A novel Betaretrovirus discovered in cattle with neurological disease and encephalitis

Case information

The BoRV CH15–positive cattle investigated in this study (cases 1–7) originated from different regions of Switzerland and Austria, and were identified by postmortem neuropathological diagnostics conducted over > 20 years (1996–2020) within the framework of surveillance programs for neuroinfectious diseases. Cases 1, 4, 5, 6, and 7 were clinically suspicious for bovine spongiform encephalopathy (BSE) and were thus submitted to reference laboratories (= BSE suspect cases). Cases 2 and 3 died on the farm or were euthanized for reasons other than human consumption (= fallen stock). All animals were dairy or suckler cows of various breeds. These animals were relatively old, with ages ranging from 8 to 18 years. In case 3, information about clinical signs was not available. In the remaining cases, neurological signs were reported, but with variable manifestations and severities. In animals with reported neurological signs, non-suppurative encephalitis was diagnosed by histopathological examination (Table 1).

Table 1 Overview of bovine retrovirus CH15–positive casesMolecular diagnostics and sequencing results

The determination of the coding-complete BoRV CH15 genomes in cases 1 and 2 using HTS has been described previously [8, 9]. The resulting scaffolds showed a typical retrovirus genome structure with open reading frames (ORFs) for the putative group-specific antigen (gag), the putative protease (pro), the putative reverse transcriptase, RNase H and integrase (pol), and the putative envelope protein (env), and with sequence similarities of encoded proteins to members of the genus Betaretrovirus. For the remaining cases, diagnostic HTS was performed on brain-tissue RNA extracts. Generated read numbers ranged from ~ 73 Mio to ~ 150 Mio; in cases 4–7, nearly the full BoRV CH15 coding sequence was covered (Additional file 1). Remaining sequencing gaps were filled by RT-PCR and subsequent Sanger sequencing to obtain coding-complete BoRV CH15 genomes.

HTS of case 3 did not yield scaffolds with similarities to BoRV CH15 in our de novo assembly pipeline, which analyzes scaffolds of > 499 base pairs (bp). However, mapping of the reads to the BoRV CH15 genome showed that ~ 30% of the genome was covered, with reads mapping primarily to seven regions of 130–280 bp (three in the gag ORF, one in the pro ORF, two in the pol ORF, and one in the env ORF). Thus, viral RNA was present, but in a relatively small amount.

With rapid amplification of cDNA ends (RACE) on RNA in cases 1 and 2, we determined the authentic 3' end of the BoRV CH15 RNA genome in these cases. 5' RACE did not yield robust results.

To confirm that the BoRV CH15 genomes were integrated into the host genome, we performed PCRs targeting the gag, pol, and env ORFs in extracted DNA with previously published primer pairs [8]. In all cases, including case 3, all three PCR runs yielded clearly positive results, whereas those for non-template controls and negative control animals with non-suppurative encephalitis but BoRV CH15 negativity remained negative. We then determined the entire coding sequence of the BoRV CH15 proviral genome in brain tissue DNA extracts of case 3 by Sanger sequencing of overlapping PCR amplicons.

To complete the long terminal repeats (LTRs) at the 5' and 3' ends of the proviral DNA genomes, sites of BoRV CH15 integration into the Bos taurus genome in case 1 were analyzed using the DNA HTS paired-end dataset generated previously. We identified six read pairs with one mate mapping to the Bos taurus genome (at six different sites) and the other mate mapping to the BoRV CH15 genome (Additional file 2). PCR amplification of regions between paired reads with read-specific primers failed. However, one HTS read bridged the 5' integration site (Additional file 2), which allowed us to determine the authentic 5' end of the integrated virus. With this information, we could establish the authentic 3' end of the integrated virus, as both LTRs of the provirus have identical sequences in retroviruses (Fig. 1A).

