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Decrypting old transcriptomics data from Latrodectus hesperus spyders uncovers a novel Nege-like virus.

Written by: Alston Lo

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Hespivirus rivalense

Hespivirus is a combination of Hesperus, spider, and virus. It seems common to name Negeviruses after their respective geographic locations (Negev is a desert in Israel, for example). My index strain came from Riverside, California, USA. Hence, rivalense is a combination of Riverside, California, and -ense, following [1].

Table of Contents

Abstract

Insects are among the most diverse groups of organisms, yet the study of their viromes is considerably biased, such as towards hematophagous insects for their medical significance. Fortunately, modern bioinformatics tools and databases have enabled the cheap discovery and analysis of novel viruses. Herein, we mine the SRA database and discover a novel RNA virus infecting the Western black widow (Latrodectus hesperus) spider, which we name Hespivirus rivalense. Our putative virus is distantly related to Nege- and virga-like viruses and has a Negevirus-like genome organization. The detection of H. rivalense in spider egg tissue further suggests that it could be vertically transmitted, something observed only once so far in spider-infecting viruses. Our computational results serve as a foundation for future experimental work to validate and refine our model of H. rivalense. More broadly, black widows are also urban pests and of academic interest for their strong venom and silk. Studying their virome can not only contribute to deeper insights into their ecology and biology but also shed light on more neglected areas of the insect virome.

Results

Viral ecology

H. rivalense was detected by the presence of an RNA-dependent RNA polymerases (RdRp) palmprint sequence across eight SRA runs: SRR1725029(1,2,3,5), SRR5285123 [2], SRR1853323, SRR1539570 [3], and SRR12918481 [4].

Black widow spiders. The first seven are of various tissues (e.g., cephalothorax, venom and silk glands, ovaries) from female Latrodectus hesperus (Western black widow spider) or L. hesperus eggs. Many of the SRA runs were created to study the genomics or transcriptomics of L. hesperus venom and silk, which are of medical and technological interest due to their potency and strength. The only geographically annotated samples SRR1725029(1,2,3,5) were collected in Riverside, California, USA. Indeed, L. hesperus can be found throughout western North America and especially thrive in urban areas [5]. Beyond these seven runs, there are 31 RNA-seq runs in the SRA annotated to L. hesperus. These are further organized into four BioProjects in all of which H. rivalense palmprints are detected. Only 1 of 31 L. hesperus samples comes from an adult male spider but is palmprint-negative.

The top hits are primarily Negeviruses [6] or related to Negeviruses, with the first being a putative polyprotein gene from the Bofa virus [7] with 62% identity (E value 2e-34). The subsequent hits include, in no particular order, the Beult virus [8], Buckhurst virus [7], Castlerea virus [9], and Mekrijärvi negevirus [10]. Negeviruses are a taxon of insect-specific viruses that have a broad host and geographic range, but have been primarily reported in hematophagous insects [6]. They are positive-sense single-stranded RNA viruses with an enveloped, spherical structure resembling a "hot air balloon" [6], [9]. Considering the moderate similarity of H. rivalense to Negeviruses and the presence of H. rivalense across multiple L. hesperus tissue samples, we hypothesize that H. rivalense is a novel virus infecting L. hesperus, an endogenous viral element (EVE), or an artifact from sample contamination.

To rule out the EVE hypothesis, we map the detected H. rivalense palmprint sequence to the assembled contigs from the highest estimated coverage run SRR17250291. Doing so recovered a single 10924 nt contig Hriv_contig. Next, we performed BLASTN (megablast) [11] searches against the WGS collection, restricting to only L. hesperus entries (taxid: 256737), which included a reference genome (JJRX02). This returned no significant hits, while repeating the process ad hoc for other palmprint-negative contigs recovered multiple hits annotated as genomic L. hesperus DNA. This suggests that H. rivalense is not an EVE. In general, while EVEs are common in insects [12], expressed EVEs are rare [7]. Another possibility is that Hriv_contig is a contaminant; for example, it could arise from a virus that infects an organism contaminating the H. rivalense palmprint-positive runs. We are unable to find other runs positive for H. rivalense through BLASTN searches in the Nucleotide and TSA collections. Given the above information, a novel virus seems most likely.

