Generation of a pure HISV-1 isolate and genome sequencing

Previously we used next-generation sequencing (NGS) for the characterization of reptarenavirus isolates [4]. This led to the identification of HISV-1, a putative representative for novel arenavirus genus, in addition to several reptarenavirus S and L segments [10]. Even though we had fairly high coverage of the S (11–27092 fold) and L (357–4493 fold) segments of HISV-1, we were unsure whether these represented the full length segments since the L segment comprised an ORF for the RdRp [10], but the ORF for the ZP found in other arenaviruses was missing. Since the original HISV-1 preparation contained also a reptarenavirus (UHV-2, University of Helsinki virus-2), we used serial dilution to obtain single virus isolates of both UHV-2 and HISV-1 (S1 Fig). Successful production of a clean HISV-1 isolate indicated that even though an ORF for the ZP, which functions as the matrix protein, cannot be found in the L segment, HISV-1 is able to replicate without a co-infecting reptarenavirus. For most subsequent comparative experiments we used UHV-2 as the reference reptarenavirus, since it was the co-infecting reptarenavirus of the original HISV-1 isolate. For a few experiments we used UGV-1 (University of Giessen virus-1) instead, as this is a reptarenavirus grows to high titers in our cell culture model. For the sequence analyses we chose to use the type species of each arenavirus genera.

To confirm the lack of an ORF for the ZP in the L segment of HISV-1, we isolated RNA from a batch of purified HISV-1 and sequenced the ends of the S and L segments using T4 RNA ligase to generate cyclic RNAs. The latter then served as templates for RT-PCR over the genome ends (S2 Fig) and yielded several sequences that indeed covered the genome ends of both the S and L segment. In addition to the S and L segment specific primer pairs, we also successfully applied primer combinations, i.e. L segment forward-S segment reverse and S segment forward-L segment reverse, in RT-PCRs, which suggested that T4 RNA ligation also produced S and L segment chimeras. This was confirmed by sequencing. The genome end sequencing then confirmed that the L segment of HISV-1 indeed lacks the matrix protein/ZP found in other arenaviruses. Subsequent NGS and de novo genome assembly for the purified HISV-1 preparation did not identify additional genome segments. The consensus sequence of the S segment revealed a nucleotide insertion (a stretch of 7 instead of 6 adenines at 575–581) in the GP1 ORF of our original submission [10], which led to incomplete “in silico” translation of the GPC.

Comparison of the HISV-1 genome and proteome with those of other arenaviruses

Obtaining the full length genome segments of HISV-1 allowed us to compare the genomes of the three arenavirus genera, Mammarenavirus, Reptarenavirus, and Hartmanivirus. Since this is the first time that full length genome segments of all arenavirus genera are available, we decided to perform a bioinformatics-based comparison of their genomes and proteomes, and selected the type species of each genus for these “in silico” comparisons. The S segments of all three genera, schematically depicted in Fig 1A, are identical in their coding strategy and similar in size. However, while the L segments of mammarenaviruses and reptarenaviruses share the same coding strategy and are similar in size, the L segment of hartmaniviruses lacks the ZP ORF (Fig 1A).

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Fig 1. The genome organization of the family Arenaviridae members.

A) The genome segments with respective sizes for the type species of the genera Hartmanivirus (HISV-1), Mammarenavirus (LCMV) and Reptarenavirus (GGV). The ORFs (ZP = Z protein, RdRp = RNA-dependent RNA polymerase, GPC = glycoprotein precursor, NP = nucleoprotein) are shown as arrows to demonstrate the direction of translation respective to genome orientation. B) Panhandle structures formed by the genome ends as predicted using DuplexFold Web Server of RNA structure (available at https://rna.urmc.rochester.edu/RNAstructureWeb/Servers/DuplexFold/DuplexFold.html) for LCMV, GGV, and HISV-1. C) Alignments of the 5´ and 3´ (shown in 5´ = >3´ orientation) genome ends for L and S segments of LCMV, GGV, and HISV-1. The nucleotide residues conserved throughout the L and S segments of family Arenaviridae genera are shown in bold.

https://doi.org/10.1371/journal.ppat.1007415.g001

As shown in Fig 1B, the genome ends–represented by 21 terminal nucleotides–of all arenavirus species have the ability to form a panhandle structure. The panhandles of LCMV and Golden Gate virus (GGV) L segments comprise 18 consecutive complementary nucleotides, while the corresponding region in the HISV-1 L segment contains two non-paired nucleotides. Comparison of the genome segment ends revealed a conserved CGCACxGxGxA motif at the 5´ end of mammarena-, reptarena-, and hartmanivirus S and L segments (shown in bold in Fig 1C). Similarly, the 3´ends show conserved nucleotides, GCGUGxCxCCU (shown in bold in Fig 1C), complementary to those found at the 5´ end with the underlined residue making an exception. While the predicted panhandle structures may differ even between viruses of the same species, the overall panhandle structure is maintained throughout arenavirus genera by the aforementioned conserved nucleotides. The terminal complementarity of the RNA is essential for replication and transcription [21] which, at least partially, may explain the conservation of the segment ends throughout the family. The non-conserved sites at both ends are speculated not to contribute to sequence-specific interaction with the RdRp [22], thus explaining the observed variation at these sites.

The proteomes and the amino acid identities between the corresponding proteins (based on MAFFT alignment) of the three genera are presented in S2 Table. The major difference in the proteomes is the lack of ZP in hartmaniviruses. While the ZPs of reptarenaviruses and mammarenaviruses share only 16% amino acid identity, their functions are assumed to be similar [2]. Interestingly, reptarenavirus ZPs have an N-terminal transmembrane helix (TM) [2, 4], whereas the N-terminus of mammarenaviruses is myristoylated [2]. The structural proteins of HISV-1 are all 20 to 23% identical to their LCMV and GGV counterparts. The RdRp and NP of GGV are slightly closer to LCMV (28% and 32% identical) than to HISV (20% and 20% identical). However, the GPCs of HISV-1 and LCMV (23% identity) are more similar than the GPCs of LCMV and GGV (16% identity). See S2 Table for more detail.

