Relatives of rubella virus in diverse mammals.

Since 1814, when rubella was first described, the origins of the disease and its causative agent, rubella virus (Matonaviridae: Rubivirus), have remained unclear1. Here we describe ruhugu virus and rustrela virus in Africa and Europe, respectively, which are, to our knowledge, the first known relatives of rubella virus. Ruhugu virus, which is the closest relative of rubella virus, was found in apparently healthy cyclops leaf-nosed bats (Hipposideros cyclops) in Uganda. Rustrela virus, which is an outgroup to the clade that comprises rubella and ruhugu viruses, was found in acutely encephalitic placental and marsupial animals at a zoo in Germany and in wild yellow-necked field mice (Apodemus flavicollis) at and near the zoo. Ruhugu and rustrela viruses share an identical genomic architecture with rubella virus2,3. The amino acid sequences of four putative B cell epitopes in the fusion (E1) protein of the rubella, ruhugu and rustrela viruses and two putative T cell epitopes in the capsid protein of the rubella and ruhugu viruses are moderately to highly conserved4-6. Modelling of E1 homotrimers in the post-fusion state predicts that ruhugu and rubella viruses have a similar capacity for fusion with the host-cell membrane5. Together, these findings show that some members of the family Matonaviridae can cross substantial barriers between host species and that rubella virus probably has a zoonotic origin. Our findings raise concerns about future zoonotic transmission of rubella-like viruses, but will facilitate comparative studies and animal models of rubella and congenital rubella syndrome.

Rubella, first described in 1814[7], is an acute, highly contagious human infectious disease typically characterized by rash, low-grade fever, adenopathy, and conjunctivitis[1]. Research from the 1940s to 1960s revealed that rubella (also called “German measles”) contracted during the first trimester of pregnancy was directly associated with severe congenital birth defects, miscarriage, and stillbirth[8,9]. Rubella virus (RuV), currently the sole recognized member of the riboviriad family Matonaviridae (genus Rubivirus), is the etiologic agent of rubella[10,11] and causes fetal pathology after transplacental transmission[12]. Extensive rubella epidemics have occurred worldwide due to the high airborne transmissibility of RuV (R = 3.5–7.8)[13]. Safe, efficacious, live-attenuated RuV vaccines, including the measles/mumps/rubella (MMR) vaccine, are now deployed worldwide and have successfully decreased global rubella incidence. However, ≈100,000 cases of congenital rubella syndrome still occur annually[1], and RuV can persist in immunologically privileged anatomic sites (e.g., the eye) for years[14]. Furthermore, RuV infection of adults is generally underreported, with 30–50% of adult cases being subclinical[15]. High-priority areas for rubella vaccination include the Western Pacific, Eastern Mediterranean, and African regions, where RuV circulates widely and primarily infects young children[16]. RuV elimination is considered rapidly achievable because of the effectiveness of available vaccines and the lack of known animal reservoirs[17,18].

Here we report the discovery of ruhugu virus (RuhV) and rustrela virus (RusV), the first known relatives of RuV. RuhV was found in 10 of 20 oral swabs from apparently healthy cyclops leaf-nosed bats (Hipposideridae: Hipposideros cyclops Temminck, 1853) in Kibale National Park, Uganda (Fig. 1). RusV was found in brain tissues of three acutely ill animals at a zoo in Germany, all of which succumbed to severe, acute neurologic disease (Extended Data Table 2): a donkey (Equus asinus Linnaeus, 1758), a capybara (Hydrochoeris hydrochaeris [Linnaeus, 1766]), and a Bennett’s tree-kangaroo (Dendrolagus bennettianus [De Vis, 1886]). RusV was subsequently detected in brain tissues of 8 of 16 yellow-necked field mice (Muridae: Apodemus flavicollis [Melchior, 1834]) on the zoo grounds and within 10 km of the zoo (Fig. 1; Extended Data Table 1).

Fig. 1 |

Geographic locations of viruses and their hosts. a) Summary map of estimated cyclops leaf-nosed bat distribution in Africa (red) and Uganda (blue box). b) Cyclops leaf-nosed bat in Kibale National Park, Uganda (photo credit: Caley Johnson). c) Location of bat sample collection and discovery of ruhugu virus (Kibale National Park, Uganda, green star). d) Summary map of estimated yellow-necked field mouse distribution in Eurasia (orange) and Germany (blue box). e) Yellow-necked field mouse in northeastern Germany (photo credit: Ulrike M. Rosenfeld). f) Location of zoo animals and discovery of rustrela virus in Germany (southern Baltic Sea region, green star).

