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. 2019 Jun 8;11(6):532. doi: 10.3390/v11060532

Unravelling the Single-Stranded DNA Virome of the New Zealand Blackfly

Simona Kraberger 1, Kara Schmidlin 1, Rafaela S Fontenele 1, Matthew Walters 2, Arvind Varsani 1,2,3,*
PMCID: PMC6630596  PMID: 31181730

Abstract

Over the last decade, arthropods have been shown to harbour a rich diversity of viruses. Through viral metagenomics a large diversity of single-stranded (ss) DNA viruses have been identified. Here we examine the ssDNA virome of the hematophagous New Zealand blackfly using viral metagenomics. Our investigation reveals a plethora of novel ssDNA viral genomes, some of which cluster in the viral families Genomoviridae (n = 9), Circoviridae (n = 1), and Microviridae (n = 108), others in putative families that, at present, remain unclassified (n = 20) and one DNA molecule that only encodes a replication associated protein. Among these novel viruses, two putative multi-component virus genomes were recovered, and these are most closely related to a Tongan flying fox faeces-associated multi-component virus. Given that the only other known multi-component circular replication-associated (Rep) protein encoding single-stranded (CRESS) DNA viruses infecting plants are in the families Geminiviridae (members of the genus Begomovirus) and Nanoviridae, it appears these are likely a new multi-component virus group which may be associated with animals. This study reiterates the diversity of ssDNA viruses in nature and in particular with the New Zealand blackflies.

Keywords: blackfly, sandfly, Austrosimulium sp., CRESS DNA virus, Circoviridae, Genomoviridae

1. Introduction

Hematophagous insects are responsible for vectoring a wide range of pathogens. Vectors of important mammalian and avian pathogens, such as mosquitoes, are members of the order Diptera (suborder: Nematocera). One group of blood sucking insects, commonly referred to as blackflies or sandflies, are members of the Simuliidae family [1]. Although blackflies are found globally, only some species consume a blood meal. For those species that feed on blood, it is the adult females that do so, whereas the males feed primarily on nectar. Aquatic environments are essential for the life cycle of blackflies, with the egg, larval and pupae stages all occurring in flowing water, followed by the emergence of a winged adult form.

Blackflies are known to transmit a handful of parasites, predominantly protozoa and parasitic worms. In humans, the most important blackfly vectored pathogen is the parasitic nematode, Onchocerca volvulus which causes river blindness. Although rare, globally O. volvulus has a significant impact on human health in Africa, affecting over 18 million people [2,3]. Avian haemazoan parasites, such as those in the genus Leucocytozoon, are also commonly transmitted by blackflies, with different blackfly species showing preference to the bird species they bite and harbouring a variety of parasite linages [4,5]. The most studied arbovirus transmitted by blackflies is vesicular stomatitis virus (family Rhabdoviridae, genus Vesiculovirus) that typically infects livestock, however, zoonotic events have also been reported [6]. Invertebrate iridoviruses (family Iridoviridae), which are double-stranded DNA viruses, have been identified in blackflies across the globe, often causing a covert infection [7,8].

Several species of blackfly are endemic to New Zealand, two of which, the New Zealand blackfly (Austrosimulium australense) and the west coast blackfly (A. ungulatum), consume blood meals [9,10]. They are notorious for their persistence in pursuit of a blood meal and in various locations are present in overwhelming swarms. Despite having some knowledge on the ecology of New Zealand blackflies, we know very little about the viruses that are circulating in these insects. No human pathogens vectored by blackflies in New Zealand have been documented and, with the exception of protozoal transmission in some avian species, little is known about the microorganisms circulating in these insects.

In recent years, studies have used viral metagenomics as a non-biased approach for identifying viruses circulating in hematophagous and phytophagous insects [11,12,13,14,15]. This has resulted in the identification of a vast number of viruses, circulating in the hosts these insects are feeding on, from the surrounding environment, and those that infect the insects themselves. Arthropods have been broadly shown to harbour a wide range of viruses with circular replication-associated (Rep) protein encoding single-stranded (CRESS) DNA genomes [11,13,14,16,17]. At present there are many established CRESS DNA virus families; Bacillidnaviridae, Circoviridae, Geminiviridae, Genomoviridae, Nanoviridae, and Smacoviridae, all of whose members encode Reps that share conserved replication associated motifs, an origin of replication and are usually <6 kb in size [18]. Also, viruses in the family Microviridae which infect bacteria, typically encode a major capsid, minor capsid and a replication initiation protein, and range in size from 4–7 kb [19]. In addition, numerous novel CRESS DNA viruses have been identified in arthropods which are yet to be taxonomically classified [11,20].