The GPC of each arenavirus genera are schematically presented in Fig 2A. By prediction the GPCs contain several N-glycosylation sites: 7 in HISV-1 (4 in GP1 and 3 in GP2) and LCMV (5 in GP1 and 2 in GP2) and 9 in GGV (7 in GP1 and 2 in GP2) (Fig 2A). The cleavage between GP1 and GP2 is mediated by subtilisin-kexin isozyme-1/site-1 protease (SKI-1/S1P) for mammarenaviruses (Fig 2A). Using ELM (eukaryotic linear motif resource, http://elm.eu.org/, [23]) we identified a potential SKI-1/S1P cleavage site in GGV GPC, and the GPC alignment of known reptarenavirus species suggests that the site is conserved (Fig 2A). For HISV-1 we could not detect a SKI-1/S1P cleavage site but rather identified a potent furin cleavage site in the same region. By comparing the region to GPCs of the other hartmanivirus species (described later in the manuscript) we could show that the furin cleavage site is preserved among the hartmaniviruses found thus far (Fig 2A).

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Fig 2. Comparison of the GPC ORF between the three arenavirus genera.

A) The GPCs of the genera mammarenavirus, reptarenavirus, and hartmanivirus as exemplified by the respective type species LCMV, GGV and HISV-1. The highlighted features are: cleavage sites (presented by vertical line and number), glycosylation sites (presented by Y-shape) and transmembrane region (TM, presented by grey shading) are based on LCMV, GGV, and HISV-1 sequences. The abbreviations are: SSP = stable signal peptide, SP = signal peptide, GP1 = glycoprotein 1, GP2 = glycoprotein 2, TM = transmembrane. The myristoylation site of mammarenavirus and hartmanivirus SSP is shown by an arrow and below is an alignment of the site in other species of the genus. SKI-1/S1P cleavage site in mammarenavirus and reptarenavirus GPC is depicted by an arrow at around the center of GPC, and below is an alignment around the cleavage site for other species of the genus. For hartmaniviruses furin performs the GP1-GP2 cleavage instead of SKI-1/S1P. B) Alignment of mammarena- and hartmanivirus SSP and GP2, the residues essential for SSP-GP2 intermolecular zinc-binding motif are depicted by grey highlighting. The conserved myristoylation in the SSP is highlighted by a box, as well as the positive charge at around the center of SSP. C) A schematic representation of the glycoprotein complex as inspired by the illustration of Nunberg and York[17].

https://doi.org/10.1371/journal.ppat.1007415.g002

A more thorough investigation of the GPCs indeed shows similarities between HISV-1 and LCMV. To begin with, both HISV-1 and LCMV have long (55 and 58 residues, respectively) signal peptides (SP) that by prediction are myristoylated at position 2, while the SP of GGV is only 23 residues and lacks the myristoylation site (Fig 2A). For mammarenaviruses the SP remains in the virion and is known as a stable signal peptide (SSP) that interacts with GP2 via an intermolecular zinc-binding motif [24] (Fig 2C). Sequence comparison between mammarenavirus and hartmanivirus SSPs and GP2s (Fig 2B) shows conservation at the histidine and cysteine residues required for the SSP-GP2 interaction. Additionally, the GP2s of both mammarenaviruses and hartmaniviruses have a relatively long cytoplasmic tails (48 and 42 residues respectively) while the GP2 tail of reptarenaviruses only comprises a few (two in GGV) residues (Fig 2A and 2B). The above suggests that there might be differences in the organization of the spikes complex between reptarenaviruses and the other two arenavirus genera. Of note, the GP2 is the most conserved reptarenavirus protein, sharing 68–99% sequence identity between all the known species and 87–99% identity when CASV and UHV-1 are excluded. The GP2s of both mammarenaviruses and hartmaniviruses appear to be more variable.

In vitro characterization of HISV-1 infection

To monitor and characterize HISV-1 infection in cell culture, we produced an antiserum against the HISV-1 NP which we refer to as anti-HISV NP antiserum throughout the manuscript. We used the C-terminal half of the NP as the antigen, since this strategy had produced a broadly reactive anti-UHV-1 NP antiserum [10] referred to as anti-UHV NP antiserum throughout the manuscript. To test the anti-HISV NP antiserum, we performed infection and co-infection experiments with UHV-2 and HISV-1 on cultured boid kidney cells (I/1Ki), and analyzed the infected cells by western blotting, immunofluorescence (IF) staining, and immunohistology (IH) (Fig 3). The western blot shows that the anti-HISV NP antiserum does not cross-react with UHV-2 NP, and vice versa (Fig 3A). The IF staining of infected cells concurs with the western blotting results, and also indicates that neither of the antisera reacts with cellular proteins at the concentrations applied (Fig 3A). A comparison of the IF staining patterns in infected cells showed that HISV-1 infection produces large fluorescent foci (i.e. infection of several cells in close proximity to each other), whereas UHV-2 infection resulted in scattered individual positive cells (100x magnification in Fig 3B). At a higher magnification, UHV-2 infected cells were found to exhibit the punctate NP staining pattern typical for reptarenavirus infected cells with inclusion bodies (IB)[4, 25], whereas HISV-1 NP appeared to be more diffusely distributed in the cells (400x magnification in Fig 3B). A similar staining pattern was seen in HISV-1 infected I/1Ki cell pellets, indicating that the antiserum is suitable for immunohistology (IH) (Fig 3C).

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Fig 3. Western blot (WB), immunofluorescence (IF), and immunohistology (IH) of cultured cells using antiserum against C-terminal portion of HISV-1 (anti-HISV NP) or UHV-1 (anti-UHV NP) NP.

