Viruses in unexplained encephalitis cases in American black bears (Ursus americanus).

Viral infections were investigated in American black bears (Ursus americanus) from Nevada and northern California with and without idiopathic encephalitis. Metagenomics analyses of tissue pools revealed novel viruses in the genera Circoviridae, Parvoviridae, Anelloviridae, Polyomaviridae, and Papillomaviridae. The circovirus and parvovirus were of particular interest due to their potential importance as pathogens. We characterized the genomes of these viruses and subsequently screened bears by PCR to determine their prevalence. The circovirus (Ursus americanus circovirus, UaCV) was detected at a high prevalence (10/16, 67%), and the chaphamaparvovirus (Ursus americanus parvovirus, UaPV) was found in a single bear. We showed that UaCV is present in liver, spleen/lymph node, and brain tissue of selected cases by in situ hybridization (ISH) and PCR. Infections were detected in cases of idiopathic encephalitis and in cases without inflammatory brain lesions. Infection status was not clearly correlated with disease, and the significance of these infections remains unclear. Given the known pathogenicity of a closely related mammalian circovirus, and the complex manifestations of circovirus-associated diseases, we suggest that UaCV warrants further study as a possible cause or contributor to disease in American black bears.

Introduction

American black bears (Ursus americanus) are the most common and widely distributed bear species in North America. Categorized as a species of “Least Concern” by the International Union for Conservation of Nature, their total population in North America is estimated at approximately 850,000–950,000 and growing [1]. Their adaptability to suburban and urban environments—and the expansion of those environments—brings them into relatively frequent contact and conflict with human and domestic animal populations. A clear understanding of the diseases affecting black bears is critical to the management of these populations, including mitigation of outbreaks and potential pathogen spillovers. Black bears may be susceptible to various pathogens infecting domestic animals, including several important viruses such as Canine distemper virus, Canine adenovirus 1, and Canine parvovirus. Serology-based surveillance has established that these exposures are not infrequent [2-6]. However, efforts to correlate infection status with clinical disease have been much more limited. Sporadic case reports have described encephalitis in ursid species due to viral etiologies including Canine adenovirus 1, Canine distemper virus, or cross-species transmission of Equine herpesvirus 1 and 9 [7-10]. Cases of clinical disease from Rabies infection appear to be exceedingly rare in bears. In general, disease threats in this species are not well characterized, and the full range of viruses infecting black bears has yet to be thoroughly explored.

A series of cases of idiopathic encephalitis was observed in American black bears from Nevada (2014–2019). Lesions were suggestive of viral etiologies, but diagnostic efforts ruled out known etiologic agents based on histopathology and molecular techniques. Because viral disease was still considered most likely based on histopathologic lesions, we investigated potential viral etiologies by metagenomics analyses. We identified novel viruses in the families Circoviridae, Parvoviridae, Anelloviridae, Polyomaviridae, and Papillomaviridae. Of these, the circovirus and parvovirus were considered plausible causes of the observed neurologic lesions based on patterns of disease recognized in other species. Encephalitis and cerebellar vasculitis have been associated with Porcine Circovirus infections in pigs, and the human Parvovirus B19 has been associated with a spectrum of neurologic inflammatory lesions in human patients [11-13]. Anelloviruses are highly prevalent, but their association with disease has not been well-established and they are commonly considered to be commensal, asymptomatic infections. Viruses in the Polyomaviridae and Papillomaviridae families were considered unlikely causes of the observed lesions, and were not pursued. Given their potential pathogenicity, the novel circovirus and parvovirus were further investigated for prevalence by PCR. The novel circovirus was detected at high prevalence, and was further evaluated for tissue distribution and possible lesion association by in situ hybridization (ISH).

Materials and methods

Animals

The investigation included 17 yearling to adult black bears. Cases 1–8 were free-ranging animals from the Reno and Lake Tahoe areas of Nevada. Case 1 was euthanized because severe clinical disease was observed, and cases 2–7 were euthanized due to behavioral problems (human-bear conflict). Case 8 was killed by a car. For these cases, postmortem examinations took place at the Nevada Department of Agriculture, Animal Disease Lab in Sparks, Nevada. Collected tissues were submitted to the Oregon Veterinary Diagnostic Laboratory in Corvallis, Oregon as part of an investigation of a cluster of cases of non-suppurative encephalitis. That investigation identified a novel gammaherpesvirus, but a specific cause for neurologic disease was not identified [14].

Cases 9–12 were free-ranging bears either found dead or euthanized due to severe disease or human-wildlife conflict from El Dorado, Mendocino, and Santa Barbara counties in California. These cases were necropsied at the California Department of Fish and Wildlife’s Wildlife Investigations Laboratory, and tissues were submitted to the California Animal Health and Food Safety laboratory in Davis, CA for histopathology and ancillary testing.

Cases 13–16 had been housed in a wildlife sanctuary in northern California for 10–15 years prior to their deaths. Cases 13–15 were geriatric animals euthanized for progressive mobility difficulties associated with degenerative joint disease, and case 16 died spontaneously with non-suppurative encephalitis. These cases were necropsied at the UC Davis School of Veterinary Medicine. Case information and significant postmortem findings are summarized in Table 1.

Table 1

American black bear cases included in this investigation.

Case	Location (county)	Sex	Age	Significant post-mortem findings	Euthanized	Date of death or euthanasia
1	Washoe, NV	M	1y	Encephalitis	Y	30 Jan 2014
2	Washoe, NV	F	1y	Encephalitis	Y	17 Mar 2014
3	CA, near Reno, NV#	M	1y	Encephalitis	Y	7 Apr 2017
4	Washoe, NV	F	1y	Encephalitis, tooth root abscess	Y	21 Feb 2018
5	Douglas, NV	M	3y	Encephalitis	Y	9 Jul 2014
6	Reno/Tahoe, NV#	M	1y	Mild dermatitis	Y	24 Apr 2017
7	Douglas, NV	F	1y	Encephalitis	Y	2 Jul 2018
8	Reno/Tahoe, NV#	M	1y	Trauma (hit by car)	N	22 Aug 2018
9	Mendocino, CA	M	adult	Meningeal lymphoma	Y	8 Mar 2018
10	El Dorado, CA	M	11m	Hepatic necrosis, encephalitis	N	25 Dec 2018
11	El Dorado, CA	M	11m	Hepatitis, encephalitis (Sarcocystis)	N	15 Dec 2018
12	Santa Barbara, CA	M	adult	Nasal tumor	Y	20 Aug 2017
13	Calaveras, CA*	F	adult	Degenerative joint disease, nasal tumor	N	26 Dec 2017
14	Calaveras, CA*	M	adult	Degenerative joint disease	Y	9 Jul 2018
15	Calaveras, CA*	M	adult	Degenerative joint disease	Y	31 Jul 2017
16	Calaveras, CA*	F	adult	Encephalitis	Y	20 Jan 2011

*Sanctuary-housed.

#Specific county of origin unknown.