Extended Data Table 2 |

Rustrela virus distribution in zoo animal tissues assessed by RT-qPCR.

		Cq value
	Source	Donkey	Capybara	Bennett’s tree-kangaroo
Central nervous system	Cerebrum (I)[a]	22.9	-	30.2
	Cerebrum (II)[b]	29.2	26.0	-
	Cerebrum (III)[b]	29.5	26.6	-
	Cerebrum (IV)[b]	-	30.9	-
	Brain stem[b]	30.5	29.1	-
	Cerebellum[b]	30.6	-	-
	Medulla oblongata[b]	33.9	-	-
	Medulla[b]	-	34.6	-
	Spinal cord[b]	-	30.7	-
Peripheral organs	Liver (I)[a]	-	-	-
	Liver (II)[b]	35.9	-	-
	Kidney[b]	neg	neg	-
	Spleen[b]	neg	neg	-
	Small intestine[b]	-	neg	-
	Organ pool (I)[a]	neg	-	35.5
	Organ pool (II)[a]	-	-	-

fresh, unfixed tissues

formalin-fixed paraffin-embedded tissues

- = no material available; neg = negative. Cells are shaded in proportion to relative viral concentration (Cq value).

Extended Data Table 1 |

Rustrela virus in small mammals from northeastern Germany assessed by RT-qPCR.

		Capture location
Common name	Species	Zoo	Within 10 km of zoo	Total
Yellow-necked field mouse	Apodemus flavicollis [Melchior, 1834]	6/11 (54.5 %)	2/5 (40 %)	8/16 (50 %)
Striped field mouse	Apodemus agrarius [Pallas, 1771]	0/4	0/2	0/6
Bank vole	Myodes glareolus [Schreber, 1780]	0/3	n/a	0/3
Brown rat	Rattus norvegicus [Berkenhout, 1769]	0/13*	n/a	0/13
House mouse	Mus musculus Linnaeus, 1758	0/3*	0/13	0/16

- = no material available.

Two brown rats and all three house mice were animals housed at the zoo.

In the case of RuhV in Uganda, all bats were captured and sampled from five tree roosts (hollow cavities in trees) each containing between 1 and 8 bats. Using molecular and metagenomic methods (Supplementary Methods), RuhV RNA was detected in 5/9 (55.6%) males and 5/11 (45.5%) females in 4 of 5 (80.0%) of roosts (50%; 95% confidence interval 29.9–70.1%). This high prevalence and frequency of positive roosts suggest that apparently healthy cyclops leaf-nosed bats are reservoir hosts, rather than incidental hosts, of RuhV. Cyclops leaf-nosed bats are insectivorous microbats primarily found in lowland rainforests from Senegal to Tanzania but also in coastal, montane, and swamp forests and disturbed and agricultural landscapes[19-21] (Fig. 1a), and they host Plasmodium cyclopsi, an apicomplexan “bat malaria” parasite[22,23]. Whether RuhV can infect animals other than cyclops leaf-nosed bats remains unknown.

In the case of RusV in Germany, the donkey, capybara, and Bennett’s tree-kangaroo were submitted for post-mortem evaluation and testing (Supplementary Methods), which led to the identification of the virus (see below). Subsequent testing of rodents housed at the zoo and wild rodents on the zoo grounds and at two other locations within 10 km of the zoo revealed 8/16 (50%; 95% confidence interval 6.7–39.1%) yellow-necked field mice to be positive for RusV RNA in brain tissue. Surprisingly, the mice had no histologic evidence of encephalitis (7/8 mice investigated) and had only low concentrations of RusV RNA in peripheral organs (Extended Data Table 3). RusV RNA was not detected in any other small mammals collected simultaneously (n=38; Extended Data Table 1). Yellow-necked field mice are omnivorous rodents native to parts of Europe and Asia, occupying mature forests to agricultural and peridomestic habitats[24] (Fig. 1d). They host tick-borne encephalitis virus (Flaviviridae: Flavivirus)[25], Dobrava-Belgrade virus, genotype Dobrava (Hantaviridae: Orthohantavirus)[26-28], Akhmeta virus (Poxviridae: Orthopoxvirus)[29], and hepatitis E virus (Hepeviridae: Orthohepevirus)[30]. Routes of transmission of RuhV and RusV within reservoir hosts and to spillover hosts (in the case of RusV) remain unknown, but positive oral swabs and feces (Extended Data Table 3) suggest that contact with oral secretions and excreta could play a role.