Interestingly, we find the putative virus in L. hesperus egg samples, suggesting a possible route of vertical transmission. To our knowledge, this would make H. rivalense the second vertically transmitted virus discovered in spiders, with the first found in spinybacked orbweaver spiders [13].

Ants. The last run SRR12918481 is from a pool of five Pheidole megacephala (big-headed ant) collected at Molokai, Hawaii [4]. However, its palmprint only has 91% identity with the other runs, which otherwise agree perfectly, and an estimated coverage of 1, which falls short of the coverage quality recommended by [14]. Thus, we discard the SRR12918481 hit for our analyses, both for simplicity and due to its low confidence. It could be worthwhile to revisit SRR12918481 in future work. Brettell et al. [4] found that P. megacephala , along with other apiary pests, may act as viral reservoirs for bee-associated viruses. Given that P. megacephala is a highly invasive pest beyond apiaries, a similar inter-species transmission could be a possibility with L. hesperus.

Viral genome

Our contig Hriv_contig is 10924 nt long with a 39.4% GC content. We observe a 42 nt A stretch towards the 3’ end of the contig, which we hypothesize to be a poly(A) tail. This is due to the recorded presence of poly(A) tails in related viruses, such as Negeviruses [6], and the use of Oligo-dT selection in SRR17250291. However, the stretch is followed by a 41 nt suffix of non-repeating bases. To check for potential assembly errors, we realign the reads from SRR17250291 to our contig using bowtie2 [19], resulting in a mean 1026x coverage. We observe high coverage (~300x) of the bases following the poly(A) stretch, which decays to 1x over the next 20 bases. However, there is a coverage bias of ends of contigs due to the assembly process, and a similar decay is observed in the 5’ end. Hence, our analysis remains inconclusive.

We find 10 ORFs in our viral contig with ORFfinder and restricting to 75 nt non-nested ORFs. InterproScan [20] predicts domains in 3 of 10 ORFs, ignoring disorder predictions, coils, and SignalP-noTM, which is not annotated further but seems to indicate a signal peptide. The three ORFs lie on the positive strand, so we will name them in downstream order: ORF1 (522-9416 nt), ORF2 (9417-9509 nt), and ORF3 (9906-10388 nt). These ORFs are predicted to have the following domains:

  • An Alphavirus-like methyltransferase (MTase) domain (IPR002588) in ORF1.
  • An rRNA MTase FtsJ domain (IPR002877) in ORF1.
  • An alpha-ketoglutarate-dependent dioxygenase AlkB-like domain (IPR027450) in ORF1.
  • A RNA virus helicase core domain (IPR027351) in ORF1.
  • An RdRp from Alsuviricetes (IPR001788) in ORF1.
  • A transmembrane helix (TMh) that is part of a membrane-bound protein in ORF2.
  • A Tobacco mosaic virus-like coat protein (TMVC) (IPR001337) in ORF3.

We are unable to find any elements encoding for non-coding RNAs in Hriv_contig, using Infernal cmscan 1.1.2. The following figure summarizes the contig-wide coverage, ORFs, predicted domains, and RdRp A, B, C motifs.

Figure 1. The genomic contig Hriv_contig of H. rivalense. The read coverage from realigning the reads from SRR17250291 to the contig, using bowtie2 [19], is depicted in the topmost graph (blue). The contig ORFs (grey), predicted domains (orange) from InterproScan [20], and RdRp A, B, C motifs (rgb) are indicated below. Plots were created in Python using the matplotlib and seaborn libraries.

AlphaFold2: RdRp structure

We use the putative RdRp sequence above and ColabFold [21] with its default settings.

drawing

Figure 2. The ColabFold-predicted structure of H. rivalense's RdRp. One seed structure is visualized with Py3DMol [22].