A) The panels on right show WBs comparing I/1Ki cells infected with UHV-2 or HISV-1 alone to I/1Ki cells co-infected with UHV-2 and HISV-1. The molecular weight marker (Precision Plus Protein All Blue Prestained Protein Standards, Bio-Rad) is shown in red, and the staining with anti-HISV NP (left) and anti-UHV NP (right) is shown in green. The figure was obtained using Odyssey infrared imaging system (LI-COR). The panels on left show IF staining of I/1Ki cells infected with UHV-2 alone or HISV-1 alone using anti-HISV NP (top panels) and anti-UHV NP (bottom panels). The cells were stained at two days post infection (dpi). The red channel shows NP staining and the blue channel nuclei (DAPI staining). The figures were captured at 400x magnification. B) I/1Ki cells infected with HISV-1 or UHV-2 IF stained at 2 dpi. The top panels show HISV-1 infected cells stained with anti-HISV NP and the bottom panels show UHV-2 infected cells stained with anti-UHV NP. The red channel shows NP staining and the blue channel nuclei (Hoechst staining). The panels on left side were captured at 100x and the panels on right side at 400x magnification. The yellow square indicates the individual cells shown at higher magnification on the rightmost panels. C) IH of non-infected and HISV-1 infected I/1Ki cells. The HISV-1 infected cells were collected and fixed at 6 dpi, the figures were captured at 400x magnification.

https://doi.org/10.1371/journal.ppat.1007415.g003

Since we did not see a marked difference in the amount of viral NP in western blots when comparing single-virus infection to co-infection (Fig 3A), we decided to study the effect of co-infection on virus replication by quantifying the amount of viral RNA released from infected cells, using qRT-PCR. We initially used UHV-2 and HISV-1 for the experiment, since these viruses originated from the same isolate. Fig 4A shows representative results of one of the three consecutive experiments. We found that the amount of UHV-2 RNA released in co-infection was not reduced as compared to the single virus infection (Fig 4A), indicating that co-infection does not markedly affect the replication rate of reptarenaviruses and hartmaniviruses. To provide further evidence for this observation, we performed a similar co-infection experiment using HISV-1 and UGV-1. We decided to use UGV-1, the virus of which S segment we most often find in snakes with BIBD (other authors call this S segment S6 [11]). Also, UGV-1 grows to high titers in our cell culture model. We obtained results comparable to those obtained with UHV-2 (Fig 4A and 4B), indicating that reptarenaviruses and hartmaniviruses do not interfere with each other’s replication during co-infection. We also tried infecting cultured mammalian cell lines (Baby hamster kidney, BHK-21; African green monkey kidney, Vero E6) with HISV-1 at 30°C and 37°C, however, we could not detect viral antigen or replication.

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Fig 4. Virus production in I/1Ki cells during single virus infection vs. reptarenavirus-hartmanivirus co-infection.

A) The supernatants of I/1Ki cells infected with UHV-2 or HISV-1 alone or co-infected with HISV-1 and UHV-2 was collected at 0, 2, 4, 6, and 8 dpi, and analyzed for the amount of viral RNA using Taqman primers and probes. The amount of S and L segment was determined individually for both viruses. The left axis shows the relative amount of virus based on delta Ct calculation. The blue curve represents the relative amount of viral RNA released in the case of UHV-2 or HISV-1 infection alone, and the red curve represents the amount of viral released RNA in co-infection. B) The supernatants of I/1Ki cells infected with UGV-1 or HISV-1 alone or co-infected with HISV-1 and UGV-1 was collected at 0, 1, 2, 3, 5, and 7 dpi, and analyzed for the amount of viral RNA using Taqman primers and probes. The amount of S and L segment was determined individually for both viruses. The left axis shows the relative amount of virus based on delta Ct calculation. The blue curve represents the relative amount of viral released RNA in the case of UGV-1 or HISV-1 infection alone, and the red curve represents the amount of viral RNA released in co-infection.

https://doi.org/10.1371/journal.ppat.1007415.g004

As indicated by IF staining (Fig 3B) the reptarenavirus NP forms large IB in infected cells, which we and others have described in detail [2, 4]. However, the IF staining suggested that such IB would not be present in HISV-1 infected cells. Therefore, we performed an ultrastructural study of HISV-1 infected snake cells. Indeed, we could not demonstrate IB in HISV-1 infected cells by electron microscopy (EM). Instead, the HISV-1 infected cells generally exhibited extensive cytoplasmic vacuolization and blebbing of the plasma membrane (PM) (Fig 5A to 5C). We also observed tubular structures within the cells (Fig 5B), which appeared to contain electron electron-dense material similar to the blebs at the PM (Fig 5B to 5D). For comparison, an example of reptarenavirus infection-associated electron dense IB is presented in Fig 5E. We assumed that the electron-dense structures observed in HISV-1 infected cells would contain and/or consist of viral proteins. Indeed, immuno-EM revealed that both the tubular structures and the blebs at plasma membrane contain HISV-1 NP (Fig 6); the latter often appeared to be continuous between two adjacent cells (Fig 6E, inset) suggesting direct cell-to-cell contact. We found HISV-1 NP also associated with vacuoles (Fig 6B and 6C) and in the nucleai/along the nuclear membrane (Fig 6G), which is in line with the IF staining (Fig 3B). The HISV-1 NP was found to be abundant in the tubular structures with the infected cells (Fig 6D, 6F and 6G). Viral antigen expression in the blebs at the PM and in the intracellular tubular structures together with the detection of large foci of infected cells in IF staining could suggest that cell-to-cell spreading plays a greater role in the spread of hartmaniviruses than of reptarenaviruses. For comparison, the NP of the reptarenavirus (UGV-1) accumulated in the cytoplasmic inclusion bodies (Fig 7).