Extended Data Table 3 |

Rustrela virus distribution in tissues of positive Apodemus flavicollis assessed by RT-qPCR.

	Cq value
	KS19/923	KS20/926	KS19/928	KS20/1296	KS20/1340	KS20/1341	KS20/1342	KS20/1343	Mu09/1341
Cerebrum	28.1	neg	22.9	24.1	26.3	21.1	20.8	20.4	25.9
Heart	neg	neg	neg	neg	31.9	neg	neg	neg	-
Lungs	neg	neg	neg	neg	36.7	35.0	neg	neg	-
Liver	neg	neg	neg	neg	neg	neg	neg	neg	-
Kidneys	neg	neg	neg	neg	neg	neg	neg	neg	-
Spleen	neg	neg	neg	neg	neg	neg	neg	neg	-
Intestine/feces	neg	36.7	neg	neg	neg	neg	neg	neg	-
Thoracic lavage	neg	neg	neg	neg	37.5	neg	neg	neg	-
Oral swab	-	-	-	-	36.2	37.5	neg	neg	-

- = no material available; neg = negative. Cells are shaded in proportion to relative viral concentration (Cq value).

Using molecular methods and in situ hybridization (Supplementary Methods), we confirmed the presence of RusV in brain tissues of all German zoo animals and in the liver of the donkey (Extended Data Table 2; Extended Data Figure 1). RusV RNA was detected within neuronal cell bodies and their processes by in situ RNA hybridization on brain tissue sections of the donkey (Extended Data Figure 1a), Bennett’s tree-kangaroo (Extended Data Figure 1b), and capybara (Extended Data Figure 1c). Histopathology revealed a non-suppurative meningoencephalitis in all 3 animals, characterized by perivascular cuffing (Fig. 2a–c), meningeal infiltrates (Fig. 2d), and glial nodules (Fig. 2e). Neuronal necrosis and degeneration with satellitosis were detected in the brain stem of the donkey (Fig. 2f). Immune cells in brain tissue consisted mainly of CD-3-positive T-lymphocytes, Iba-1-positive microglial cells and macrophages and CD79a-immunoreactive B-lymphocytes (Fig. 2g–l). In general, apoptosis was not a significant feature, with few active caspase-3-labelled cells distributed perivascularly and scattered within the gray and white matter (Fig. 2m–n). Multifocal perivascular red blood cells in brain samples from the donkey and Bennett’s tree-kangaroo were positive for iron in the Prussian Blue reaction, indicating intravital hemorrhages (Fig. 2o). In yellow-necked field mice, detection of viral RNA in samples collected between 2009 and 2020 and absence of inflammation (Extended Data Figure 1d–e) suggest this broadly distributed rodent to be the RusV reservoir.

Extended Data Figure 1 |

RNA a-e) Detection of rustrela virus RNA in brain tissues of a donkey (a), Bennett’s tree-kangaroo (b), capybara (c) and yellow-necked field mice (d, e). Chromogenic labelling (fast red) with probes to rustrela virus NSP-coding region are visible in neuronal cell bodies (arrow) but not in adjacent glial cells (arrow head). Mayer’s hematoxylin counter stain. Scale bar = 50 μm f). Negative control probe to bacterial gene dapB encoding dihydrodipicolinate reductase. Lack of chromogenic labelling (fast red). Mayer’s hematoxylin counter stain. Scale bar = 100 μm. RNAscope results were evaluated on at least 3 slides per animal, yielding comparable results in all cases. In situ hybridization was performed according to the manufacturer’s instructions, including a positive control probe, peptidylprolyl isomerase B (cyclophilin B, ppib), and a negative control probe, dihydrodipicolinate reductase (DapB). Evaluation and interpretation were performed by a board certified pathologist (DiplECVP) with more than 13 years experience.