Phylogenetic analysis

We use the putative RdRp sequence from above. For BLASTP [23], we search against the nr database restricted to RNA viruses (taxid:2559587), and we set the maximum number target sequences to 1000. Filtering the candidates with >80% query coverage and >40% identity leaves 191 sequences (including the H. rivalense RdRp). We further remove entries with duplicate scientific names or named "ssRNA positive-strand virus sp." and "Virgaviridae sp.", tie-breaking by E score. On the remaining 81 sequences, we perform a multiple sequence alignment (MSA) using MUSCLE [24]:

Figure 3. The MSA of H. rivalense's RdRp and its top BLASTP hits visualized using Jalview [25]. For visual clarity, we have trimmed the MSA to H. rivalense's RdRp palmprint and included only the top 19 BLASTP hits. The A, B, and C motifs of H. rivalense's palmprint are annotated in the last row, and by inspection, we can see that they are well-conserved. The trimmed and full MSAs are uploaded.

We pass the full MSA into IQTree [26], with the default parameters (i.e., VT+F+I+G4 substitution model via ModelFinder [27], 1000 ultrafast bootstrap replicates [28]), to construct a maximum-likelihood phylogenetic tree.

Figure 4. The maximum-likelihood phylogenetic tree of H. rivalense's RdRp and its top BLASTP hits visualized using the iTOL webtool [29]. H. rivalense is highlighted in green and the smallest clade containing H. rivalense and the top Negevirus or Nege-like virus BLAST hits from above are lightly shaded. The raw IQTree output is uploaded.

Discussion

Our phylogenetic analysis places H. rivalense more closely to Negeviruses than the Nege-like Bofa, Buckhurst, and Beult viruses obtained from BLAST. We also observe similarity to virga-like viruses. Indeed, we observe a Negevirus-like genome organization in H. rivalense. Both ORF1s in H. rivalense and Negeviruses contain similar domains of nonstructural proteins: viral MTase, rRNA MTase, helicase, RdRp in the same order [6]. Similarly, both ORF2s have a transmembrane domain and both ORF3s encode for a structural membrane protein. Although ORF1&2 are not separated by an intergenic region in H. rivalense as in the prototypical Negevirus, we note that this or an ORF1&2 overlap have been observed in some species [10], [16]. However, ORF2 in H. rivalense appears much smaller than expected; ColabFold predicts its structure to be a single helix. We attempted to BLAST search the ORF2 protein, the ORF2 genomic interval, and the ORF2 genomic interval expanded by 50 nt on both ends, but all results were inconclusive.

Interestingly, we found one paper [30] that found a viral-like sequence in L. hesperus. Specifically, they obtained a contig (GBCS01005187) from the TSA dataset, which is in the same bioproject (PRJNA242358) as SRR1539570. GBCS01005187 has a 98% identity (MUSCLE) with Hriv_contig, but is shorter (4745 nt) and only captures the helicase, RdRp, and TMVC domains. In addition, their RdRp domains are split over two ORFs. Inspecting the alignment, we find that this arises due to an T insertion in GBCS01005187 that causes a frameshift, producing an early stop codon 17 nt downstream. It is unclear whether this is an assembly error or a defective viral variant. Nonetheless, the genomic sequence analyzed herein can be considered a refinement and extension upon GBCS01005187.

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Viral Short Story

Written in the style of Lovecraftian horror:

The spiders were acting weirdly. Now and then, people would see the odd one
scurry out of a dark crevice, but always too fast for a spider, as if it were
searching for something. Dr. Parker, a geneticist at UC Riverside, was among
the first to notice something wrong. A black widow spider had made its way
into a hamster cage, which would not have been a strange occurrence if not for
the spider being the size of the rodent that it devoured. After making it to
Parker’s lab, it was clear that this was some kind of RNA viral affliction.
The virus permeated the glands, eggs, and other spider tissues, making them
grow and mutate uncontrollably. Soon, the spider grew into a monstrous
creature. Perhaps it became much stronger and smarter than Parker could
recognize because it escaped one day. It wasn’t soon after that eggs started
showing up, from which baseball-sized hatchlings sprung. The virus could be
inherited. Worse yet, massive bugs began to appear in neighbouring states. A
giant ant was found in Hawaii. Did the moderate proximity of the virus to
other insect viruses allow it to hop between hosts? Nonetheless, while the
world began to celebrate the appearance of these strange creatures, Parker
could only watch on with dread at what he released into the world. He could
only hope that they never got a taste for humans...