Fig. 1figure1

The bovine retrovirus CH15 proviral genome, sequencing strategy, and long terminal repeat (LTR) characteristics. A The coding-complete BoRV CH15 genome was determined by high-throughput sequencing (HTS) and Sanger sequencing. With 3' rapid amplification of cDNA ends (RACE), the LTR was sequenced to the redundant (R) region. With an HTS read pair overspanning the proviral 5' end and mapping to the Bos taurus genome, the missing bases of the 5' LTR could be determined. Because the LTRs have an identical sequence, the full-length proviral 3' LTR also could be determined. Green arrow-boxes represent the open reading frames of the group-specific antigen (gag), protease (pro), reverse transriptase, RNase H and integrase (pol), and envelope (env) proteins. Numbers represent bases in the viral genome. Purple, orange, and blue boxes depict the unique 3' (U3), R, and unique 5' (U5) regions of the LTRs, respectively. B Gray arrow-boxes represent the flanking regions of the LTRs: the primer-binding site (PBS) and polypurine tract (PPT). Pink boxes represent regulatory elements in the U3 region, the TATA-box (TATA) and the poly(A) signal (poly(A)). Inverted repeats (IR) are depicted by red triangles

With primers binding the determined LTRs and the conserved primer-binding site (PBS) region, we were able to define the complete LTRs for the BoRV CH15 provirus genomes in cases 4–7. Some uncertainty remained regarding the LTRs of the BoRV CH15 genomes in cases 5 and 7. Sanger sequencing data suggest no uniform length for the integrated unique 3' (U3) region in these animals, with four possible insertions of 42–58 and 67–167 bp, respectively, in the BoRV CH15 genomes (Additional file 3). Insertions with good sequencing quality suggest duplications of U3 sequence elements. On blastn analysis, none of these inserts revealed a similarity to sequences of the bovine reference genomes or other available bovine sequences. As the LTRs did not show these insertions in the majority of PCR and Sanger sequencing runs of these cases, the proviral sequences without insertions in the LTRs were reported to GenBank.

Because the binding of cellular transcription factors is a basic principle in retrovirus replication [26, 27], the U3 regions of the LTRs in all cases were analyzed in silico for putative transcription factor–binding sites. This analysis revealed the presence of a nuclear factor 1 (NF-1) binding site shared by all of the Swiss BoRV CH15 sequences (Additional file 4). The sequence of the Austrian case 5 contained an insertion potentially disrupting this NF-1 site. An NF-1 site was also present in this sequence, but it was farther downstream than those in the Swiss sequences.

To assess sequence heterogeneity in different BoRV CH15 genomes (case 1 and cases 4–7), we remapped the raw reads to the complete genomes. Between 91 and 100% of the genomic sequences were covered by reads, with some uncovered nucleotides at the 5' and 3' ends in BoRV CH15 genomes in cases 1 and 4. The average coverage depth was > 65× and the average pairwise identities calculated over all reads per position was in all cases > 98.4%, indicating a high level of sequence similarity within the different animals.

Bovine retrovirus CH15 strain genomes

In the seven BoRV CH15 strains sequenced, the proviral genomes ranged from 7′661 to 8′180 nucleotides (nt) in length, with a genomic organization typical of members of the genus Betaretrovirus (Table 2). All ORFs (gag, pol and env) were conserved and without apparent premature stop codons. Whereas the ORFs encoding the putative Gag, Pol, and Env proteins showed only minor variation in length, the pro-encoding ORF lengths ranged from 507 to 636 nt. The env and pol ORFs overlapped by 110 nt in all strains. We annotated the LTRs based on annotations published by Cousens et al. [28] for the enzootic nasal tumor virus. The total lengths were 348–607 nt. The U3 region lengths differed among viral genomes, ranging from 252 to 511 nt and containing important regulatory elements such as the TATA box and the poly(A) signaling sequence. The U3 region overlapped with the env ORF by 23 nt at the viral 3' end. In all BoRV CH15 genomes, the redundant (R) and unique 5' (U5) region lengths were 36 and 60 nt, respectively, and the LTR-flanking inverted repeats (IRs) were TTG and CAA. The LTRs were flanked by the PBS (TGGCGCCCGAACAGGGAC) at the 5' end and by the polypurine tract (PPT, AAAAAGAAAAAAGGGGGAA) at the 3' end (Fig. 1B).