Fig. 2 |

Histopathology and immune reaction of rustrela virus in the brain of a capybara, Bennett’s tree-kangaroo and donkey. a–c) Non-suppurative meningoencephalitis with mononuclear, perivascular cuffing, brain, capybara (a), Bennett’s tree-kangaroo (b), and donkey (c). d) Mononuclear meningeal infiltrates, brain, donkey. e) Glial nodules, brain, donkey. f) Neuronal necrosis (arrow) and degeneration with satellitosis (arrow head), brain, donkey. HE stain; scale bar 20 μm (a–c, e–f), 50 μm (d). g–l) Immune reaction by immunohistochemistry, perivascular, brain, Bennett’s tree kangaroo; and in glial nodules, brain, donkey (j–l), numerous CD-3 labelled T- lymphocytes (g, j), Iba-1 positive microglial cells and macrophages (h, k), CD79a immunoreactive B- lymphocytes (i, l). Immunohistochemistry, AEC chromogen, Mayer’s haematoxylin counter stain, scale bar 20 μm. m–n) Apoptosis, few active Caspase-3 labelled cells (arrows), perivascular and scattered throughout the neuropil, brain, Bennett’s tree-kangaroo (m), brain, capybara (n). Immunohistochemistry, AEC chromogen, Mayer’s haematoxylin counter stain; scale bar 20 μm. o) Hemorrhage, Prussian Blue reaction demonstrates multiple iron deposits (arrows) within mononuclear cells found perivascularly, admixed with red blood cell accumulations, indicating intra-vital haemorrhage; scale bar 20μm. Immunohistochemistry was performed on at least 4 slides per animal, yielding comparable results in all cases. In each run, positive control slides and negative control primary antibodies were included. Evaluation and interpretation were performed by a board certified pathologist (DiplECVP) with more than 13 years experience

The genome organizations of RuV, RuhV, and RusV are identical, consisting of two large open reading frames (ORFs), two untranslated regions (UTRs) at 5′ and 3′ termini, and an intergenic region (IR) between the two ORFs (Fig. 3a). Across the nonstructural and structural coding regions, RuhV is more similar to RuV than is RusV (Extended Data Table 4). Genetic similarity varies within coding regions and is generally highest in a hyper-conserved region within the Y domain of P150[2,31,32] (Extended Data Fig. 2). RusV contains a notably long IR (366 nt, versus 46 nt and 75 nt in RuV and RuhV, respectively) and a correspondingly short C protein (205 aa, versus 300 aa and 317 aa in RuV and RuhV, respectively; Extended Data Table 4). In addition, RuV and RuhV share a Gly-Gly-Gly amino acid sequence at the p150/p90 cleavage site, whereas RusV has a Gly-Gly-Ala amino acid sequence at this same site, which may impair cleavage in the case of RusV[3].

Fig. 3 |

Evolutionary relationships among viruses. a) Comparative genome architecture of RuV, RuhV, and RusV, showing five open reading frames (colored), two untranslated regions at the 5′ and 3′ termini (white), and an intergenic region (white) between the ORFs encoding the non-structural (nsPP) and structural (sPP) polyproteins. b) Maximum likelihood phylogenetic tree of rustrela virus, ruhugu virus, and rubella virus genotypes 1A–1J and 2A–2C. Black silhouettes represent natural hosts of each virus, and red silhouettes represent spillover hosts in the case of RusV. Numbers beside nodes indicate bootstrap values (%; only values for major branches are shown); the scale bar indicates amino acid substitutions per site.

Extended Data Table 4 |

Genomic features of ruhugu virus (RuhV; GenBank MN547623) and rustrela virus (RusV; GenBank MN552442).

	Nucleotide position (5′→3′)		Amino acid residues		Amino acid sequence identity (%)			GC content (%)
Genome featurec	RuhV	RusV	RuhV	RusV	RuhV[a]	RusV[a]	RuhV–RusV	RuhV	RusV	RuV[b]
Complete genome	1–9621	1–9322	6296	5876	56.4	43.0	43.3	63.5	70.6	69.6
Non-structural polyprotein	44–6190	68–5833	2049	1921	59.0	45.9	47.5	62.2	70.2	70.0
p150 protease	44–3754	68–3391	1237	1108	48.6	34.5	35.7	63.1	72.0	71.4
p90 replication complex	3755–6190	3392–5830	812	813	75.7	65.5	66.6	60.9	67.7	67.8
Structural polyprotein	6266–9562	6193–9246	1099	1017	51.4	41.1	39.5	66.1	71.4	69.4
Capsid protein	6266–7216	6193–6807	317	205	51.7	46.6	43.0	66.6	74.5	73.1
E2 envelope protein	7217–8101	6808–7785	295	326	43.6	31.4	23.9	67.9	72.7	71.0
E1 envelope protein	8102–9562	7786–9243	487	486	56.3	51.0	50.6	64.8	69.3	66.3

Ruhugu virus and rustrela virus inferred amino acid sequence identities are compared to rubella virus strain F-Therien (RuV; RefSeq #NC_001545).