Viral metagenomic studies reveal a great deal about viruses circulating in arthropods. For example, a study on mosquitoes [14] identified viral sequences with similarities to animal-infecting ssDNA viruses (families: Anelloviridae, Circoviridae, Parvoviridae), double-stranded (ds) DNA viruses (families: Herpesviridae, Poxviridae and Papillomaviridae), plant-infecting ssDNA viruses (families: Geminiviridae and Nanoviridae), and three bacteria-infecting dsDNA viruses (families: Myoviridae, Podoviridae and Siphoviridae). Viral metagenomic studies allows for a snapshot of the viruses circulating in the vectors hosts, the insect and surrounding ecosystem to be identified. Based on the lack of information on the widespread New Zealand hematophagous insect commonly known as the blackfly, we undertook a metagenomics approach to investigate the associated ssDNA virome.

2. Materials and Methods

2.1. Collection of Blackflies and Isolation of Viral Nucleic Acid

For this project, 40 individual blackflies were collected from North Canterbury, New Zealand in 2015. The 40 individuals were collected from a single site. The sex of the individuals, and whether they had consumed a blood meal, was determined. These samples were pooled and homogenized using a pestle in 2 mL of SM buffer (0.1 M NaCl, 50 mM Tris/HCl-pH 7.4, 10 mM MgSO4). The homogenized sample was centrifuged for 10 min at 10,000 rpm and the resulting supernatant was filtered through a 0.2 µM filter. The viral particles in the filtrate were precipitated overnight at 4 °C with 15% PEG and following this the solution was centrifuged at 14,000 rpm for 10 min and resulting pellet resuspended in 500 µL of SM buffer. Following this, 200 µL of the resuspended material was subsequently used to isolate viral DNA using the High Pure Viral Nucleic Acid Kit (Roche Diagnostics, USA) according to the manufacturer’s specifications. The viral nucleic acid was then used in a rolling circle amplification reaction with the TempliPhi™ kit (GE Healthcare, USA) to preferentially amplify circular DNA molecules.

2.2. High-Throughput Sequencing and Viral Genome Verification

Rolling circle amplified DNA was used to prepare 2 × 150 bp paired-end libraries for sequencing on an Illumina 2500 platform at Macrogen Inc. (Korea). The paired-end reads were de novo assembled using metaSPAdes v 3.12.0 [21] and resulting contigs (>750 nts) were filtered for viral-like sequences using BLASTx [22] against viral protein database generated from the GenBank RefSeq depository. For contigs with similarities to viruses in the Microviridae family, full de novo assembled genomes were confirmed by mapping raw reads using BBMap [23], and deemed credible with a coverage level of greater than 10×. For viral contigs with similarities to other ssDNA viruses, abutting primers (Table S1) were designed to recover the complete genomes by PCR using Kapa HiFi HotStart DNA polymerase (KAPA Biosystems, USA). Amplicons were resolved on a 0.7% agarose gel, the correct size amplicons were excised, gel purified and cloned into pJET1.2 cloning vector (ThermoFisher Scientific, USA). Recombinant plasmids containing the viral genomes were purified from transformed XL blue E. coli competent cells and Sanger sequenced at Macrogen Inc. (Korea) by primer walking. The Sanger sequences were assembled using Geneious software V11.1.5.

2.3. Network Construction, Phylogenetic and Similarity Comparison Analyses

The blackfly viral rep (CRESS DNA viruses) and the major capsid protein (mcp) gene (microviruses) together with those available in GenBank were extracted, translated and used to build a Rep and MCP protein sequence dataset. The Rep of circoviruses, geminiviruses, smacoviruses, genomoviruses and nanoviruses and the MCP of viruses in the subfamily Gokushovirinae (family Microvividae) clustered first with CD-HIT [24] using 0.9 sequence identity cut off and a representative from each cluster was included in the final dataset. The Rep and MCP protein sequences were used separately to build sequence similarity networks (E-value = 1 × 10−5) using the EFI-Enzyme similarity tool server [25,26]. The MCP similarity network was constructed using minimum similarity score 10−175 and the Rep with 10−60. The score is the similarity threshold for connected nodes, i.e., proteins, with each other and thus those with scores below this value are not connected. Protein similarity networks were visualised using Cytoscape V3.7.0 [27].