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Fig 5. In vitro effects of hartmanivirus.

Primary cell line derived from juvenile B. constrictor kidney (I/1Ki). A-D) HISV-1 infection, 6 dpi. A) Overview; cells with extensive cytoplasmic vacuolization(arrows) and multiple electron dense nodules along the cell membrane (blebbing; arrowheads). B) Individual cell with cytoplasmic lamellar bodies (arrows), microtubuli (arrowheads) and cytoplasmic vacuolization. C) Individual cells with blebbing along the cell membrane (arrowheads) and large cytoplasmic vacuoles (asterisks). D) Higher magnification of cell membrane blebs. E) UGV-1 infection, 6 dpi. Cells with characteristic cytoplasmic electron dense inclusion bodies (asterisks) and mild cytoplasmic vacuolization. There is no evidence of blebbing along the cell membrane.

https://doi.org/10.1371/journal.ppat.1007415.g005

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Fig 6. Immuno-electron microscopy (immuno-EM) of HISV-1 infected I/1Ki cells.

HISV NP was visualized by anti-HISV NP antiserum and 18 nm gold-conjugated goat anti-rabbit IgG antibody. A-C) Cells after brief trypsin treatment prior to collection. A) Cells showing HISV-1 NP at cytoplasmic microtubules (arrows) and at the plasma membrane blebs (arrowheads). Cytoplasmic vacuoles are indicated by asterisks, N: nucleus. B) A shrunken infected cell with marked cytoplasmic vacuolization (asterisks). C) Higher magnification of B). The cytoplasm shows deposits of HISV-1 (arrows) adjacent to vacuoles (asterisks). D) A cell without trypsin treatment prior to collection. HISV-1 NP is found along cytoplasmic microtubules (arrowheads) and in association with cell protrusions (arrows). E-G) Cells after brief trypsin treatment prior to collection. E) A cell with small, partly contracted HISV-1 NP positive “blebs”at the plasma membrane (arrows). Inset: Higher magnification of the blebs, showing evidence of tubular cell-to-cell connections (arrows). F) A cell with abundant cytoplasmic vacuoles (asterisks) and cytoplasmic tubules containing HISV-1 NP. G) A higher magnification of HISV-1 NP positive tubuli in the cell presented in F. HISV-1 NP is also found within vacuoles (arrows) and around the nuclear membrane (arrowheads). N: nucleus.

https://doi.org/10.1371/journal.ppat.1007415.g006

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Fig 7. Immuno-EM of UGV-1 infected I/1Ki cells, 6 dpi.

UGV-1 NP was visualized by anti-UHV NP antiserum and 18 nm gold-conjugated goat anti-rabbit IgG antibody. A) A cell with small and large cytoplasmic inclusion bodies containing UGV-1 NP (asterisks). B) A cytoplasmic inclusion body at a higher magnification demonstrating abundant UGV-1 NP (arrows).

https://doi.org/10.1371/journal.ppat.1007415.g007

Molecular and morphological characterization of HISV-1 virions

Since we could not find evidence of ZP at genome level, we decided to analyze purified HISV-1 virions in comparison to those of purified reptarenavirus (UGV-1). We used UGV-1 for this approach, as it produces abundant virions in our cell culture model. For the structural study, we purified the virions by density gradient ultracentrifugation (Fig 8A). The major band in the main fractions containing HISV-1 (F6-F8 in Fig 8A) represents the NP, as demonstrated by western blotting (Fig 8B). We then compared the SDS-PAGE patterns of purified UGV-1 and HISV-1 to find more evidence on the lack of ZP in hartmaniviruses. The NP appears as the most abundant protein for both UGV-1 and HISV-1 with an approximate size of 70 kDa by SDS-PAGE mobility (Fig 8B). The exact sizes of GP1 and GP2 are not known, however, by comparing the concentrated supernatants produced by the same cell line infected with reptarenavirus vs. hartmanivirus, the indicated bands most likely represent the viral glycoproteins (Fig 8B). A doublet band at around 16–17 kDa is evident in the UGV-1 preparation, which is slightly larger than the calculated molecular weight, 12.7 kDa, of the ZP. The observed difference between predicted molecular weight (M.W.) and SDS-PAGE mobility could be due to palmitoylation, since the ZP is by prediction palmitoylated at 3 sites (http://csspalm.biocuckoo.org/online.php). As expected, the corresponding bands are missing from the HISV-1 preparation (Fig 8B), providing further evidence that hartmaniviruses lack the ZP. The result also suggests that the lack of ZP is not complemented by a similar(-sized) cellular protein. We then studied the HISV-1 virions in EM under negative staining. The virions appeared pleomorphic to roundish with an approximate diameter of 120–150 nm and most of the particles seemed not to be intact (top panels in Fig 8C). We then analyzed HISV-1 virions in cryo-EM, which provided, in agreement with the SDS-PAGE analysis, no evidence of continuous density lining the inside of the membrane (Fig 8C). A comparison of the appearance of HISV-1 and UGV-1 virions in cryo-EM confirmed that the overall morphology and size (roughly 120–150 nm in diameter) of the virions is similar (Fig 8C).

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Fig 8. Ultrastructural characterization of HISV-1 virions.