Metagenomic analyses

Frozen tissues were mechanically homogenized with a handheld rotor in 1 mL of PBS buffer and the homogenate was centrifuged at 9,000 rpm for 5 min. Supernatant (500 μl) was placed in a microcentrifuge tube with 100 μl of zirconia beads and quickly frozen on dry ice, thawed, and vortexed five times, then centrifuged for 5 minutes at 9,000 rpm. Tissue samples were pooled according to animal and not by tissue type. The supernatants were then passed through a 0.45 μm filter (Millipore, Burlington, MA, USA) and digested for 1.5 hours at 37°C with a mixture of nuclease enzymes consisting of 14U of Turbo DNase (Ambion, Life Technologies, USA), 3U of Baseline-ZERO (Epicentre, USA), 30U of Benzonase (Novagen, Germany) and 30U of RNase One (Promega, USA) in DNase buffer (Ambion, Life Technologies, USA) to enrich for viral particles. Nucleic acids were extracted immediately afterwards using the MagMAX™ Viral RNA Isolation kit (Applied Biosystems, Life Technologies, USA) according to the manufacturer’s instructions. Nucleic acids were incubated for 2 min with 100 pmol of random primer A (5’GTTTCCCACTGGANNNNNNNN3’) followed by a reverse transcription step using Superscript III (Invitrogen) with a subsequent Klenow DNA polymerase step (New England Biolabs). cDNA was then further amplified by a PCR step using AmpliTaq Gold™ (ThermoFisher Scientific) DNA polymerase LD with primer B (similar to primer A but minus the randomized 3’ end, or 5’GTTTCCCACTGGATA3’). The reaction (25 μL) contained with 2 μM of primer B, 1.85U of AmpliTaq Gold® DNA Polymerase (Applied Biosystems, Thermo Fisher, Waltham, MA, USA), 0.25 of mM dNTPs, 4 mM of MgCl2, 1× PCR Buffer, and 5 μL of cDNA template. The thermal profile for amplification was composed of 95°C for 5 min, 5 cycles of 95°C for 30 s, 59°C for 60 s, and 72°C for 90 s, 35 cycles of 95°C for 30 s, 59°C for 30 s, and 72°C for 90 s (+2 s per cycle) followed by 72°C for 10 min and hold at 4°C. The amplified product was checked by gel electrophoresis (approximately expected size 300–1000 bp), and one ng was used as target for Illumina library generation. The randomly amplified DNA products were quantified by Quant-iT™ DNA HS Assay Kit (Invitrogen, USA) using Qubit fluorometer (Invitrogen, USA). The library was generated using the transposon-based Nextera™ XT Sample Preparation Kit (Illumina, San Diego, CA, USA) and the concentration of DNA libraries was measured by Quant-iT™ DNA HS Assay Kit. The libraries were pooled at equal concentration and size-selected for a range of 300–1,000 bp using the Pippin Prep (Sage Science, Beverly, MA, USA). The library was quantified using the KAPA library quantification kit for Illumina platforms (Kapa Biosystems, USA) and a 10 pM concentration was loaded on the MiSeq sequencing platform for 2x250 cycles pair-end sequencing with dual barcoding. Human and bacterial reads were identified and removed by comparing the raw reads with human reference genome hg38 and bacterial genomes release 66 (collected from ftp://ftp.ncbi.nlm.nih.gov/blast/db/FASTA/, Oct. 20, 2017) using local search mode. The filtered sequences were de-duplicated if base positions 5 to 55 were identical. One random copy of duplicates was kept. The sequences were then trimmed for quality and adaptor and primer sequences by using VecScreen [15]. After that, the reads were de novo assembled by EnsembleAssembler [16]. Assembled contigs and all singlet reads were aligned to an in-house viral protein database (collected from ftp://ftp.ncbi.nih.gov/refseq/release/viral/, Oct. 20, 2017) using BLASTx (version 2.2.7) with E-value cutoff of 0.01. The significant viral hits were then aligned to an in-house non-virus-non-redundant (NVNR) universal proteome database using DIAMOND [17] to eliminate false positive viral hits. Hits with more significant E-value to NVNR than to viral database were removed. Remaining singlets and contigs were compared to all eukaryotic viral protein sequences in GenBank’s non-redundant database using BLASTx. The genome coverage of the target viruses was further analyzed by Geneious R11.1.4 software (Biomatters, New Zealand). Genome features were identified by visualization of expected protein sequences of canonical domains, and splice sites were predicted based on expected RNA transcripts.

PCR detection

For UaCV DNA detection in fresh tissue, DNA was extracted from tissue samples that were collected at necropsies and stored frozen (-80°C) until use, using the DNEasy Blood and Tissue Kit (Qiagen) according to the manufacturer’s instructions. A nested PCR (“Assay 1”) was used, consisting of two separate PCR reactions wherein the PCR product from the first round was used as template for the second reaction. The assay targeted part of the Rep gene, based on the contig discovered through metagenomic analysis, with the first-round primers S6_circo_322F (5’-GGCGGGGATTCAAGTGCTAT-3’) and S6_circo_651R (5’-TGGGTTCCCACAGGTAAAGC-3’) to amplify a 329-nt first round product, and second round primers S6_circo_356FN (5’-GGGGTAATTGGTGGGATGGG-3’) and S6_circo_625RN (5’-GCCCTTCATCCCAGGTAAGG-3’) to amplify a 269-nt second-round product. The PCR [containing a final concentration of 0.2 μm of each primer, 0.2 mM of dNTPs, 0.625 U of Amplitaq Gold® DNA polymerase (Applied Biosystems, Waltham, MA, USA), 1× PCR Gold buffer II, 1.5 mM of MgCl2 and 1 μL of DNA template in a 25 μl reaction] proceeded as follows: 95°C for 5 min, 40 cycles of (95°C for 30 s, (52°C for the first round and 54°C for the second round of primers) for 30 s, and 72°C for 30 s), followed by a final extension at 72°C for 7 min. PCR products of the correct size were verified by gel electrophoresis and Sanger sequencing.

For formalin-fixed, paraffin-embedded (FFPE) tissues, a separate, single-amplification assay (“Assay 2”) was used to amplify a 276-nt segment of the Rep gene using primers BearCV.92F (5’-CTGACCTTGAAGATGCCTGTAG-3’) and BearCV.368R (5’-CATACCCATCCCACCAATTACC-3’). This one-step assay amplifies a slightly smaller product than the first round of Assay 1, and was utilized to circumvent problems of viral genomic degradation due to formalin fixation. Multiple serial sections (scrolls) or single 5-μm-thick unstained sections scraped from slides of FFPE liver tissue were deparaffinized in xylene and graded alcohol washes, and DNA was extracted using the DNEasy Blood and Tissue Kit (Qiagen) according to manufacturer instructions. PCR targeting a housekeeping gene (GAPDH) was used to confirm successful DNA extractions. For UaCV “Assay 2,” reactions consisted of HotStarTaq Plus Master Mix (Qiagen), 5 pmol of each primer, 2.5 μL of dye, and approximately 25–50 ng of template DNA, diluted to a final volume of 25 μL. Cycling conditions were: 95°C for 5 minutes, followed by 40 cycles of 94°C (30s), 52°C (30s), 72°C (30s), and a final 72°C elongation step for 10 minutes. For visualization, products were run on 1.4% agarose gels containing Gel Red (Biotium).

In situ hybridization

To demonstrate viral genome in tissue sections, colorimetric in situ hybridization (ISH) was performed on 5-μm-thick sections of formalin-fixed, paraffin-embedded tissues on Superfrost Plus slides (Fisher Scientific, Pittsburgh, PA) using the RNAscope 2.5 Red assay kit (Cat #322360, Advanced Cell Diagnostics, Inc., Hayward, CA). We designed V-UaCirV, (ACD Cat #555001) as 26 ZZ-paired probe sets targeting a 1409 nt segment of the viral genome corresponding to nucleotide positions 478–1887 of the reference sequence (GenBank accession MN371255). Selected tissues included livers and spleens, as these are established sites of circoviral distribution in related species, as well as brains in order to evaluate the possible association with neurologic disease in these cases. Each 5-μm-thick tissue section was pretreated with heat and protease prior to probe hybridization for 2 hours at 40°C. Negative controls used for validation of signal included an unrelated (GC-content matched) probe on serial sections. Slides were counterstained with hematoxylin and mounted with EcoMount (Biocare Medical, Concord, CA).