Based on the network clusters, unique cluster sequences datasets were built for each major cluster (comprised of ten or more members) that contained a blackfly derived sequence. The datasets included genomoviruses, circoviruses and five additional clusters labelled cluster group 1–5 for the unclassified CRESS DNA viruses and two microvirus groups labelled cluster MV group 1 and 2. The sequences in each of these cluster groups were aligned using MUSCLE [28] and maximum likelihood phylogenetic trees inferred using PHYML [29] with the best fit models, determined using ProtTest [30]. The substitution models used are genomoviruses, LG+I+G; circoviruses, rtREV+G+I; CRESS group 1, rtREV+G+I; CRESS group 2, WAG+G+I; CRESS group 3, rtREV+G; CRESS group 4, WAG+G+I; CRESS Group 5, WAG+G+I, MV group 1, rtRev+G+I; MV group 2, rtREV+G+I+F. Branches with aLRT support of <0.8 were collapsed using TreeGraph2 [31]. The Maximum likelihood phylogenetic trees were midpoint rooted with the exception of the genomoviruses which were rooted using the geminivirus Reps and the cycloviruses with circoviruses Reps.

BLASTx [22] comparisons were undertaken for any singletons to determine the most closely related sequences in GenBank. For clusters comprised of less than four sequences an amino acid pairwise comparison using SDT V1.2 [32] was undertaken.

3. Results and Discussion

In a pool of 40 individual blackflies, diverse CRESS DNA viruses were identified. Cluster analyses using the Rep protein, which is the most conserved gene among CRESS DNA viruses, provides a broad overview of the extensive range of viruses recovered in the blackfly samples (Table 1 and Figure 1). The network analyses results reveal (Figure 1) that the Reps from the blackfly derived viruses or DNA molecules cluster with those in the Genomoviridae family (n = 9), Circoviridae family (n = 1), unclassified CRESS DNA viruses (n = 15) or as singletons (n = 6).

Table 1.

Summary of the genomes of blackfly associated viruses including genome organisation and GenBank Accession numbers.