A) Analysis of fractions from sucrose density gradient ultracentrifugation of HISV-1 by SDS-PAGE, the proteins were visualized using Coomassie staining. The fractions were collected from the bottom of the gradient (from higher to lower density). The fractions F6-F8 contain the majority HISV-1 as judged by the intensity of ~70 kDa NP band, these fractions were pooled, concentrated and analyzed by WB (shown in panel B) to confirm the presence of NP. The molecular weight marker (Precision Plus Protein All Blue Prestained Protein Standards, Bio-Rad) is at leftmost lane. The figure was obtained using Odyssey infrared imaging system (LI-COR). B) The panel on right shows comparison of the proteins in purified preparation of UGV-1 (reptarenavirus) and HISV-1 (hartmanivirus), as seen by Coomassie staining. The arrows indicate the viral proteins as identified by mobility in SDS-PAGE, the label GP1 and GP2 shows the region where the processed GP1 and GP2 would likely migrate. The molecular weight marker (Precision Plus Protein All Blue Prestained Protein Standards, Bio-Rad) is at leftmost lane. The panels on right show Ponceau S (total protein) staining and WB of purified HISV-1 (pooled fractions F6-F8, panel A) and UGV-1 with anti-HISV NP antiserum. Ponceau S staining was recorded with regular flatbed scanner, the Coomassie stained gel and WB were obtained using Odyssey infrared imaging system (LI-COR). C) Top panel shows HISV-1 examples of virions in EM under negative staining, the scale bar is 100 nm. The bottom panel shows HISV-1 and UGV-1 as seen in cryoelectron microscopy, the scale bar is 200 nm.

https://doi.org/10.1371/journal.ppat.1007415.g008

Identification and sequencing of additional hartmanivirus species

We recently studied several Boa constrictor clutches born to parental animals with BIBD, aiming to demonstrate or rule out vertically transmission of reptarenaviruses [12]. We used NGS to initially define the reptarenavirome of each clutch, and confirmed our findings by species-specific RT-PCRs. However, for one of the five clutches we used only RT-PCR to demonstrate the virus transmission, since we already had analyzed three clutches from the same breeder by NGS [12]. Some of our species-specific RT-PCRs designed based on the NGS results of other clutches did not work optimally with the samples from clutch #5 [12], which prompted us to study some of the animals (snakes 2.1–2.5, Table 1) by NGS and de novo genome assembly. We found a hartmanivirus, designated as Veterinary Pathology Zurich virus-1 (VPZV-1), accompanied by three reptarenavirus L and two S segments in the liver of the father of this clutch (snake 2.1, Table 1) and in some of the 12 to 20-month-old offspring (snakes 2.2–2.5, Table 1) as well as one additional reptarenavirus L segment in a pooled sample of the offspring (2.2, Table 1). The GenBank accessions for the hartmaniviruses and reptarenaviruses identified in this study are provided in S1 Table. These findings suggest that also hartmaniviruses can be vertically transmitted, although we cannot entirely rule out the transmission after birth for these juvenile snakes. We also identified VPZV-1 by NGS in liver and brain samples of an adult snake from a different breeder (2.6, Table 1), again together with several reptarenavirus L and S segments. By studying some of the cell culture isolates of an earlier study [4] (animal 2.7, Table 1) with the NGS approach, and found a virus which according to ICTV criteria represents a genetically distinct lineage of the VPZV species (designated ad VPZV-2). This virus was accompanied by three reptarenavirus L and one S segments.

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Table 1. Animals included in the study.

The results of NGS and/or RT-PCR studies performed, the means of diagnosis of BIBD, age and weight, immunohistology (IH) results with anti-HISV NP antiserum, and comments/references pointing to earlier studies that included the same animal are provided.

https://doi.org/10.1371/journal.ppat.1007415.t001

In another snake (1.4, Table 1) from the breeder of snake 2.6, we found a virus that by sequence comparison represents the same species as HISV-1 and thus named it HISV-2. The next hartmaniviruses we identified in a pooled blood sample of snakes (3.1, Table 1) with confirmed BIBD. According to ICTV criteria the identified viruses represent yet another hartmanivirus species, and were designated as Old Schoolhouse virus-1 and -2 (OScV-1 and -2). Subsequently, we found another representative of OScV-2 in a pooled blood sample (3.2, Table 1) of snakes with BIBD from another breeding colony. Finally, we identified a putative representative of a fourth hartmanivirus species (4.1, Table 1), which we named Dante Muikkunen virus-1 (DaMV-1), in a snake with mild neurological signs suspected to be associated with BIBD. In addition to identifying DaMV-1 and reptarenavirus segments, we also found the snake to carry a novel deltavirus, which we describe outside this report [26].

Congruently with PASC analysis (S3 Table), the phylogenetic analyses suggested that the novel hartmaniviruses clustered according to their tentative species designations on the basis of their L and S segments (Fig 9). The phylogenetic analysis of the RdRp amino acid sequences of the representatives of all known arenavirus species suggested that the genus Hartmanivirus forms an outlying group separate from mammarenaviruses and reptarenaviruses, whereas recently found Wenling frogfish arenaviruses [27] form an outlying group separate from the hartmani-, reptarena- and mammarenaviruses (Fig 9).

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Fig 9. Phylogenetic analysis of hartmaniviruses.

A) Maximum clade credibility tree of the polymerase region of arenaviruses. The tree was constructed from amino acid alignment using Bayesian MCMC method with LG model of substitution. Posterior probabilities are shown in each node. B) Maximum clade credibility tree of S segment of hartmaniviruses. The tree was constructed from Bayesian MCMC method with HKY model of substitution with gamma distributed rate variation among sites and proportion of invariable sites. Posterior probabilities are shown in each node.

https://doi.org/10.1371/journal.ppat.1007415.g009

To gain some information on the prevalence of hartmaniviruses in larger populations, we designed primers based on the L segments of OScV-1 and -2, and used RT-PCR to screen a set of 71 blood samples collected from the same breeding colony, in which we initially detected these viruses. Interestingly, 44/71 snakes were RT-PCR positive for OScV-1 and/or -2; of these, one snake was positive for both OScV-1 and -2. Thirty-four snakes in the collection were diagnosed with BIBD as they exhibited IBs in blood cells, of these, 23 had hartmanivirus infection. The fact that close to 70% of snakes with BIBD had an accompanying hartmanivirus infection indicates that the role of hartmanivirus infection in the pathogenesis of BIBD needs to be further investigated.