Results

Metagenomics

Metagenomics analysis was performed on tissues from seven black bears (cases 1–6, 9). The analyzed tissues and results are summarized in Table 2. Metagenomic analysis revealed the presence of a novel circovirus in all but one pool, a polyomavirus in two pools, and a novel papillomavirus and a novel parvovirus each present in one pool. Anellovirus reads were detected in all sample pools. The 812 bp polyomavirus contig in case 3 showed 95% aa identity to the large T antigen of the giant panda polyomavirus (NC_035181.1), while the polyomavirus in case 9 had 74% identity to the Leptonychotes wedellii (Weddell seal) polyomavirus large T antigen (NC_032120). The 887 bp papillomavirus contig present in case 9 had 55% identity to L2 Canis familliaris papillomavirus (NC 013237). All raw reads were submitted to the short read archive under PRJNA564639.

Table 2

Viruses detected by metagenomics analyses.

Case	Tissue pools	Virus family	Reads per million
1	Kidney	Anelloviridae	17950
		Circoviridae	149
		Parvoviridae	169
2	Cerebrum	Anelloviridae	28
	Lymph node	Circoviridae	3
3	Cerebrum	Anelloviridae	774
		Circoviridae	39
4	Cerebrum	Anelloviridae	507
		Circoviridae	29
5	Cerebrum	Anelloviridae	3432
		Circoviridae	4
6	Cerebrum	Anelloviridae	243
	Lymph node	Polyomaviridae	12
9	Cerebrum	Anelloviridae	20827
		Circoviridae	2
		Papillomavirus	27
		Polyomaviridae	1

New parvovirus

A novel parvovirus, Ursus americanus Parvovirus (UaPV, accession MN166196) in the new genus Chaphamaparvovirus, was identified in the kidney and liver of case 1. The nearly complete genome is 3,787 bp containing three major open reading frames (ORFs), including a 660 aa non-structural protein (NS1), a 165 aa NP, and the 490 aa viral capsid (VP) (Fig 1). The NP was detected in two potential isoforms, including NS2-L (165 aa) and after splicing NS2-P (490 aa). BLASTp of these proteins revealed 73% identity in NS1, 76% identity in VP, 85% identity in NS2-L, and 76% identity in NS2-P to the recently described mouse kidney parvovirus (MKPV) that induces kidney disease in laboratory mice (MH670587) [18]. UaPV is therefore currently the closest relative of MKPV. The in silico predicted splicing is similar to that of MKPV based on the putative promotors, predicted splice sites, and polyadenylation signals. There is a conserved acceptor site just before the start of the VP ORF. Two polyadenylation sites were identified based on the conserved nature of these sites in other chaphamaparvoviruses. There are four putative promoter sites reported in this genome, however, the promoter located upstream of the VP start codon is conserved among chaphamaparvoviruses [19]. Putative exons include the NS1, two isoforms of the NS2, and VP.

Fig 1

A. The genome schematic of UaPV. Clear boxes represent untranslated regions, while the dashed lines represent splicing. The arrows represent ORFs. B. Maximum likelihood tree of NS1 aa chaphamaparvovirus sequences. Bar, 0.2 amino acid substitutions per site. Bootstrap values below 60 were removed.

New circovirus

The Ursus americanus circovirus (UaCV) genome (GenBank accession MN371255) is 2,054 bp with two ambisense open reading frames encoding capsid and replication-associated proteins (Fig 2). Based on full nucleotide sequence, it is most similar to a circovirus identified in a masked palm civet (Paguma larvata; Pl-CV8, GenBank accession LC416890.1), with which it shares 75.42% nt sequence identity. A BLASTp showed the Cap (260 aa) had 80% identity to Pl-CV8, and the Rep (287 aa) had a 72% identity to another masked palm civet circovirus sequence, Pl-CV9 (GenBank accession LC416391.1). UaCV contains a stem-loop motif between the intergenic region of the two ORFs consisting of a palindromic 15 bp stem, an 11 bp loop for the initiation of rolling-circle replication, and a 9 bp canonical nonamer (5’-TACTATTAC-3’) on the apex of the loop. The Rep contains 3 rolling circle replication motifs at the N-terminus including, motif I [CFTVNN], motif II [PHLQG], and motif III [YCKK]. The superfamily 3 helicase motifs located at the C-terminus of the UaCV replication-associated protein displayed a Walker-A motif [GPPGCGKT], a Walker–B motif [CLDD], and motif C [ITSN]. Similar to the PlCV, two introns were identified in the Rep encoding ORF at locations 47–96 and 363–505. A BLASTx of the Rep showed a 235 aa region that has a 51% identity to porcine circovirus 3 (PCV3-CN-AHB004-2018, accession MK178285.1).

Fig 2

A. Genome map of Ursus americanus circovirus (UaCV) including Rep (blue) and Capsid (purple) genes. B. Maximum likelihood trees of the Rep (left) and Capsid (right) aa circovirus sequences. Bar, 0.2 (Rep) and 0.5 (Capsid) amino acid substitutions per site. Bootstrap values below 60 were removed.

Prevalence

Having detected UaPV in one case and UaCV in multiple cases by metagenomics, we elected to screen additional black bears for these viruses by PCR. In total, tissues from 16 black bears were tested by PCR for circovirus and parvovirus. For cases 1–6, the same fresh tissue samples used for metagenomics were screened. For cases 7–16, only formalin-fixed, paraffin-embedded (FFPE) tissues were available. Parvovirus was detected in one case (index case 1). Circovirus was detected in 10/16 cases (62.5%) by one or both PCR assays. Results of PCR detection are summarized in Table 3.

Table 3

PCR detection of circovirus and parvovirus in black bear tissue samples.

Case	Encephalitis	Tissue	Fresh/FFPE	Circovirus PCR		Parvovirus PCR
				Result	Assay
1	Y	Kidney	Fresh	+	1	+
		Liver	Fresh	+	1	+
2	Y	Lymph node	Fresh	+	1	-
		Cerebrum	Fresh	-	1	-
3	Y	Lymph node	Fresh	-	1	-
		Cerebrum	Fresh	-	1	-
		Liver	FFPE	-	2	-
4	Y	Cerebrum	Fresh	+	1	-
		Liver	FFPE	-	2	-
5	Y	Cerebrum	Fresh	+	1	-
		Liver	FFPE	-	2	-
6	N	Cerebrum	Fresh	+	1	-
7	Y	Liver	FFPE	+	2	-
8	N	Liver	FFPE	-	2	-
9	N	Liver	FFPE	+	2	-
10	Y	Liver	FFPE	-	2	-
11	Y	Liver	FFPE	+	2	-
12	N	Liver	FFPE	-	2	-
13	N	Liver	FFPE	+	2	-
14	N	Liver	FFPE	+	2	-
15	N	Liver	FFPE	-	2	-
16	N	Liver	FFPE	-	2	-

Circovirus ISH

Given the high frequency of UaCV detection, we evaluated possible tissue targets of infection by in situ hybridization. Selected tissues from 12 cases were examined for circovirus by ISH. Positive probe hybridization was detected in 6 cases. Tissues examined included liver, spleen, and brain sections (5 cases), brain only (2 cases), liver only (2 cases), liver and spleen (2 cases), or lymph node only (1 case). Individuals and specific tissues tested were limited by availability of tissue blocks with adequate tissue preservation. Sections examined and results are summarized in Table 4.