Family/Subfamily Genus Virus Name Accession Number Size (nts) ORF Orientation
Genomoviridae Gemycircularirus Blackfly genomovirus-1 SF02 506 MK433242 2290 Bidirectional
Blackfly genomovirus-2 SF02 631 MK433234 2131 Bidirectional
Blackfly genomovirus-3 SF02 1766 MK433235 2138 Bidirectional
Blackfly genomovirus-4 SF02 836 MK433236 2181 Bidirectional
Blackfly genomovirus-5 SF02 599 MK433237 2182 Bidirectional
Blackfly genomovirus-6 SF02 459 MK433238 2186 Bidirectional
Blackfly genomovirus-7 SF02 767 MK433239 2195 Bidirectional
Blackfly genomovirus-9 SF02 507 MK433241 2217 Bidirectional
Genomoviridae Gemyduguvirus Blackfly genomovirus-8 SF02 579 MK433240 2226 Bidirectional
Unclassified CRESS DNA virus Unassigned Blackfly DNA virus-1 SF02 666 MK433215 1805 Unidirectional
Blackfly DNA virus-2 SF02 583 MK433216 1936 Bidirectional
Blackfly DNA virus-3 SF02 402 MK433217 2005 Unidirectional
Blackfly DNA virus-4 SF02 664 MK433218 2047 Unidirectional
Blackfly DNA virus-5 SF02 839 MK433219 2058 Unidirectional
Blackfly DNA virus-6 SF01 308 MK433220 2172 Bidirectional
Blackfly DNA virus-7 SF02 462 MK433221 2306 Bidirectional
Blackfly DNA virus-8 SF02 1137 MK433222 2337 Bidirectional
Blackfly DNA virus-9 SF02 881 MK433223 2389 Bidirectional
Blackfly DNA virus-10 SF02 899 MK433224 2444 Bidirectional
Blackfly DNA virus-11 SF02 963 MK433225 2449 Bidirectional
Blackfly DNA virus-12 SF02 422 MK433226 2466 Unidirectional
Blackfly DNA virus-13 SF02 413 MK433227 2501 Bidirectional
Blackfly DNA virus-14 SF02 295 MK433228 2583 Bidirectional
Blackfly DNA virus-15 SF02 403 MK433229 2697 Bidirectional
Blackfly DNA virus-16 SF02 377 MK433230 2704 Bidirectional
Blackfly DNA virus-17 SF02 1426 MK433231 2835 Unidirectional
Blackfly DNA virus-18 SF02 66 MK433232 3003 Bidirectional
Blackfly DNA virus-19 SF02 380 MK433233 2002 Bidirectional
Unclassified Circular DNA molecules Unassigned Blackfly DNA molecule 1 - rep MK561604 1157 Unidirectional
Unclassified Multi-component virus Unassigned Blackfly multicomponent virus 1 - rep MK561605 1163 Unidirectional
Blackfly multicomponent virus 1 - cp MK561606 1154 Unidirectional
Blackfly multicomponent virus 2 - rep MK561607 1136 Unidirectional
Blackfly multicomponent virus 2 - cp MK561608 1133 Unidirectional
Microviridae/Gokushovirinae Unassigned Blackfly microvirus-1_ SF02_SP_9 MK249160 4873 Unidirectional
Blackfly microvirus-2_ SF02_SP_11 MK249161 4544 Unidirectional
Blackfly microvirus-3_ SF02_SP_12 MK249162 4625 Unidirectional
Blackfly microvirus-4_ SF02_SP_13 MK249163 4660 Unidirectional
Blackfly microvirus-5_ SF02_SP_14 MK249164 4594 Unidirectional
Blackfly microvirus-6_ SF02_SP_15 MK249165 4675 Unidirectional
Blackfly microvirus-7_ SF02_SP_17 MK249166 4379 Unidirectional
Blackfly microvirus-8_ SF02_SP_18 MK249167 4483 Unidirectional
Blackfly microvirus-9_ SF02_SP_20 MK249168 4324 Unidirectional
Blackfly microvirus-10_ SF02_SP_21 MK249169 4656 Unidirectional
Blackfly microvirus-11_ SF02_SP_22 MK249170 4565 Unidirectional
Blackfly microvirus-12_ SF02_SP_24 MK249171 4231 Unidirectional
Blackfly microvirus-13_ SF02_SP_25 MK249172 4582 Unidirectional
Blackfly microvirus-14_ SF02_SP_27 MK249173 4694 Unidirectional
Blackfly microvirus-15_ SF02_SP_28 MK249174 4557 Unidirectional
Blackfly microvirus-16_ SF02_SP_30 MK249175 4689 Unidirectional
Blackfly microvirus-17_ SF02_SP_31 MK249176 4474 Unidirectional
Blackfly microvirus-18_ SF02_SP_32 MK249177 4841 Unidirectional
Blackfly microvirus-19_ SF02_SP_34 MK249178 4727 Unidirectional
Blackfly microvirus-20_ SF02_SP_36 MK249179 4498 Unidirectional
Blackfly microvirus-21_ SF02_SP_38 MK249180 4461 Unidirectional
Blackfly microvirus-22_ SF02_SP_44 MK249181 6565 Unidirectional
Blackfly microvirus-23_ SF02_SP_45 MK249182 4565 Unidirectional
Blackfly microvirus-24_ SF02_SP_46 MK249183 4633 Unidirectional
Blackfly microvirus-25_ SF02_SP_49 MK249184 4521 Unidirectional
Blackfly microvirus-26_ SF02_SP_50 MK249185 4731 Unidirectional
Blackfly microvirus-27_ SF02_SP_51 MK249186 4581 Unidirectional
Blackfly microvirus-28_ SF02_SP_52 MK249187 4667 Unidirectional
Blackfly microvirus-29_ SF02_SP_54 MK249188 4637 Unidirectional
Blackfly microvirus-30_ SF02_SP_57 MK249189 4980 Unidirectional
Blackfly microvirus-31_ SF02_SP_67 MK249190 4901 Unidirectional
Blackfly microvirus-32_ SF02_SP_69 MK249191 4633 Unidirectional
Blackfly microvirus-33_ SF02_SP_71 MK249192 4901 Unidirectional
Blackfly microvirus-34_ SF02_SP_73 MK249193 4870 Unidirectional
Blackfly microvirus-35_ SF02_SP_74 MK249194 4765 Unidirectional
Blackfly microvirus-36_ SF02_SP_75 MK249195 4789 Unidirectional
Blackfly microvirus-37_ SF02_SP_78 MK249196 4774 Unidirectional