Target cells of hartmanivirus in infected snakes

Production of the anti-HISV NP antibody enabled us to study the tissue and cell tropism of hartmaniviruses, using immunohistology. We initially screened the tissues of the snake from which UHV-2 and HISV-1 originate (animal 1.1, Table 1). In comparison to reptarenavirus NP, which can be detected in most tissues as cytoplasmic IB [28], HISV NP expression was very limited and most consistent in the brain. The neurons exhibited a diffuse, finely granular, cytoplasmic and/or axonal reaction (Fig 10A), which clearly differed from the reptarenavirus NP expression pattern (Fig 10B). Additionally, HISV-1 NP expression was occasionally seen in a range of other cell types: smooth muscle cells in the lung and respiratory epithelial cells (Fig 10C), endothelial cells and medial smooth muscle cells in arteries (Fig 10D), ependymal cells in the brain (Fig 10E), and axons of peripheral nerves (Fig 10F). Examination of another three snakes that harbored HISV-1 or -2 (animals 1.2–1.4) revealed a similar HISV-1 NP expression pattern, though with some variation in the range of cell types (Table 1). In animal 1.4, infected with HISV-2, a broader range of epithelial cells was found to be occasionally positive (pulmonary epithelial cells (Fig 10G), glandular epithelial cells in trachea and stomach (Fig 10H), intestinal epithelial cells, acinar epithelial cells in the exocrine pancreas (Fig 9I) and tubular epithelial cells in the kidney (Fig 10J)). Also, smooth muscle cells in the muscular layers of stomach and intestine as well as dendritic cells in the spleen were found to express the viral antigen (Figs 9 and 10). In animal 1.3, the liver exhibited HISV-1 NP expression in sinusoidal endothelial cells. In none of the hartmanivirus negative animals was there any evidence of NP expression (Table 1). We tested some of the snakes found to be infected with the above HISV-like viruses (VPZV-1: 2.1, 2.3–2.6; DaMV-1: 4.1) by IH for HISV-1 NP. The antibody appeared to cross react only minimally, as the reaction was restricted to a few individual cells in two animals (Table 1). We then tested six snakes that had been HISV-1 negative by RT-PCR (5.1–5.5) or hartmanivirus negative by NGS (5.6) for the expression of HISV-NP, all with a negative result (Table 1).

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Fig 10. Identification of the hartmanivirus target cells in vivo.

A-F) Snake 1.1. A, B) Brain. A) HISV-NP expression is seen as a granular reaction in the cytoplasm (arrows) and axons (arrowheads) of intact neurons. B) In contrast, expression of reptarenavirus NP is invariably seen in association with cytoplasmic, BIBD-typical inclusion bodies (arrows) in neurons. C) Lung. The HISV NP is abundant in smooth muscle cells (arrows). Inset: respiratory epithelium with several positive cells, exhibiting a granular cytoplasmic reaction. D) Large artery with smooth muscle cells positive for HISV-NP (arrow) and endothelial cell (arrow head). E) Brain, ependyma with several cells strongly positive for HISV-NP. F) Peripheral nerve, exhibiting HISV-NP expression in axons (arrowhead). G-K) Snake 1.4. G) Lung. There are HISV-NP positive epithelial cells. Arrowhead: smooth muscle cells. H) Stomach. The mucosa exhibits numerous glandular epithelial cells positive for HISV NP (arrows). Smooth muscle cells in the muscular layers are also occasionally positive (arrowheads). I) Pancreas with several HISV-NP positive acinar epithelial cells (arrows). J) Kidney with HISV-NP expression in epithelial cells of one tubule (T). K) Spleen. Several dendritic cells exhibit HISV-NP expression (arrowheads).

https://doi.org/10.1371/journal.ppat.1007415.g010

Ethics statement

The samples included in this study originated from animals submitted by their owners either to the Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, Switzerland, or to the Department of Veterinary Biosciences, Faculty of Veterinary Medicine, University of Helsinki, Finland, for a diagnostic post mortem examination. An Animals Scientific Procedures Act 1986 (ASPA) schedule 1 (appropriate methods of humane killing, http://www.legislation.gov.uk/ukpga/1986/14/schedule/1) procedure was applied to euthanize the snakes. Full diagnostic post mortem examination, blood sampling and diagnostic testing of collected samples were performed with full owners' consent. Ethical permissions for euthanasia and diagnosis-motivated necropsies (both routine veterinary procedures) were not required due to suspicion of a lethal disease, BIBD.

Animals

The study was performed on tissues or full blood of 23 snakes that were suspected to suffer from BIBD. A further 68 blood samples from snakes in a private breeding collection which previously had animals dying with BIBD were screened for hartmanivirus infection upon the owner’s request. All animals were captive snakes from breeding collections in Germany and Switzerland, ranging in age from juvenile to more than 12 years (Table 1).