Table 4

Summary of circovirus in situ hybridization testing and results.

Case	PCR result	PCR-positive tissue	In situ hybridization
			Liver	Spleen/Lymphoida	Brain
1	+	Kidney, liver	+	+	+*
2	+	Lymph node	-	-	-*
3	-		-	-	-*
4	+	Cerebrum	+	+	+*
5	+	Cerebrum	+	+	NT*
7	+	Liver	+	+	-*
8	-		-	-	+
9	+	Liver	NT	NT	-
10	-		-	NT	NT*
11	+	Liver	-	NT	NT*
12	-		NT	+ (LN)	NT
13	+	Liver	-	-	-

Positive UaCV ISH probe hybridization was detected in various tissues from a subset of cases. Positive results are highlighted in green.

NT = not tested due to lack of available tissue blocks or lack of adequate tissue preservation.

*Histologic diagnosis of encephalitis.

aSpleen tissue was tested whenever possible. In case 12, spleen tissue was unavailable but positive ISH probe hybridization was observed in other lymphoid tissues (lymph nodes).

Livers that were positive by ISH (4/10) exhibited punctate to diffuse cytoplasmic hybridization in individual cells in hepatic sinusoids (presumed Kupffer cells and/or endothelial cells). This pattern was seen diffusely throughout liver sections (Fig 3), and is consistent with patterns of circovirus detection in the livers of PCV-infected pigs [20] and in dogs infected with Canine circovirus (Pesavento lab, unpublished data).

Fig 3

ISH detection of UaCV nucleic acid in tissue sections.

Case 1. UaCV probes (A, D) and negative control (DapB) probes (B, E); hematoxylin counterstain. A. In sections of liver, UaCV ISH signal is detected in scattered polygonal cells (presumed Kupffer cells or endothelial cells) in hepatic sinusoids. Inset: higher magnification of a cell exhibiting positive probe hybridization. B. No signal is detected with negative control (DapB) probes. C. Immunohistochemistry for PCV2 in the liver of a pig demonstrates a similar pattern of immunoreactivity. (UC Davis SVM archives. IHC performed at California Animal Health and Food Safety Laboratory, Davis CA.) D. Sections of brain exhibit punctate UaCV ISH signal in and around small-caliber blood vessels. E. No signal is detected with negative control probes. Bars = 25μm.

In 5/9 cases, lymphoid tissues (spleen or lymph node) exhibited similar but more sparse punctate hybridization in individual cells. In spleens, signal was distributed throughout the tissue in the cytoplasm of scattered individual round cells (presumed lymphocytes and/or macrophages). Lymph nodes exhibited a similar pattern, with signal appearing most prominently in cells in the medullary sinuses and occasionally in cortices.

Brains were of particular interest due to a series of cases of unexplained encephalitis in black bears in the region. Ten of the cases in this study had postmortem diagnoses of mild to severe cerebral cortical inflammation characterized by multifocal to coalescing zones of mononuclear perivascular cuffing, edema, gliosis, hemorrhage, and necrosis. Sections of cerebrum from 8 cases were examined by ISH. Five of these cases had histologic evidence of encephalitis, and three did not. For animals with encephalitis, sections including inflammatory lesions were selected. Sparse positive hybridization was observed in brains from 3 cases—two with encephalitis and one without—occurring as scattered punctate intracellular or extracellular foci. To confirm positive probe hybridization above spurious background staining, two authors (CEA, TT) evaluated ISH slides and negative controls for these sections and tallied individual punctate foci of hybridization, noting their location with respect to blood vessels: vessel-associated (including luminal, endothelial, mural, and Virchow-Robbins space signal) or neuroparenchymal. Counts from the two reviewers were averaged to compare signal from circovirus-specific ISH slides with their corresponding negative controls. Cases were interpreted as positive when the number of puncta of specific probe hybridization far exceeded (>10x) background signal. In all three cases, most hybridization signal was seen in the lumen, endothelium, wall, or Virchow-Robbins spaces of small-caliber blood vessels, suggesting the possibility of endothelial or vascular mural infection. Affected vessels were distributed throughout the examined brain sections, without apparent co-localization to lesions of encephalitis. No clear association between infection and disease was established. No other tissues exhibited similar vessel-associated ISH signal. Counts of ISH signal foci by brain region in these cases are summarized in S1 Table.

Discussion

A metagenomic investigation of black bears identified novel viruses belonging to five genera: Circoviridae, Parvoviridae, Anelloviridae, Polyomaviridae, and Papillomaviridae. Of these, circovirus and anellovirus genomes were detected at high prevalence in the study cohort, while parvovirus, polyomavirus, and papillomavirus genomes were identified in 1–2 cases each. Although a definitive association of viral infection with encephalitis lesions was not established, our results expand the known diversity of viruses infecting black bears, and several of the identified viruses warrant further consideration as potential pathogens.

Circovirus infection was detected in 10/16 (62.5%) of cases tested, but the clinical significance of these infections remains to be established. Within Circoviridae, an 80% nucleotide sequence identity threshold is used for species demarcation [21]. Thus UaCV, which shares 75.42% nt sequence identity with its closest known relative (Pl-CV8), warrants classification as a novel viral species. While both UaCV and Pl-CV8 were identified in carnivore hosts (the latter in a masked palm civet), the two host species are not closely related and do not share a geographic range. There is no evidence to suggest a recent viral spillover from palm civet to black bear; rather, we speculate that UaCV has been enzootic but previously undetected in black bears. Retrospective studies of archived black bear cases from across their geographic range would be useful to clarify the evolutionary history and historical prevalence of UaCV in this population, as well as any association with disease.

The presence and distribution of UaCV sequences in spleens and livers, as demonstrated by PCR and ISH, is consistent with the behavior of known circoviruses in other mammalian hosts, including pathogenic circoviruses of pigs. However, the “classic” histologic lesion of circovirus infection derived from PCV2 studies, characterized by lymphoid depletion with histiocytic replacement and botryoid cytoplasmic inclusions [22], was not evident in these cases. Circoviral nucleic acid was detected by ISH in brains of 3 bears—two with encephalitis and one without—and the distribution in these sections was overwhelmingly vascular or perivascular. It is plausible that the positive ISH signal we observed in these sections indicates a viral tropism for endothelial cells, as has been demonstrated for other circoviruses, but no association could be made between the distribution of ISH signal and histologic brain or vascular lesions. In pigs, PCV2 is known to be endotheliotropic and has been associated with vascular lesions including lymphohistiocytic vasculitis and fibrinoid vascular necrosis, with PCV2 genome demonstrable in endothelium, mural myocytes, and perivascular/infiltrating leukocytes [12]. Although affected vessels may be found anywhere in the body, neurovascular (particularly cerebellar) involvement has been reported in a subset of cases [13]. Similarly, canine circovirus was demonstrated by ISH in (histologically normal) presumed endothelial cells in at least two cases that had evidence of necrotizing vasculitis in other tissues [23]. We speculate that the observed distribution of UaCV in bear brains could be consistent with infection of endothelial cells or vascular mural cells. Alternatively, in encephalitic bears, a compromised blood-brain barrier could allow leakage of virions or virus-infected cells from circulation, accounting for an observed vascular/perivascular distribution. However, the affected blood vessels were often histologically normal by routine staining, and in cases with encephalitis ISH signal was not obviously associated with areas of inflammation.

