corresponding to the typical size of clathrin-coated endocytic vesicles in trypanosomatids [73
]. Interestingly, clathrin-mediated endocytosis is the general route for an uptake of bunyaviruses [74
]. Bunyaviruses evolved to utilize the eukaryotic endomembrane system for virus assembly and spreading. Apparently, LBVs use the same strategy in trypanosomatids.
Infectivity and formation of viral particles in bunyaviruses depend on glycoproteins, type I transmembrane proteins that are proteolytically processed and glycosylated in the ER [3
]. Their C-terminal cytoplasmic domains are thought to bind viral ribonucleoproteins and play a crucial role in genome packaging [75
], whereas the N-terminal ectodomains are involved in receptor recognition and membrane fusion [65
]. In leishbunyaviruses, the M segments and putative glycoproteins encoded within them are significantly reduced in size, extremely divergent, and sometimes contain a reduced number of transmembrane domains. We hypothesize that such a layout reflects a reduced functionality of these proteins and potential broad specificity of viral infection, which explains their facilitated host-to-host transition. It was demonstrated that extended deletions in the bunyaviral glycoprotein N-terminus (ectodomain) do not prevent cell fusion and transport to the Golgi, but lead to attenuation of viruses [80
]. This illustrates the propensity of these proteins to undergo reduction. The opportunity to be inherited vertically probably removes the need for efficient proliferation and infection and may even make such properties undesirable.
Transfer of viruses between different species of trypanosomatids is possible because of coinfections, which are quite common in these parasites [81
]. Coinfections were previously reported for Leptomonas moramango
]. Here we did not observe viral coinfection but revealed a putative consequence of such an event—re-assortment of genomic segments. This assumption arises from discordance of phylogenies of proteins from the L and S segments on one hand, and the M segment on the other. The RDRP and nucleocapsid of Lmar
LBV1 are closely related to their counterparts in DukeBV, whereas its glycoprotein is more similar to the corresponding proteins of Lepmor
LBV1a and Lepmor
LBV1 is the first non-LRV virus discovered in Leishmania.
It was found in one of the members of the most enigmatic subgenus of these flagellates—Mundinia.
Although the first species was characterized over 70 years ago, the subgenus itself was established only recently [21
]. For the moment, this taxon contains four described species: L.
, and L.
]. The first two infect guinea pigs and kangaroos, respectively, while the remaining two are isolated from humans. In contrast to other human-infecting Leishmania
, which use sand flies as their vectors, these flagellates may be transmitted by biting midges [92
]. Host switching may have shaped the genome evolution in these flagellates [94
]. While the parasitofauna of biting midges is understudied, several species of monoxenous trypanosomatids are documented in these insects [95
]. This is in agreement with our proposal that Lmar
LBV1 originates from LBVs of monoxenous trypanosomatids.
is frequently found in skin lesions of immunocompromised patients indicating that it may be an opportunistic pathogen [98
]. However, recent analysis of multiple records in Thailand and Myanmar reveals that neither the presence nor the severity of the infection is necessarily associated with HIV [87
]. Notably, the clinical manifestations range from asymptomatic infection and various types of lesions to visceral disease. Previously, it was demonstrated that LRV1 boosts virulence of Leishmania guyanensis
in humans [33
]. The discovery of a virus in L. martiniquensis
poses an important question on whether it also influences the pathogenicity of this parasite. We demonstrate that the presence of Lmar
LBV1 is slightly beneficial for Leishmania
. The molecular mechanism of such facilitation may be non-specific, since it was recently shown that simultaneous inoculation of virus-negative L. guyanensis
and Toscana virus (Bunyavirales
) increases footpad swelling and parasite burden in mice, reminiscent of the reaction to the LRV1-positive L. guyanensis
]. Although it was not shown experimentally, the presence of the membrane-bound viral particles in LBVs suggest that they can be shed by trypanosomatid cells. This way, Lmar
LBV1 can interact with the immune system of a vertebrate host, increasing the severity of leishmanial infection. Our results signify the need for a systematic exploration of trypanosomatid viromes.