Cells, viruses and purification of viruses

The Boa constrictor kidney cell line, I/1Ki, was used for virus propagation and virus isolation attempts as described [4]. A virus preparation containing HISV-1 and UHV-2 described in [10] was used as the source of pure isolates. The isolation strategy is depicted in S1 Fig. Briefly, 10-fold dilution series of the virus stock were prepared on I/1Ki cells grown on a 96-well plate, and at 14 days post infection (dpi) the cells inoculated with virus dilutions 1:10⁷ and 1:10⁸ were transferred onto a 24-well plate. The cell culture medium was collected at 7 and 14 dpi, and the pooled cell culture supernatants were analyzed by virus species specific RT-PCR as described [10, 12]. For production of HISV-1 stock, a 75-cm² flask of semi-confluent I/1Ki was inoculated with 500 μl of the pooled supernatant from the 24-well plate, the cell culture medium was collected and replaced at 2–3 day intervals until 14 dpi, and the pooled supernatants were filtered through a 0.45 μm syringe filter (Millipore) and stored at -80°C for further use. Large quantities of HISV-1 were produced by inoculating semi-confluent I/1Ki 75-cm² flasks with 1 ml of 1/50-1/200 diluted HISV-1 stock, followed by supernatant collection as described above. Viruses were concentrated by pelleting through a sucrose cushion and more pure virus preparations were obtained by sucrose density gradient ultracentrifugation as described [4, 35, 36]. For cryo-electron microscopy (cryo-EM) the virus-containing fractions were pooled and dialyzed against phosphate-buffered saline (PBS). For transmission electron microscopy and immunocytology, cells were infected and harvested six days after inoculation, pellets prepared and fixed as described [4]. For co-infection experiments I/1Ki cells were inoculated either with equal amounts of HISV-1 and UHV-2, or HISV-1 and UHV-2 alone as controls (multiplicity of infection > 1). The infection vs. co-infection experiments were done in duplicate.

Isolation of RNA

Trizol and Trizol LS isolation reagent (Life Technologies) in combination with QiaGEN RNeasy Mini Kit (Qiagen) was used for RNA isolation as described [12]. RNA isolation from cell culture supernatants was done with either the QIAamp Viral RNA Mini Kit (Qiagen) or the GeneJET RNA Purification Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. No carrier RNA was used during RNA isolation for samples analyzed by NGS. RT-PCR served to detect viral RNA from cell culture supernatants as described [10, 12].

Circularization of RNA, reverse transcription-polymerase chain reaction (RT-PCR), and sequencing of the genome ends

HISV-1 RNA isolated from pelleted virus was treated with T4 polynucleotide kinase (Thermo Fisher Scientific) according to the manufacturer’s protocol, purified using the QiaGEN RNeasy Mini Kit (Qiagen), and circularized with T4 RNA ligase (Thermo Fisher Scientific). The RNA circularization reaction (2 h at 25°C) mix (20 μl in DEPC-treated water) included: 2 μl of 10X reaction buffer, 5 μl of isolated RNA, 1 μl of T4 RNA ligase, 0.5 μl RNAse inhibitor (40 μ/μl), 10% PEG 8000, and 100 μM ATP. The reaction ligase was inactivated by heating the reaction mix to 70°C for 10 min. The circularized RNA was reverse transcribed using RevertAid H Minus Reverse Transcriptase (Thermo Fisher Scientific) following the manufacturer’s protocol for specific primers. The S segment primers were 5´-CTCCATTTACTCGAACAAGCTCAC-3´ and 5´-CAGGTTAAATTCATTGTTGGAGCA-3´, the L segment primers were 5´-GCACAACAATCTTTCTGCGAT-3´ and 5´-CAGGGCTTTGTTTTGTCCAG-3´. Phusion Flash High-Fidelity PCR Master Mix (Thermo Fisher Scientific) was used for PCR amplification. The reaction mix consisted of: 1 μl of cDNA, 10 μl of Master Mix, 1 μl of forward and reverse primer (10 μM stocks), and 7 μl of molecular grade water. The cycling conditions were: 1) 10 s at 98°C; 2) 1 s at 98°C; 3) 5 s at 60°C; 4) 7 s at 72°C; 5) 1 min at 72°C; steps 2 to 4 were repeated 35 times. The PCR products were separated by agarose gel electrophoresis, purified with the QIAquick gel extraction kit (Qiagen) following the manufacturer’s instructions, and cloned into plasmid using Zero Blunt TOPO PCR Cloning Kit (Thermo Fisher Scientific) following the manufacturer’s recommendations. Plasmid minipreps were purified using the GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific) and the purified plasmids were sent for sequencing (with M13 forward and reverse primers) to Microsynth (Zurich, Switzerland).

In silico structure prediction for genome ends

To study the secondary structures formed by the genome ends of LCMV (strain Armstrong 53b, S segment GenBank accession NC_004294, L segment NC_004291), GGV (S segment NC_018483, L segment NC_018482), and HISV-1 (S segment KR870017, L segment KR870031) we used DuplexFold Web Server of RNA structure at RNAstructure (Web Servers for RNA Secondary Structure Prediction, available at https://rna.urmc.rochester.edu/RNAstructureWeb/Servers/DuplexFold/DuplexFold.html) [37, 38]. We applied standard parametrization for the predictions, the folding free energies for the models chosen for presentation are: LCMV S segment -30.9 kcal/mol; LCMV L segment, -41.4 kcal/mol; GGV S segment -33.4 kcal/mol; GGV L segment -35.6 kcal/mol; HISV S segment -34.3 kcal/mol; and HISV L segment, -29.9 kcal/mol.

Next-generation sequencing (NGS)

NGS and de novo assembly was done as described [12, 39].

Phylogenetic analysis

The sequences were aligned with Clustal Omega algorithm [40] implemented in EMBL-EPI webserver [41]. The phylogenetic trees were constructed using Bayesian Monte Carlo Markov Chain (MCMC) method implemented in BEAST version 2.4.7 [42] using LG or HKY-G-I substitution models for amino acid and nucleotide sequences, respectively. The analyses were run for 50 million states and sampled every 5000 steps. They were carried out on the CSC server (IT Center for Science Ltd., Espoo, Finland). Posterior probabilities were calculated with a burn-in of 10% and checked for convergence using the Tracer version 1.6.