Several cases exhibited inconsistent circovirus testing results. Disparate results between PCR assays (cases 2, 4, and 5) may be attributable to differences in tissues tested (case 2) and/or degradation of genomic material in formalin-fixed, paraffin-embedded tissues (cases 4 and 5). Several PCR-positive cases were negative by ISH (cases 2, 11, and 13), possibly reflecting low viral load or variation in the tissue distribution of virus. Extended formalin fixation time or advanced autolysis are also plausible causes for loss of ISH signal in tissue sections. Two cases (8 and 12) were PCR-negative for circovirus but exhibited convincing probe hybridization by ISH. In these cases, tissues used for PCR differed from those examined by ISH, and the PCRs also utilized DNA from FFPE tissues, so tissue distribution, low viral concentration, and genomic degradation from prolonged fixation are also possible causes for these results.

The identification of a circovirus infecting black bears expands the known mammalian host range of the Circoviridae, and could provide new insight into the disease threats affecting this species. These infections were not definitively associated with pathologic findings. However, given the complicated manifestations of circovirus-associated diseases, we suggest that UaCV warrants further study as a possible cause or contributor to disease in American black bears, particularly (as with other pathogenic mammalian circoviruses) in the context of viral persistence, immunologic effects, and potentiation of/by co-infections. The pathogenic potential of UaCV and other viruses in this study could be influenced by co-pathogens and the general health and immune status of these bears.

Viruses in the genus Chaphamaparvovirus are a recent addition to the family Parvoviridae [19, 24]. Chaphamaparvovirus infections have been demonstrated to cause chronic kidney disease in laboratory mice, and a chaphamaparvovirus in domestic dogs has been suggested to play a role in diarrhea, but the full spectrum of chaphamaparvovirus-associated disease remains to be elucidated [18, 25]. Species demarcation criteria within Parvoviridae dictate that members of a given species share >85% amino acid sequence identity in the nonstructural (NS1) protein [24]. UaPV shares ~73% aa sequence identity with its closest phylogenetic neighbors (murine chaphamaparvoviruses), and thus warrants classification as a novel parvoviral species. UaPV was detected in one case in the present study, and its clinical significance is unknown. Given the known association of murine chaphamaparvoviruses with renal disease, further investigations of UaPV focused on renal tissue may be warranted. Sample availability precluded this possibility in the present study.

Anellovirus genomic material was found in all cases investigated by metagenomic analysis. Anelloviridae is a ubiquitous family of small, single-stranded, circular DNA viruses that are highly prevalent in the human population (>90%) and cause persistent, presumably life-long infections [26]. Anelloviruses are also ubiquitous in other mammals and generally considered commensal infections [27]. In pigs, Torque teno sus anellovirus viral loads are increased when co-infected with porcine circovirus 2 [28] and anellovirus loads also increase in immunosuppressed humans [29-36]. The possible contribution to neurologic lesions or other disease in these bears remains unknown, and the high rate of detection could reflect immune compromise in some of these cases.

The papillomavirus and the two distinct polyomaviruses identified in this investigation were also found in 1 and 2 cases, respectively, and were considered unlikely to have been associated with significant apparently clinical disease in this cohort.

In summary, we identified diverse novel viruses infecting black bears from California and Nevada. A clear relationship between infection status and neurologic lesions was not established for any of these viruses. However, this work expands the known diversity of viruses infecting free-ranging black bears in north America, and several of the identified viruses warrant further investigation as potential pathogens.

Foci of probe hybridization in cases exhibiting ISH signal in sections of brain.

Signal was distributed throughout sections, but predominantly identified in/around small-caliber blood vessels (lumen, endothelium, wall, or Virchow-Robbins space). Counts are averages from two reviewers.

(DOCX)

Click here for additional data file.

9 Jun 2020

PONE-D-20-14538

Viruses in unexplained encephalitis cases in American black bears (Ursus americanus)

PLOS ONE

Dear Dr. Pesavento

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

Many thanks for submitting your manuscript to PLOS One

Your manuscript was reviewed by two experts in the field, who have both made some suggestions for improvement prior to acceptance

If you could write a detailed response to reviewers, this will expedite review when it is resubmitted

I wish you all the best with your revisions

Hope you are keeping safe and well in these difficult times

Thanks

Simon

==============================

Please submit your revised manuscript by Jul 24 2020 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Simon Clegg, PhD

Academic Editor

PLOS ONE

Journal requirements:

When submitting your revision, we need you to address these additional requirements.

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. We note that you are reporting an analysis of a microarray, next-generation sequencing, or deep sequencing data set. PLOS requires that authors comply with field-specific standards for preparation, recording, and deposition of data in repositories appropriate to their field. Please upload these data to a stable, public repository (such as ArrayExpress, Gene Expression Omnibus (GEO), DNA Data Bank of Japan (DDBJ), NCBI GenBank, NCBI Sequence Read Archive, or EMBL Nucleotide Sequence Database (ENA)). In your revised cover letter, please provide the relevant accession numbers that may be used to access these data. For a full list of recommended repositories, see http://journals.plos.org/plosone/s/data-availability#loc-omics or http://journals.plos.org/plosone/s/data-availability#loc-sequencing.

3. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Partly

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: N/A

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Alex C, et al. describes in the manuscript entitled “Viruses in unexplained encephalitis cases in American black bears Ursus americanus” the identification of diverse viruses in fresh and formalized tissue samples from black bears of Nevada and California states. They have used a metagenomic approach for viral identification, and PCR and ISH for viral frequency determination, tissue distribution and possible pathological associations. Two putative new viruses from Circoviridae and Parvoviridae families have been detected and the nearly complete genome sequence described. Potential genomic structures and putative proteins have been predicted and the viral evolutionary history has been analyzed including representative viruses members.

The manuscript fits the journal scope, it is well structured, the results are clear and the figures are didactic and easy to understand, however, there are some concerns that must be addressed before proceeding.

Questions

1. Do you think that using viral RNA extraction kit in fresh samples may have affected your results, since you have described the detection of DNA viruses.

2. You are proposing the identification and description of two new viruses, the UaPV and UaCV. Can you please include the ICTV demarcation criteria for these two new viral species?

3. Don’t you need an Ethic committee approval for this work?

Minor comments

Material and Methods

Table 1. Can you include the collection date of the samples? It is a good information for time-spatial identification of viral presence;

Line 94. What dilution (weight/volume) were the samples prepared? How tissues were homogenized for tissue disruption? Mechanical, syringe, automated...? Please, include this information in the text;

Line 96. What do you mean by vortexed for five cycles? After that, was it again clarified at 9,000rpm during 5 min? If it was, please include this information in the text;

Line 101. Did you follow the manufacture instructions or and adaptation for RNA isolation? Please, include this information;

Line 103. Please, can you comment why you have used “primer A” for cDNA synthesis instead of generically random primers? Please, also comment why did you use only “primer A-short” for DNA amplification.

Line 131-134. Can you explain why did you used two round PCR for circovirus detection? What are the targets of these primers?

Line 142. Why did you use a different PCR assay (Assay 2) for circovirus detection in formalin-fixed tissue?

Results

Line 204 – 209 and 220 - 227. How did you predict all those features? Splicing sites, polyadenylation signals, promoter sites, ATP biding motif, stem-loop motif, rolling circle motif and all other motifs? Please, comment in Material and Methods.

Figure 2. Can you please include which color represents the Rep and Capsid ORF (Blue/purple)?