Blackfly microvirus-38_ SF02_SP_79 MK249197 4711 Unidirectional
Blackfly microvirus-39_ SF02_SP_80 MK249198 4858 Unidirectional
Blackfly microvirus-40_ SF02_SP_82 MK249199 4799 Unidirectional
Blackfly microvirus-41_ SF02_SP_83 MK249200 4825 Unidirectional
Blackfly microvirus-42_ SF02_SP_84 MK249201 4779 Unidirectional
Blackfly microvirus-43_ SF02_SP_87 MK249202 4768 Unidirectional
Blackfly microvirus-44_ SF02_SP_88 MK249203 4615 Unidirectional
Blackfly microvirus-45_ SF02_SP_89 MK249204 4630 Unidirectional
Blackfly microvirus-46_ SF02_SP_91 MK249205 4733 Unidirectional
Blackfly microvirus-47_ SF02_SP_93 MK249206 4721 Unidirectional
Blackfly microvirus-48_ SF02_SP_97 MK249207 4646 Unidirectional
Blackfly microvirus-49_ SF02_SP_98 MK249208 4700 Unidirectional
Blackfly microvirus-50_ SF02_SP_99 MK249209 4706 Unidirectional
Blackfly microvirus-51_ SF02_SP_100 MK249210 4721 Unidirectional
Blackfly microvirus-52_ SF02_SP_104 MK249211 4692 Unidirectional
Blackfly microvirus-53_ SF02_SP_106 MK249212 4680 Unidirectional
Blackfly microvirus-54_ SF02_SP_107 MK249213 4706 Unidirectional
Blackfly microvirus-55_ SF02_SP_108 MK249214 4647 Unidirectional
Blackfly microvirus-56_ SF02_SP_110 MK249215 4618 Unidirectional
Blackfly microvirus-57_ SF02_SP_115 MK249216 4608 Unidirectional
Blackfly microvirus-58_ SF02_SP_116 MK249217 4596 Unidirectional
Blackfly microvirus-59_ SF02_SP_117 MK249218 4590 Unidirectional
Blackfly microvirus-60_ SF02_SP_118 MK249219 4621 Unidirectional
Blackfly microvirus-61_ SF02_SP_120 MK249220 4617 Unidirectional
Blackfly microvirus-62_ SF02_SP_122 MK249221 4591 Unidirectional
Blackfly microvirus-63_ SF02_SP_123 MK249222 4499 Unidirectional
Blackfly microvirus-64_ SF02_SP_124 MK249223 4510 Unidirectional
Blackfly microvirus-65_ SF02_SP_125 MK249224 4507 Unidirectional
Blackfly microvirus-66_ SF02_SP_126 MK249225 4581 Unidirectional
Blackfly microvirus-67_ SF02_SP_128 MK249226 4503 Unidirectional
Blackfly microvirus-68_ SF02_SP_129 MK249227 4570 Unidirectional
Blackfly microvirus-69_ SF02_SP_130 MK249228 4498 Unidirectional
Blackfly microvirus-70_ SF02_SP_131 MK249229 4558 Unidirectional
Blackfly microvirus-71_ SF02_SP_135 MK249230 4488 Unidirectional
Blackfly microvirus-72_ SF02_SP_137 MK249231 4551 Unidirectional
Blackfly microvirus-73_ SF02_SP_138 MK249232 4335 Unidirectional
Blackfly microvirus-74_ SF02_SP_139 MK249233 4547 Unidirectional
Blackfly microvirus-75_ SF02_SP_142 MK249234 4538 Unidirectional
Blackfly microvirus-76_ SF02_SP_143 MK249127 4471 Unidirectional
Blackfly microvirus-77_ SF02_SP_145 MK249128 4529 Unidirectional
Blackfly microvirus-78_ SF02_SP_146 MK249129 4521 Unidirectional
Blackfly microvirus-79_ SF02_SP_148 MK249130 4549 Unidirectional
Blackfly microvirus-80_ SF02_SP_149 MK249131 4460 Unidirectional
Blackfly microvirus-81_ SF02_SP_150 MK249132 4460 Unidirectional
Blackfly microvirus-82_ SF02_SP_156 MK249133 4443 Unidirectional
Blackfly microvirus-83_ SF02_SP_159 MK249134 4438 Unidirectional
Blackfly microvirus-84_ SF02_SP_160 MK249135 4483 Unidirectional
Blackfly microvirus-85_ SF02_SP_162 MK249136 4350 Unidirectional
Blackfly microvirus-86_ SF02_SP_163 MK249137 4484 Unidirectional
Blackfly microvirus-87_ SF02_SP_164 MK249138 4502 Unidirectional
Blackfly microvirus-88_ SF02_SP_165 MK249139 4487 Unidirectional
Blackfly microvirus-89_ SF02_SP_166 MK249140 4410 Unidirectional
Blackfly microvirus-90_ SF02_SP_167 MK249141 4459 Unidirectional
Blackfly microvirus-91_ SF02_SP_168 MK249142 4401 Unidirectional
Blackfly microvirus-92_ SF02_SP_169 MK249143 4387 Unidirectional
Blackfly microvirus-93_ SF02_SP_174 MK249144 4445 Unidirectional
Blackfly microvirus-94_ SF02_SP_175 MK249145 4400 Unidirectional
Blackfly microvirus-95_ SF02_SP_178 MK249146 4422 Unidirectional
Blackfly microvirus-96_ SF02_SP_181 MK249147 4391 Unidirectional
Blackfly microvirus-97_ SF02_SP_183 MK249148 4368 Unidirectional
Blackfly microvirus-98_ SF02_SP_184 MK249149 4293 Unidirectional
Blackfly microvirus-99_ SF02_SP_185 MK249150 4351 Unidirectional
Blackfly microvirus-100_ SF02_SP_188 MK249151 4276 Unidirectional
Blackfly microvirus-101_ SF02_SP_189 MK249152 4318 Unidirectional
Blackfly microvirus-102_ SF02_SP_190 MK249153 4205 Unidirectional
Blackfly microvirus-103_ SF02_SP_192 MK249154 4320 Unidirectional
Blackfly microvirus-104_ SF02_SP_195 MK249155 4302 Unidirectional
Blackfly microvirus-105_ SF02_SP_206 MK249156 4238 Unidirectional
Blackfly microvirus-106_ SF02_SP_208 MK249157 4237 Unidirectional
Blackfly microvirus-107_ SF02_SP_211 MK249158 4170 Unidirectional
Blackfly microvirus-108_ SF02_SP_213 MK249159 4232 Unidirectional
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Figure 1.