Table 2 Putative genome-element lengths (nt) of seven bovine retrovirus CH15 strainsPhylogenetic analysis and sequence comparison of bovine retrovirus CH15 strains

Phylogenetic analysis was performed with the coding regions (start of the gag ORF to end of the env ORF) of selected exogenous virus genomes of the genera Betaretrovirus, Lentivirus, Gammaretrovirus, Deltaretrovirus and Bovispumavirus, as well as the env mRNA of two bovine endogenous retroviruses. In a maximum-likelihood phylogenetic tree, all BoRV CH15 genomes show an affiliation to the genus Betaretrovirus and neither to known neuroinvasive viruses belonging to genera Lentivirus, Gammaretrovirus, and Deltaretrovirus, nor to bovine endogenous retrovirus sequences (Fig. 2). Overall, the closest related virus was the Jaagsiekte sheep retrovirus (accession no. NC_001494), with sequence identities of 47.9–49.7% to the different BoRV CH15 strains. The BoRV CH15 genomes clustered closely together and showed an overall nucleotide identity of 88.5–98.8% to each other comparing the coding sequence (nucleotide sequence from the start of the gag ORF until the end of the env ORF) and an overall identity of the concatenated translated protein sequences of 91.9–99.2%. The genomes generated from cases 3 and 4 had the closest relationship and those generated from cases 5 and 6 were most diverse (Table 3). Comparison of individual ORF sequences and elements of the LTRs similarly reflected these relationships (Additional file 5).

Fig. 2figure2

Phylogenetic analysis showed the affiliation of all bovine retrovirus CH15 genomes to the Betaretrovirus genus. The maximum-likelihood tree was based on selected coding-complete exogenous virus sequences of the genera Betaretrovirus, Lentivirus, Gammaretrovirus, Deltaretrovirus, and Bovispumavirus, as well as the env mRNA of bovine endogenous retroviruses. Branches are labeled by GenBank accession number and virus name. The scale indicates the number of substitutions per site, reflected by the branch lengths. Unclassified betaretroviruses are marked with an asterisk. CH, Switzerland; AT, Austria

Table 3 Pairwise sequence identity (%) of nucleotide coding sequences and concatenated protein sequences of bovine retrovirus CH15 strains [nt/aa]Prevalence of bovine retrovirus CH15 in the brains of healthy slaughtered animals

To assess the association of BoRV CH15 infection to disease, 130 fresh-frozen medulla oblongata samples from healthy slaughtered cattle without histopathological lesions in the brain were tested for the presence of BoRV CH15 provirus. These control animals were of different breeds (Fleckvieh n = 51, Brown Swiss n = 32, Holstein n = 26, Simmental n = 6, Limousin n = 4, mixed breed n = 5, unknown breed n = 6) and had a mean age of 6 years. The minimum and maximum age of the control animals were 3 and 16 years, respectively, with the 1st quartile of 4 years and the 3rd quartile of 8 years. After DNA extraction, we assessed the DNA extraction efficiency and the presence of putative PCR inhibitors by qPCR targeting the bovine housekeeping gene 12s rDNA. DNA extracts had Cq values between 10.3 and 24.4 and thus extraction was evaluated as successful. On these DNA extracts (n = 130), we performed a PCR assay with a previously published primer pair amplifying a 500-bp-long fragment of the gag ORF [8] of BoRV CH15. All 130 DNA extracts were negative.