Table 4. Which tissue are you considering as Positive/negative for PCR in this table? Since we have different PCR results from different tissues in Table 3, it is not clear which tissue you are referring to. Please, clarify;

Writing suggestions

Line 53. “…We identified novel viruses from Circoviridae, Parvoviridae, Anelloviridae, Polyomaviridae, and Papillomaviridae families….”;

Line 56. Remove the sentence “This report describes the results of these investigations.” ;

Line 62. “… due to behavioral problems (human-bear conflict) or due to severe clinical disease observation (Case 1)…”;

Line 100. Remove “1x”;

Line 105. Please, include manufacturer of AmpliTaq Gold;

Line 112. “…using BLASTx (version 2.2.7) with E-value cutoff…”

Line 123. “The significant viral hits were then…”;

Line 124. “…to eliminate false positive viral hits.”;

Line 130. “For UaCV DNA detection in fresh tissue,…”;

Line 145. Can you change “Scrolls” for a more formal word? In addition, specify what is FFPE, since it is the first time of appearance;

Line 149. Please, include the Celsius degree symbol “°” in all temperatures;

Line 196. First time appearance of UaPV, please describe the whole name;

Line 379. “…increased when co-infected with pig circovirus 2…”;

Line 385. “…associated with significant apparently clinical disease…”;

Reviewer #2: Alex and Fahsbender et al., present a manuscript detailing an investigation of the virome of American black bears resident in Nevada and California using a high through put sequencing approach. Using this technique the authors found novel members of the Circo-, Parvo-, Anello-, Polyoma- and Papillomaviridae in the American black bear. The authors then examined the novel Circovirus and Parvovirus in more detail including experiments to evaluate if the novel Circovirus may contribute to cases of encephalitis with unknown causes. This paper contributes to our understanding of viral pathogens of this species, the data is freely available via NCBI, and the paper is generally well presented and readable.

Points to Address:

While the paper is readable and well presented the structure and language used infers a link between the novel ursine Circovirus and encephalitis that the data does not support and the authors state this conclusion clearly towards the end of the manuscript. For me the presentation of the data needs revision to remove this inferred link except where it is directly addressed experimentally to avoid ambiguity for the reader.

Could the authors comment more on why they chose to focus on the circovirus and parvovirus to the exclusion of the anello/polyoma/papillomaviruses? Circovirus, with sequences detected in many individuals was an obvious candidate to follow up, anellovirus sequences were detected in all tested individuals (often with the highest number of reads) but were not followed up, why? Polyomavirus sequences were detected in two individuals with papillomavirus and parvovirus in 1 case each. With this in mind could the rationale of choosing parvovirus rather than papillomavirus or polyomavirus be made clear? To this end revision of the introduction to present existing data on which this choice may have been based would be helpful, as would indication of if any of these agents have known links to encephalitis. Similarly more detail on known ursine viral pathogens (with or without links to encephalitis) would aid the reader in interpreting the significance of the authors work and choice in further investigating the novel ursine circo- and parvovirus.

The methods section would benefit from revision also:

Within the animals section it may aid the reader if the animals and handling of samples is presented in order ie origin of cases 1-8, then handling/processing, followed by the same for 9-16 to make this section easier to read.

Within the metagenomics section additional detail concerning sample preparation would assist in reproduction and interpretation. Additional detail on how samples were initially homogenised and why a second homogenisation with zircon beads was performed would be helpful. Clarity is also needed on if these are fresh, frozen or FFPE preserved samples, and if fresh how rapidly after isolation treatment occurred and any steps taken to inhibit endogenous nucleases.

Can the authors explain why they incubated their homogenate with a cocktail of 2 DNases, one broad spectrum nuclease and an RNase when seeking to purify viral RNA? Repeated freeze thaws and use of RNases may have decreased the author’s ability to isolate the genomes of novel ursine RNA viruses, and decreased transcript abundance of the DNA viruses identified. Also why isolate only RNA which for DNA viruses would not be protected at all from nucleases by encapsidation?

With all viruses except anelloviruses having low reads/million counts additional controls are necessary to show these are not contamination. Procedural control with blank samples to test for contamination from kits would be important in this context as would those to assess the survival of spiked in known (uncoated) RNAs/DNAs through homogenisation and nuclease treatment to see if these process account for the low read counts of most identified viruses.

Additional detail is needed for the cDNA synthesis, DNA amplification and library preparation stages, for example how much DNA is used, the source etc. How was library cDNA isolated and what concentrations were the pools, what sizes were selected?

For the PCR assays please make clear if the given primers are based on the authors own UaCV sequence or are degenerate based on other known viruses. Are the Nested primers used in a single reaction or in separate reactions, this isn’t clear – and if used in separate reactions how has the template DNA been treated between reaction with primer set 1 and 2? What is the source of tissue DNA in this context and how much is used to template the PCR? When using FFPE samples how much input material is used for DNA extraction?

For the ISH probes can coordinates for the target region be given with reference to the authors’ genomic sequence?

Results:

Please define what is meant by pools (L173/174). Can sequence comparison of the anellovirus data also be given here, and can data for the coverage of genomes be given? In table 2 does reads per million relate to short reads or contigs?

Can the authors make clear that Pl-CV9 is from a masked palm civet as Pl-CV8, and increase the size of the stem loop structure in Fig 2A to make it readable. Would the authors comment on the proximity of their novel ursine Circovirus to a circovirus of masked palm civet, do these animals share a geographical range? Is there brain tissue (as FFPE) available for performing the UaCV PCR on in cases 7-16 to investigate the putative link with encephalitis further?

In table 4 does not tested (NT) mean not available or not done?

For the ISH data where regions of brain were examined were these taken near to or from zones of encephalitis? The probe detects genomic DNA, probes targeting viral mRNAs may be more appropriate here to show active infection. Why is DapB used as a control and with multiplexing possible with this technology can an internal positive ISH control be used (against a host gene) to better assess the validity of the ISH detection of vDNA/viral mRNA. This technique can also be used quantitatively assess the levels of nucleic acid, have the authors considered using this approach particularly on their brain tissue sections to further support their conclusions?

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

25 Nov 2020

Thank you for the opportunity to revise this manuscript. Our specific responses to reviewers' questions and suggestions are below, and were also included with the submission as a separate Word document (from which the responses below were copied and pasted).

Reviewer #1: Alex C, et al. describes in the manuscript entitled “Viruses in unexplained encephalitis cases in American black bears Ursus americanus” the identification of diverse viruses in fresh and formalized tissue samples from black bears of Nevada and California states. They have used a metagenomic approach for viral identification, and PCR and ISH for viral frequency determination, tissue distribution and possible pathological associations. Two putative new viruses from Circoviridae and Parvoviridae families have been detected and the nearly complete genome sequence described. Potential genomic structures and putative proteins have been predicted and the viral evolutionary history has been analyzed including representative viruses members.

The manuscript fits the journal scope, it is well structured, the results are clear and the figures are didactic and easy to understand, however, there are some concerns that must be addressed before proceeding.

Questions

1. Do you think that using viral RNA extraction kit in fresh samples may have affected your results, since you have described the detection of DNA viruses.

The kit used (MagMAXTM Viral RNA Isolation kit) is based on both RNA and DNA binding to silica. Both RNA and DNA are purified despite the kit name with the DNA typically being removed by DNAse digestion. From one of the vendor’s web site:

The MagMAX™ Viral RNA Isolation Kit can efficiently isolate viral RNA and DNA from samples as large as 400 µl. RNA and DNA recovery is typically greater than 75%, but may vary depending on sample type. The RNA recovered with the kit is of high quality and purity and is suitable for real-time RT-PCR.

2. You are proposing the identification and description of two new viruses, the UaPV and UaCV. Can you please include the ICTV demarcation criteria for these two new viral species?

Species demarcation criteria for both viral families are now included in the discussion section of the revised manuscript.