Production of recombinant HISV NP, purification, and production of antiserum

Full-length NP (amino acids 1–582), and N- (aa 1–295) and C-terminal (aa 296–582) parts of it were PCR cloned for E. coli expression using primers (forward for full length and N-terminal portion 5´-CACCATGTCCTTGAACAAGGACCTT-3´; reverse for N-terminal portion 5´- TCTGTCGCTGGTGCAACC-3´; forward for C-terminal portion 5´- CACCATGATCTCATCTCAAAACATACC-3´; reverse for full length and C-terminal portion 5´- GTTGTTCATTATGTAGTTGAA-3´) designed according to the Champion pET101 Directional TOPO Expression Kit with BL21 Star (DE3) One Shot Chemically Competent E. coli manual (Thermo Fisher Scientific). Protein production and purification was done as described [39]. Full length HISV NP could not be recovered by this strategy, but the N- and C-terminal portions were recovered in moderate and good amount, respectively. The purified C-terminal portion of HISV NP was dialyzed against PBS. A rabbit polyclonal antiserum against C-terminal portion HISV NP (anti-HISV NP-C) was produced by Biogenes GmbH (Berlin, Germany).

Immunofluorescence (IF) staining

IF staining was done on cells grown on 24-well Glass Bottom Plates (In Vitro Scientific) as described [39]. The primary antibodies, anti-UHV NP-C or anti-HISV NP-C antisera, were used at 1:2,000 dilution in PBS.

SDS-PAGE and western blotting

Routine protocols, described in [43], were utilized for SDS-PAGE and western blotting, the results were recorded with the Odyssey Infrared Scanning System (LI-COR).

Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)

A Taqman qRT-PCR assay for quantifying the S and L segments of UHV-2 and HISV-1 served to monitor the growth of UHV-2 and HISV-1 in cell culture. The primers and probes were: UHV-2 S segment forward primer (FWD) 5´-GCAAAACAGAACTGCTGCAGTC-3´, reverse primer (REV) 5´-TGCGATACAGACATAATTAGAGACATTG-3´, and probe 5´-6-Fam(carboxyfluorescein)-GTCACCATGTGTCCCTCAGAACTCATTCA-3´-BHQ-1 (Black Hole Quencher); UHV-2 L segment FWD 5´-TTGGGGAGTTTGTTACCAATGT-3´, REV 5´- GTGGGCCCAAATAACAAACCT-3´, and probe 5´-6-Fam- CTCTCTCGGACCTCCCACTTGTTCCTTTATG-3´-BHQ-1; UGV-1 S segment forward primer (FWD) 5´- CAAGAAAAACCACACTGCACA-3´, reverse primer (REV) 5´- AACCTGTTGTGTTCAGTAGT-3´, and probe 5´-6-Fam(carboxyfluorescein)- CTCGACAAGCGTGGGCGGAGG-3´-BHQ-1 (Black Hole Quencher); UGV-1 L segment FWD 5´- TCATAAAAGCTTCTAGCTATTCTTTTCAT-3´, REV 5´- CAAGTTGGAGGCCCAAGAG-3´, and probe 5´-6-Fam- TGAAGTCTCCTCCAAGACCCTGGTTATCAG-3´-BHQ-1; HISV-1 S segment FWD 5´-CTCAAAATCTTACCGAAGTTGTATGTAC-3´, REV 5´-CACTTTCCCTTTTGGATCTTTG-3´, and probe 5´-6-Fam-GTGACGACCAAGTGTCGGGTCACAC-3´-BHQ-1; HISV-1 L segment FWD 5´-GAGTCTTTGTTTGATAATGGTGGTT-3´, REV 5´-ATTGAAGACTACAGAACCATATC-3´, and probe 5´-6-Fam-TCATTTGATTCAAGTGTTCTCAGGGCA-3´-BHQ-1 (Metabion International Ag). RNA isolation for Taqman assays was done with the GeneJET RNA purification kit (Thermo scientific) with carrier RNA following the manufacturer’s protocol. Taqman Fast Virus 1-step master mix (Thermo scientific) was used for qRT-PCR, 10 μl reactions were run with the AriaMX real-time PCR System (Agilent) in duplicate with recommended cycling conditions: 1) 50°C 5 min; 2) 95°C 20 s; 3) 95°C 3 s; 4) 60°C 30 s (steps 3 and 4 were repeated 39 times).

Transmission electron microscopy (EM) and immune-EM

Pellets from HISV-1 or UGV-1 infected I/1Ki cells were fixed in 1.5% glutaraldehyde, buffered in 0.2 M cacodylic acid buffer, pH 7.3 for 12 h at 5°C and embedded in epoxy resin. Toluidin blue stained semithin (1.5 μm) sections and, subsequently, ultrathin (100 nm) sections were prepared and the latter contrasted with lead citrate and uranyl acetate and examined with a Philips CM10 transmission electron microscope at 80kV.

For immuno-EM, cell pellets were fixed in 2.5% glutaraldehyde in 0.5xPBS and epoxy resin embedded. Thin sections (100 nm) were prepared and incubated for 30 min at RT in PBS with 1% BSA, followed by overnight incubation with rabbit anti-HISV NP antiserum (diluted 1:1,000 in PBS with 1% BSA) at 4°C. After washing with PBS, sections were incubated with 18 nm gold-conjugated goat anti-rabbit IgG antibody (Milan Analytica AG, Rheinfelden, Switzerland; diluted 1:20 in PBS with 1% BSA) for 2 h at RT. Sections were then contrasted and examined as descrived above.

Histology and immunohistology

Immunohistology with anti-HISV NP antiserum at 1:6,000 dilution was performed on formalin-fixed, paraffin embedded (FFPE) tissue sections, following previously published protocols [4, 12, 39]. Refer to Table 1 for animals examined. Consecutive sections incubated with a non-reactive rabbit polyclonal antibody instead of the specific primary antibody served as negative controls. A further section of each block was stained for reptarenavirus NP as described [4]. A FFPE pellet prepared from each an HISV- and reptarenavirus-infected cell sample served as positive control for the immunohistological stains. All snakes were also examined for any histopathological changes, using hematoxylin-eosin stained consecutive sections.

Accession numbers

The virus sequences obtained in this study are made available via GenBank, the accession numbers are provided in S1 Table.