Figure 1

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A network and phylogenetic analyses of the circular replication-associated (Rep) protein encoding single-stranded (CRESS) DNA virus replication-associated (Rep) protein sequences. The network analyses shows the clustering of the blackfly CRESS DNA viruses (shown as red circles) within taxonomic family groupings, larger and smaller groupings of unclassified CRESS DNA viruses (only those groups containing CRESS DNA viruses from this study), and singletons from this study that do not cluster with other known Reps. Closest Rep comparisons and percentage similarity are shown for the smaller groups and singletons. Maximum likelihood phylogenetic trees of the Rep sequence of circovirus (showing only the Cyclovirus genus clade) and the five larger unclassified CRESS DNA virus groupings. Blackfly originating sequence names are shown in red. The maximum likelihood phylogenetic tree of the Rep sequence for the genomoviruses is shown in Figure 2.

3.1. Blackfly CRESS DNA Viruses Clustering with Genomoviruses

Genomoviruses have been frequently found to be associated with various arthropods [11,14,16,17,20,33,34] in addition to various other samples. Genomoviruses are typically ~2 kb in genome size and likely infect fungi based on a study which showed a genomovirus species, Sclerotinia sclerotiorum hypovirulence virus 1, is able to replicate and cause hypovirulence in Sclerotinia sclerotiorum [35]. Taxonomically, genomoviruses group in nine genera; Gemycircularvirus, Gemyduguivirus, Gemygorvirus, Gemykibivirus, Gemykolovirus, Gemykrogvirus, Gemykronzavirus, Gemytondvirus and Gemyvongvirus. Here we describe nine novel genomoviruses hereby referred to as blackfly genomovirus (BfGV) 1–9 (Table 1, Figure 1 and Figure 2). Eight of these (BfGV 1–7, -9) can be more broadly classified into the Gemycircularvirus genus and BfGV-8 Rep lies basal to that of the members of Gemyduguivirus genus (Figure 2). Based on the 78% full genome species demarcation [36] the seven of the genomoviruses, BfGV-8 whose Rep is basal to those of gemyduguiviruses and six (BfGV-1, -2, -4, -5, -9) assigned to the Gemycircularvirus genus, are new species. The genome of BfGV-3 shares 79% identity with a genomovirus from snowshoe hare faeces (MG611211; [37]) and BfGV-6 shares 81% identity with a genomovirus from porcine faeces (KF371640; [38]. All share a conserved nonanucleotide “TAATATTAT” sequence which is nicked by Rep to initiate replication. The Reps of these genomoviruses share between 33–95% amino acid identities with those of all genomoviruses whereas the capsid proteins (CPs) share 29–79% identity. (Supplementary Data 1). Replication associated motifs in the Rep are conserved throughout the nine genomovirus genomes (Table S2).

Figure 2.

Figure 2

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Network and phylogenetic analysis of the Rep sequences of genomovirus, each genus with a corresponding colour and blackfly originating genomoviruses shown in red.

3.2. Divergent CRESS DNA Viruses

A large cohort of Rep-encoding viruses exist that are divergent and do not, at present, fall into classified viral families. These vary in genome organisation but all encode a Rep and putative CP. Several novel CRESS DNA viruses were also discovered in this study, all but one cluster outside major virus family groups and are distributed across five larger network clusters and three smaller clusters contain two to three sequences (Table 1, Figure 1). Nine do not cluster with any other Reps therefore are represented as singletons (Table 1, Figure 1). These range in genome size from ~1.8 to 3 kb and are referred to as blackfly DNA virus (BfV) 1–19 (Table 1). Genome organisation is highly variable, see Table 1 for details on open-reading frame orientations. Interestingly, BfV-18 appears to have an unusual rep which contains two predicted intron regions. All Rep encoding molecules harbour RCR and SF3 helicase motifs, with the exception of BfV-1 and -2 which are apparently missing motif C and RCR motif II, respectively.