3. Don’t you need an Ethic committee approval for this work?

An ethics statement has been added to the submission. This retrospective study was performed on stored/archived samples from diagnostic cases at the behest of the California Department of Fish and Wildlife (CDFW). No live animals were used to support this study; all samples were taken post-mortem from dead or dying animals. When euthanasia was performed it was either done under the auspices of the Wildlife Resource Agency for public safety or animal welfare concerns or by an attending veterinarian for animal welfare concerns. For tissues from CDFW cases, all were taken as part of the normal duties performed by the Wildlife Health staff and processed and examined in collaboration with other state agencies, laboratories (e.g. CAHFS), and research institutions (e.g. UCD).

Minor comments

Material and Methods

Table 1. Can you include the collection date of the samples? It is a good information for time-spatial identification of viral presence;

This information has been added to the table.

Line 94. What dilution (weight/volume) were the samples prepared?

How tissues were homogenized for tissue disruption? Mechanical, syringe, automated...? Please, include this information in the text;

We have now indicated in the text that homogenization was performed mechanically.

Line 96. What do you mean by vortexed for five cycles? After that, was it again clarified at 9,000rpm during 5 min? If it was, please include this information in the text;

Additional detail has been added to Materials and Methods to clarify this process: “Frozen tissues were mechanically homogenized with a handheld rotor in 1 mL of PBS buffer and the homogenate was centrifuged at 9,000 rpm for 5 min. Supernatant (500 µl) was placed in a microcentrifuge tube with 100 µl of zirconia beads and quickly frozen on dry ice, thawed, and vortexed five times, then centrifuged for 5 minutes at 9,000 rpm. The supernatants were then passed through a 0.45 µm filter (Millipore, Burlington, MA, USA) and digested for 1.5 hours at 37°C...”

Line 101. Did you follow the manufacture instructions or and adaptation for RNA isolation? Please, include this information;

Manufacturer suggestions were followed; this has now been indicated in the text.

Line 103. Please, can you comment why you have used “primer A” for cDNA synthesis instead of generically random primers? Please, also comment why did you use only “primer A-short” for DNA amplification.

Primer-A behaves in a very similar fashion to random hexamers due to its randomized 3’ end. “Primer-A short” is the same primer minus the randomized 3 prime and is used to further amplify the products of primer-A. “Primer-A-short” was renamed “Primer B” in the revised manuscript for clarity.

Line 131-134. Can you explain why did you used two round PCR for circovirus detection? What are the targets of these primers?

The nested approach was utilized for sensitive detection in tissues in which viral genetic material may have been present at low copy number. We have clarified in the manuscript that these primers target the Rep gene.

Line 142. Why did you use a different PCR assay (Assay 2) for circovirus detection in formalin-fixed tissue?

Assay 2 amplifies a smaller first-round product than the first step of Assay 1, so is potentially more sensitive for use in FFPE tissues where viral genomic DNA degradation could have impaired detection. A statement of this rationale was added to the methods sections.

Results

Line 204 – 209 and 220 - 227. How did you predict all those features? Splicing sites, polyadenylation signals, promoter sites, ATP biding motif, stem-loop motif, rolling circle motif and all other motifs? Please, comment in Material and Methods.

These features were detected based on visualization of the expected sequences and canonical domains. Splice sites were similarly detected based on expected RNA transcripts. A statement to this effect has been added to the Materials and Methods section.

Figure 2. Can you please include which color represents the Rep and Capsid ORF (Blue/purple)?

The figure legend was updated to include this information.

Table 4. Which tissue are you considering as Positive/negative for PCR in this table? Since we have different PCR results from different tissues in Table 3, it is not clear which tissue you are referring to. Please, clarify;

This information has been added to the table.

Writing suggestions

Line 53. “…We identified novel viruses from Circoviridae, Parvoviridae, Anelloviridae, Polyomaviridae, and Papillomaviridae families….”;

Line 56. Remove the sentence “This report describes the results of these investigations.” ;

Line 62. “… due to behavioral problems (human-bear conflict) or due to severe clinical disease observation (Case 1)…”;

Line 100. Remove “1x”;

Line 105. Please, include manufacturer of AmpliTaq Gold;

Line 112. “…using BLASTx (version 2.2.7) with E-value cutoff…”

Line 123. “The significant viral hits were then…”;

Line 124. “…to eliminate false positive viral hits.”;

Line 130. “For UaCV DNA detection in fresh tissue,…”;

Line 145. Can you change “Scrolls” for a more formal word? In addition, specify what is FFPE, since it is the first time of appearance;

Line 149. Please, include the Celsius degree symbol “°” in all temperatures;

Line 196. First time appearance of UaPV, please describe the whole name;

Line 379. “…increased when co-infected with pig circovirus 2…”;

Line 385. “…associated with significant apparently clinical disease…”;

All of these changes have been implemented in the revised manuscript.

Reviewer #2: Alex and Fahsbender et al., present a manuscript detailing an investigation of the virome of American black bears resident in Nevada and California using a high through put sequencing approach. Using this technique the authors found novel members of the Circo-, Parvo-, Anello-, Polyoma- and Papillomaviridae in the American black bear. The authors then examined the novel Circovirus and Parvovirus in more detail including experiments to evaluate if the novel Circovirus may contribute to cases of encephalitis with unknown causes. This paper contributes to our understanding of viral pathogens of this species, the data is freely available via NCBI, and the paper is generally well presented and readable.

Points to Address:

While the paper is readable and well presented the structure and language used infers a link between the novel ursine Circovirus and encephalitis that the data does not support and the authors state this conclusion clearly towards the end of the manuscript. For me the presentation of the data needs revision to remove this inferred link except where it is directly addressed experimentally to avoid ambiguity for the reader.

We appreciate and share the reviewer’s concern for clarity regarding disease causation. We have tried to be very cautious in asserting any link between viral infection and disease in these cases. We have included statements in the abstract and results of the revised manuscript to further clarify that no definitive disease association has yet been established.

Could the authors comment more on why they chose to focus on the circovirus and parvovirus to the exclusion of the anello/polyoma/papillomaviruses? Circovirus, with sequences detected in many individuals was an obvious candidate to follow up, anellovirus sequences were detected in all tested individuals (often with the highest number of reads) but were not followed up, why? Polyomavirus sequences were detected in two individuals with papillomavirus and parvovirus in 1 case each. With this in mind could the rationale of choosing parvovirus rather than papillomavirus or polyomavirus be made clear? To this end revision of the introduction to present existing data on which this choice may have been based would be helpful, as would indication of if any of these agents have known links to encephalitis. Similarly more detail on known ursine viral pathogens (with or without links to encephalitis) would aid the reader in interpreting the significance of the authors work and choice in further investigating the novel ursine circo- and parvovirus.

We have included more detail in the introduction regarding our rationale for pursuing the circovirus and parvovirus, and not the others. Additional detail was also included regarding viral pathogens in bears, including those linked to encephalitis.

The methods section would benefit from revision also:

Within the animals section it may aid the reader if the animals and handling of samples is presented in order ie origin of cases 1-8, then handling/processing, followed by the same for 9-16 to make this section easier to read.

This section has been re-structured according to this recommendation.

Within the metagenomics section additional detail concerning sample preparation would assist in reproduction and interpretation. Additional detail on how samples were initially homogenised and why a second homogenisation with zircon beads was performed would be helpful. Clarity is also needed on if these are fresh, frozen or FFPE preserved samples, and if fresh how rapidly after isolation treatment occurred and any steps taken to inhibit endogenous nucleases.