Neuropathological features in bovine retrovirus CH15–positive animals

All cases showed clear non-suppurative encephalitis, with the exception of case 3, which had no histological abnormality in the caudal brainstem, the only tissue available. Non-suppurative encephalitis was characterized by gliosis and mononuclear cell infiltrates, the latter presenting mainly as perivascular cuffs (Fig. 3A, C, D). These lesions had a multifocal distribution. In one animal (case 1), neuronotropic lesions comprised of glial nodules around neurons, a typical manifestation of neuronal viral infections, were present (Fig. 3B). In two animals (cases 1 and 7), mononuclear cells were not only located perivascularly in the Virchow-Robin space, but also infiltrated the vessel wall, i.e., these animals exhibited lymphohistiocytic vasculitis (Fig. 3C). Additionally, severe and acute ecchymotic and ring hemorrhages were seen multifocally in the brain of one of these animals (case 7; Fig. 3D). The cause of these lesions could not be determined clearly; they could have occurred secondary to vascular changes, but also partly due to captive-bolt stunning of the animal. In two animals (cases 2 and 6), severe diffuse neuronal degeneration was present, manifesting with chromatolytic, swollen, and eosinophilic neurons (Fig. 3E). In case 6, for which multiple brain regions were available for histopathological examination, degenerated neurons were found in the motoneurons of cranial nerve, basal, red, and thalamic nuclei, as well as in the Purkinje cells. Additionally, peri- and intraneuronal vacuolization was observed in the cerebral cortex. Neuronal changes were multifocal, but not always associated with perivascular cuffs, and gliotic nodules surrounding the altered neurons were absent. Taken together, these findings suggest a metabolic-toxic event as the cause of the widespread neuronal degeneration. In two other animals (cases 5 and 7), hippocampal sclerosis with chromatolysis and the degeneration of pyramidal cells surrounded by hypertrophic astrocytes was observed (Fig. 3F). These lesions were not in association with perivascular infiltrates. An overview of lesion types in the animals examined in this study is provided in Fig. 4.

Fig. 3figure3

Bovine retrovirus CH15–positive animals showed non-suppurative encephalitis on histopathological examination. Formalin-fixed paraffin-embedded brain tissue slides were stained with hematoxylin and eosin. A Perivascular infiltrates of mononuclear cells forming a cuff around a vessel; case 6, hypothalamus. B Nodular gliosis (arrows) around neurons; case 1, medulla oblongata. C Perivascular cuff with infiltration of mononuclear cells in the vessel wall (arrows); case 7, medulla oblongata. D Severe ecchymotic and ring hemorrhages, often in association with perivascular cuffs (asterisk); case 7, thalamus. E Chromatolytic, swollen, and eosinophilic motoneurons (arrowheads); case 6, medulla oblongata. F Eosinophilic and shrunken pyramidal cells with loss of Nissl substance (arrows) surrounded by hypertrophic astrocytes (arrowheads); case 7, hippocampus

Fig. 4figure4

Overview of lesion types observed this study. Case numbers are in colored circles. BoRV CH15, bovine retrovirus CH15; BSE, bovine spongiform encephalopathy

We performed immunohistochemistry (IHC) with antibodies labeling CD3+ T-lymphocytes to demonstrate the involvement of the adaptive immune system in non-suppurative encephalitis in the cases under investigation. High numbers of CD3+ T-lymphocytes contributed to perivascular cuffs in four animals (cases 1, 5, 6, and 7; Fig. 5A and B) and were additionally found in the surrounding parenchyma in diffuse and nodular gliosis (Fig. 5C and D). The involvement of the adaptive immune system in case 6, in which lesion type and distribution suggest the occurrence of a metabolic-toxic event and not the involvement of pathogens (Fig. 5A), was unexpected. In the perivascular cuffs of the other two animals (cases 2 and 4), however, the predominant cell type was not T-lymphocytes, but ionized calcium-binding adapter molecule 1 (Iba-1)-positive macrophages (Fig. 5E and F).