The Rep of BfV-7 in group 1 is most closely related to that from a giant house spider (MH545537) [11], sharing 77% Rep identity (Sup data 2). In group 2, the Reps of BfV-12 and -15 share 70% identity with each other, and 68% with giant panda circovirus 1 Rep (MF327573) and 60% with dragonfly larvae-associated circular virus-2 Rep (KF738874) [39], respectively. Group 3 includes Reps of BfV-8 which shares 68% with those of Pacific flying fox faeces-associated circular DNA virus-15 (KT732834) [40] and BfV-11 which shares 70% with sewage-associated circular DNA virus-18 (KM821753) [41]. Reps of BfV-3, -4 and -5 in group 4 share 62% identity with CRESS virus from a minnow (MH617376) [42], and 76% and 68% with llama faeces-associated circular DNA virus-1 (KT862235) [43], respectively. The Rep of BfV-6 in group 5 shares 48% identity with that of Avon-Heathcote estuary-associated circular virus 24 (KM874354) [44].

Several viruses related to those in the family Circoviridae have also been identified in arthropods [11,14,15,45,46,47,48]. Here we identify a blackfly DNA virus (BfV-10) whose Rep clusters with members of the Circoviridae family and it is phylogenetically basal to the cycloviruses (Figure 1). The Rep of BfV-10 share 35–43% amino acid identity to Reps of circoviruses.

Reps of BfV-13, -19 and -16 form small clusters sharing 42–53% identities to their closest related Reps from Bromus-associated circular DNA virus 2 (KM510191) [49], Lytechinus variegatus variable sea urchin-associated circular virus (KR528569) [50], sewage-associated circular DNA virus-2 (KJ547626) [41], CRESS virus sp. isolate ctbg173 (MH617562) [42], Pacific flying fox faeces-associated circular DNA virus-2 (KT732829) [40]. BfV-1, -2, -9, -14, -17 and -18 Reps are singletons sharing 31–42% with Reps of Apis mellifera virus 15 (MH973771) [34], Circoviridae sp. isolate ctbe41 (MH617348) [42], uncultured virus clone CG155 (KY487824) [51], CRESS virus sp. isolate ctdb65 (MH616925) [42], Lake Sarah-associated circular virus-18 (KP153428) [44] and sewage-associated circular DNA virus-1 (KJ547620) [41] (Figure 1).

3.3. Multi-Component Viruses and Circular Rep-Encoding DNA Molecule

Viruses in the Nanoviridae family infect plants and are comprised of up to eight separate circular DNA components, all encoding a different functional gene including a Rep. Members of another plant infecting virus family, Geminiviridae, are known to have satellite molecules which enhance replication and pathogenicity. More recently a multi-component virus was isolated from faecal samples of Pacific flying fox, this, like other multi-component DNA viruses shares sequence recognition regions in the intergenic regions. In this study, three circular DNA molecules that encode a rep gene were identified. For two of these, cognate molecules encoding a cp gene were identified and therefore these represent two multicomponent viruses (Table 1, Figure 3A–C, Supplementary Data 2). These components have common regions in the intergenic region (Figure 3B), such common regions in multicomponent viruses act as recognition sites for initiation of replication [52]. These, referred to as blackfly multicomponent virus (BfMCV) 1 and 2, have Reps most similar to another multicomponent virus recovered from a Pacific flying fox multicomponent virus (KT732816) [40], sharing 65% and 67% aa similarity, respectively (Figure 3C,D). The cognate BfMCV 1 and 2 CPs share 44% with leaf-footed bug circular genetic element (MH545544) [11] and 43% with Pacific flying fox multicomponent virus (KT732816) [40]. No cognate molecule was identified for the third DNA molecule, referred to as Blackfly DNA molecule 1, however, this molecule is most closely related to the other two multicomponent viruses detected here, sharing 61–62% Rep identity, and to rodent stool associated circular genome virus (JF755415) [53] sharing 62% Rep identity. A cognate molecule encoding a CP may therefore not have been recovered, although no molecule was identified in the high-throughput sequencing data, or this genetic element may represent replication. All molecules have the same nonanucleotide sequence ‘TAGTATTAC’.

Figure 3.

Figure 3

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(A) Genome organisation of two blackfly multicomponent DNA viruses (1 and 2) and a DNA molecule indicating open reading frames and common regions (B) Alignment of the common regions from blackfly multicomponent virus 1 and 2 (C) Phylogeny of Rep for the two multicomponent viruses, DNA molecule and closest relatives (D) Pairwise comparison of the Reps show percentage similarity.