We have added details of the virus enrichment sample preparation. Procedure was used on thawed frozen samples. Here the goal is to enrich viral nucleic acids within viral particles. Both homogenization and the use of zircon beads and repeated freezing and thawing are methods we have found that help release viral particles from cells and tissues and allow then to pass through 0.45ul filter. Endogenous nucleases were not inhibited as the goal is to minimize the concentration of all nucleic acids except those protected from digestion within viral particles. We actually add nuclease enzymes before extracting particle-protected nucleic acids using kits that contain denaturants that will then denature these nucleases.

Can the authors explain why they incubated their homogenate with a cocktail of 2 DNases, one broad spectrum nuclease and an RNase when seeking to purify viral RNA? Repeated freeze thaws and use of RNases may have decreased the author’s ability to isolate the genomes of novel ursine RNA viruses, and decreased transcript abundance of the DNA viruses identified. Also why isolate only RNA which for DNA viruses would not be protected at all from nucleases by encapsidation?

The extraction kit used is based on nucleic acid binding to silica. Both RNA and DNA are purified. The RNA specificity claimed by the kit’s name (MagMax iral RNA isolation kit) requires use of DNAse AFTER extraction which we do not use. We therefore preferentially purify and extract both viral RNA AND DNA. Two DNAses and one RNAse are used to maximize digestion of host and bacterial nucleic acids in the homogenates while viral nucleic acids are protected from digestion within their viral capsids prior to their extraction. The initial use of reverse transcription to generate cDNA ensure that our methodology is able to amplify both RNA and DNA viruses as reported in prior publications.

With all viruses except anelloviruses having low reads/million counts additional controls are necessary to show these are not contamination. Procedural control with blank samples to test for contamination from kits would be important in this context as would those to assess the survival of spiked in known (uncoated) RNAs/DNAs through homogenisation and nuclease treatment to see if these process account for the low read counts of most identified viruses.

The metagenomics procedure was done to identify viral nucleic acids rather then to quantify them. We acknowledge the possibility that other viruses present at lower concentrations could have been missed. The presence of both the circovirus and the parvovirus were confirmed in new extracts by PCR.

Additional detail is needed for the cDNA synthesis, DNA amplification and library preparation stages, for example how much DNA is used, the source etc. How was library cDNA isolated and what concentrations were the pools, what sizes were selected?

Because the quantities of nucleic acids remaining after the extensive particle enrichment/purification steps are minimal we are typically unable to measure them by conventional methods and simply proceed with the random amplification as described. One ng of the random RT-PCR amplification step is used as target for the Illumina Nextera step as now added to the materials and methods.

For the PCR assays please make clear if the given primers are based on the authors own UaCV sequence or are degenerate based on other known viruses. Are the Nested primers used in a single reaction or in separate reactions, this isn’t clear – and if used in separate reactions how has the template DNA been treated between reaction with primer set 1 and 2? What is the source of tissue DNA in this context and how much is used to template the PCR? When using FFPE samples how much input material is used for DNA extraction?

We have clarified in the text that PCR primers were based on sequences generated by metagenomic analysis, and that the nested PCR consisted of two separate, sequential reactions, wherein the product of reaction 1 was used as template for reaction 2. We also clarified that DNA extracted from fresh tissue samples (stored frozen since necropsy) was used for these assays.

For assays on FFPE, DNA was extracted from tissue scrolls when possible, or from single unstained tissue sections scraped from glass slides. We confirmed successful DNA extraction using PCR targeting a housekeeping gene (GAPDH) and used a spectrophotometer to quantify extracted DNA to normalize the PCR reactions. The PCR reactions used approximately 25-50 ng of template DNA.

For the ISH probes can coordinates for the target region be given with reference to the authors’ genomic sequence?

This information was added to the Materials and Methods section.

Results:

Please define what is meant by pools (L173/174).

The text has been updated to reflect that tissue samples were pooled by animal, and not by tissue type.

Can sequence comparison of the anellovirus data also be given here, and can data for the coverage of genomes be given?

As a frequent infection generally considered to be a commensal we did not perform further analyses of the anellovirus sequences.

In table 2 does reads per million relate to short reads or contigs?

These numbers refer to short reads.

Can the authors make clear that Pl-CV9 is from a masked palm civet as Pl-CV8, and increase the size of the stem loop structure in Fig 2A to make it readable.

We have edited the text to clarify that Pl-CV9 is also from a masked palm civet. The figure has been adjusted as suggested.

Would the authors comment on the proximity of their novel ursine Circovirus to a circovirus of masked palm civet, do these animals share a geographical range?

We have included comments on the relationships between UaCV and the palm civet circovirus in the revised manuscript, and have also indicated that there is no overlap in geographic ranges of these two carnivore hosts. The palm civet sequences were detected from animals in Japan.

Is there brain tissue (as FFPE) available for performing the UaCV PCR on in cases 7-16 to investigate the putative link with encephalitis further?

Particularly for brain tissue, there were some inconsistencies among cases in terms of autolysis, samples collected, and duration of formalin fixation that could affect PCR results. Given that no obvious link was established – which we have endeavored to make clear in the manuscript – we elected to forego UaCV PCR testing of FFPE brain tissue in favor of testing prospectively collected samples in which sampling and sample handling are more controlled. We anticipate that this will allow for a more rigorous assessment of viral presence/association with lesions. This work is in progress.

In table 4 does not tested (NT) mean not available or not done?

NT indicates tissues that were either not available for testing (no available tissue blocks) or tissues with significant autolysis that would have impeded ISH detection or interpretation.

For the ISH data where regions of brain were examined were these taken near to or from zones of encephalitis? The probe detects genomic DNA, probes targeting viral mRNAs may be more appropriate here to show active infection. Why is DapB used as a control and with multiplexing possible with this technology can an internal positive ISH control be used (against a host gene) to better assess the validity of the ISH detection of vDNA/viral mRNA. This technique can also be used quantitatively assess the levels of nucleic acid, have the authors considered using this approach particularly on their brain tissue sections to further support their conclusions?

In cases of encephalitis, sections of brain including inflammatory lesions were selected for ISH, although the degree of inflammation and extent of affected tissue did vary between section. We use DapB probes as a control to evaluate levels of spurious, non-specific background staining. Our confidence in the determination of positive signal is based on detection of hybridization in the context of biologically (anatomically) sensible distribution, as evaluated by board-certified veterinary pathologists. For this initial characterization, we feel confident in our interpretation of circoviral presence in these tissues using these methods. We agree that viral mRNA probes, double-labeling, and quantitative methods would provide further support for the presence of active circovirus infection. Additional prospective circovirus studies are planned to further evaluate the possible link between circovirus and encephalitis lesions in black bears, and some of these techniques may be incorporated in that work to support any observed link.

Submitted filename: Response to reviewers.docx

Click here for additional data file.

3 Dec 2020

Viruses in unexplained encephalitis cases in American black bears (Ursus americanus)

PONE-D-20-14538R1

Dear Dr. Pesavento,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Simon Clegg, PhD

Academic Editor

PLOS ONE

Additional Editor Comments:

Many thanks for resubmitting your manuscript to PLOS One

As you have addressed all the comments, and the manuscript reads well, I have recommended the manuscript for publication

You should hear from the Editorial office soon

It was a pleasure working with you, and I wish you the best of luck for your future research

Hope you are keeping safe and well in these difficult times

Thanks

Simon

7 Dec 2020

PONE-D-20-14538R1

Viruses in unexplained encephalitis cases in American black bears (Ursus americanus)

Dear Dr. Pesavento:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

If we can help with anything else, please email us at plosone@plos.org.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Simon Clegg

Academic Editor

PLOS ONE