Fig. 5figure5

T-lymphocytes and macrophages contributed in high numbers to perivascular cuffs and gliosis. AD Immunohistochemical analysis was performed with staining with an antibody targeting CD3. Positivity (red granular staining) is visible in the cytoplasm of CD3 + T-lymphocytes in perivascular cuffs (A case 6, medulla oblongata; B case 5, thalamus), diffuse gliosis (C case 6, medulla oblongata), and nodular gliosis (D case 6, hypothalamus). E, F Immunohistochemical analysis was performed with staining with an antibody targeting ionized calcium-binding adapter molecule 1. Positivity (red granular staining) is visible in the cytoplasm of cells in perivascular cuffs and ramified cells in the neuroparenchyma (microglia; E case 2, medulla oblongata; F case 4, thalamus)

In situ detection of bovine retrovirus CH15

For in situ detection of BoRV CH15 RNA, fluorescent and chromogenic in situ hybridization (ISH) was performed on formalin-fixed, paraffin-embedded (FFPE) brain tissue slides. The probe used for ISH targeted the gag ORF; more details can be found in the materials and methods section. For cases 2 and 3, only medulla oblongata material was available. For the remaining cases, brain regions showing typical signs of non-suppurative encephalitis on hematoxylin and eosin (H&E) staining were used. Fluorescent ISH enabled the detection of viral RNA with a clear cytoplasmic distribution in five animals (cases 1, 2, 4, 6, and 7; Fig. 6A–C). In one of these animals (case 6), the morphology of the infected cells was consistent with neurons (Fig. 6B). To assess the infected cell type in the remaining positive animals, chromogenic ISH and counterstaining with Mayer's hemalum solution were performed. This analysis enabled the detection of BoRV CH15 RNA, mostly in cells morphologically compatible with neurons (Fig. 7A–C) and in cellular extensions of neurons (Fig. 7D). Labeling was located in brain areas with lesions of non-suppurative encephalitis, but with no clear topographic association of nodular or diffuse gliosis with BoRV CH15 RNA. BoRV CH15 RNA labeling was absent in highly eosinophilic and chromatolytic neurons in cases 2 and 6 (Fig. 7E), in which metabolic-toxic events were believed to have caused the pathological changes, and in pyramidal cells of the hippocampus in cases 5 and 7 (Fig. 7F), although neuronal extensions of presumably granular cells in the hippocampus were strongly positive in case 7 (Fig. 7D). In two animals (cases 3 and 5), we could not identify BoRV CH15 RNA in situ in the available material. Negative control FFPE tissue slides from animals with non-suppurative encephalitis and BoRV CH15 negativity remained negative in both ISH staining analyses.

Fig. 6figure6

Detection of bovine retrovirus CH15 (BoRV CH15) RNA in situ in the cellular cytoplasm. Fluorescent in situ hybridization of formalin-fixed, paraffin-embedded tissue slides using the BoRV CH15 RNAscope probe. Nuclei are stained in blue, BoRV CH15 RNA is stained in red. A Case 1, medulla oblongata; B case 6, cerebral cortex; C case 7, medulla oblongata. The morphology of the positive cell in B is consistent with a neuron

Fig. 7figure7

Detection of bovine retrovirus CH15 (BoRV CH15) RNA in cells morphologically compatible with neurons. Chromogenic in situ hybridization of formalin-fixed, paraffin-embedded tissue slides using the BoRV CH15 RNAscope probe. BoRV CH15 RNA (red granular staining) is visible in the cytoplasm of cells and cellular extensions morphologically compatible with neurons. The neurons appear to be morphologically normal, with the maintenance of Nissl substance, and inflammatory cells are absent from the immediate surroundings of positive neurons. A Case 1, medulla oblongata; B case 6, hypothalamus; C case 6, cerebral cortex; D case 7, hippocampus. Neurons with clear signs of necrosis (chromatolytic and eosinophilic cytoplasm, eccentric nuclei, loss of Nissl substance) on hematoxylin and eosin staining (insets) remained negative for BoRV CH15 RNA. E Case 6, medulla oblongata; F case 7, hippocampus

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