3.4. Bacteria-Infecting CRESS DNA Viruses

Microviruses, which infect bacteria, are commonly found where their host is present including the microbiome of arthropods [34,54] and harbour unidirectional genomes which encode a replication initiation protein, major and minor capsid proteins and other accessory proteins [19]. Divided into two subfamilies, Gokushovirinae and Bullavirinae (International committee for virus taxonomy: 2018 release; https://talk.ictvonline.org/taxonomy/), microviruses are largely host specific [19]. Here, we identify 108 novel microviruses from blackflies. Using the most well conserved protein, the MCP, we show the wide scope of microvirus diversity present in blackflies (Figure 4). A large proportion of blackfly microviruses (BfMVs) MCPs group with those of gokushoviruses (Figure 4). The MCPs of 88 BfMVs cluster in MV group 1, four in MV group 2 and the remaining are part of small network clusters or are singletons. Although a few BfMVs in the MV group 1 are interspersed throughout the MCP phylogeny, the majority fall in two major clades (Figure 4), sharing between 51–85% MCP identity with other microviruses (Supplementary Data 3). The MCPs of BfMVs in MV group 2, along with the other small network clusters, share 29–74% identity with those of other microviruses (Figure 4 and Supplementary Data 3). The singletons range in similarity to nearest neighbours in the public database sharing between 23–72% MCP protein sequence identity (Figure 4 and Supplementary Data 3).

Figure 4.

Figure 4

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Network analyses of the microvirus major capsid protein. Maximum likelihood phylogeny of the MCP sequences of microviruses for the two major groups (MV group 1 and 2) are shown. Smaller networks and singletons are shown with closet Rep comparisons and percentage similarities. Blackfly derived sequence are shown in red.

Many of the newly described microviruses identified in this study group with those isolated from other arthropods such as those from honey bees [34]. Taking into consideration that only 40 blackflies were sampled, the sheer number and diversity of microviruses that have been found here is remarkable. This may, in fact, reflect an equally diverse associated bacterial community in the blackfly and the animals from which they obtain a blood meal. Furthermore, this, taken with the recent recovery of many novel microviruses from arthropods [34,54], indicates the presence of distinct arthropod microvirus populations.

4. Conclusions

The ongoing expansion of our knowledge of the CRESS DNA viruses facilitated by high-throughput sequencing approaches emphasizes the breadth of diversity in various ecosystems and organisms. The diversity and number of CRESS DNA viruses associated with arthropods alone is staggering [11,14,16,20,48]. Here, we report the identification of CRESS DNA viruses associated with blackflies in New Zealand. Nine genomoviruses, 19 unclassified CRESS DNA viruses, two multicomponent viruses, a circular rep-encoding DNA molecule and 108 microviruses. To date, the only multicomponent CRESS viruses in established families are the nanoviruses and geminiviruses which both infect plants. Other than these, our group have identified novel multicomponent viruses previously in faecal matter of Pacific flying foxes [40] and now in blackflies. These appear to be related (i.e., CP and Reps) and thus eludes to the fact that there are other Rep encoding multicomponent viruses [40], given the presence of a completely unique group comprised of the BfMCVs and the pacific flying fox multicomponent virus beyond those that belong to the Geminiviridae and Nanoviridae families. Due to the blood feeding nature of blackflies, these viruses may have originated from the blackflies, the hosts they feed from or surrounding environment. Although further investigation is needed to truly determine the hosts of the CRESS viruses, we know the microviruses infect bacteria and therefore, prominent groups of related microviruses may reflect a specific blackfly bacterial profile. This study provides knowledge on viruses associated with the understudied hematophagous blackfly of New Zealand and further displays the sheer breadth of CRESS DNA viral diversity in arthropods globally.

Supplementary Materials

The following are available online at https://www.mdpi.com/1999-4915/11/6/532/s1, Table S1: Primer details for each recovered blackfly virus genomes and molecules; Table S2: Rolling circle replication and helicase motifs identified in the Reps of the eukaryotic CRESS DNA viruses; Data S1: Pairwise identity comparison of the full genome, Rep and CP sequences of the genomoviruses recovered in this study together with all previously identified.

Click here for additional data file. (1.1MB, zip)

Author Contributions

Conceptualization, S.K., M.W. and A.V.; methodology, S.K., K.S., R.S.F., M.W. and A.V.; formal analysis, S.K. and A.V.; investigation, S.K., K.S., R.S.F., M.W. and A.V.; resources, A.V.; data curation, S.K. and A.V.; writing—original draft preparation, S.K. and A.V.; writing—review and editing, S.K., K.S., R.S.F., M.W. and A.V.; visualization, S.K. and A.V.; supervision, A.V.; project administration, A.V.; funding acquisition, S.K., M.W. and A.V.

Funding

This research was funded a grant awarded to S.K., M.W. and A.V. from the Brian Mason Scientific and Technical Trust of New Zealand (grant # 2015/09).

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

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