GC content is shown for rubella virus strain F-Therien (RefSeq #NC_001545).

Extended Data Figure 2 |

Average substitution rates at non-synonymous (dN; dashed lines) and synonymous (dS; grey lines) sites, and the ratio of dN/dS (solid lines), for aligned, concatenated amino acid sequences comparing RuV and RuhV (a), RuV and RusV (b), and RuhV and RusV (c) using sliding window analysis (100 residue window length, 10 residue steps).

Protein domains are labeled on the X axes: MT=methyltransferase; Y, Q, and X=domains of unknown function; Pro=protease; Hel=helicase; RdRp=RNA-directed RNA polymerase; NT1=neutralizing epitope 1.

RuhV (named for Ruteete Subcounty, Uganda, and the Tooro word for insectivorous bat, obuhuguhugu) is an outgroup to all known RuV genotypes (Fig. 3b). RusV (named for its rubella virus-like genome and the Strela Sound of the Baltic Sea in Germany) is a close outgroup to the RuV/RuhV clade of viruses (Fig. 3b). This topology is consistent with the higher similarity of RuhV to RuV in each of the five mature polypeptides of the protein-coding viral genome (Extended Data Table 4, Extended Data Fig. 2). Nucleotide sequences of RusV were 97.4 to 100 % similar within p90 and E1 coding regions sequenced in the donkey, capybara, Bennett’s tree-kangaroo, and yellow-necked field mice in Germany (Extended Data Figure 3).

Extended Data Figure 3 |

Phylogenetic analyses of the coding sequences of envelope glycoprotein E1 (a) and helicase and RNA-directed RNA polymerase p90 (b) of RusV and RuhV and RusV, including all sequences obtained in this study (GenBank accession numbers in parentheses).

Numbers above branches represent bootstrap values; scale bars indicate amino acid substitutions per site.

The RuV E1 protein, a receptor-binding, class II fusion protein[5], contains an immune-reactive region (amino acid residue positions 202–283) with immunodominant T-cell epitopes[6] and four linear, neutralizing B-cell epitopes (NT1–4)[4] (Fig. 4a). The modeled tertiary and quaternary structures of trimeric RuhV and RusV E1 are homologous to RuV E1[33], and homology-based modeling of RuhV E1 quaternary structure predicts with high confidence that RuhV and RusV E1 proteins form homotrimers in the post-fusion state[5] (Fig. 4b and c). One neutralizing epitope maps to amino acid positions 223–239 of the E1 protein at disulfide bond 8 (NT1)[34]. The mechanism of neutralization appears to involve blocking of E1 trimerization, which is necessary for virion fusion with the host-cell plasma membrane[5]. Surprisingly, only one amino acid residue (R237Q, near the C-terminus) differs between the RuV and RuhV NT1 epitope (Fig. 4a), despite higher amino acid-level divergence across E1 (Extended Data Figure 3). By contrast, RusV differs from RuV at 5 amino acid residues within the same region (Fig. 4a). T-cell epitopes are not well conserved in the capsid (Extended Data Table 5); however; the exposed RuhV and RusV NT3 and NT4 putative linear epitopes within E1 are moderately conserved in comparison to RuV (Fig. 4; Extended Data Table 5), suggesting that they also should be evaluated for cross-neutralization by anti-RuV antibodies.

Fig. 4 |

Comparisons of the rubella (RuV), ruhugu (RuhV), and rustrela virus (RusV) E1 envelope glycoproteins. a) Amino acid alignment and sequence logo of an immunoreactive region of E1 for RuhV, RusV, and 13 RuV genotypes (GenBank accession numbers in parentheses). Lines indicate locatons of putative linear neutralizing B-cell epitopes NT1-NT4. b) Homology-based model of RuhV E1 homotrimer structure in the post-fusion state showing receptor-binding site view (left) and profile view (right). Global model quality estimates (QMEAN) indicate a good model fit relative to the crystal structure of the RuV E1 in the post-fusion form (Protein Data Bank biological assembly 4adg.1). c) Homology-based model of the RusV E1 homotrimer structure in the post-fusion state, as described above for RuhV. Key differences are seen in the modeled neutralizing epitopes NT3 and NT4 and in Fusion Loops 1 and 2 (FL1 and FL2). RuhV FL1 and FL2 residues are highly similar to those of RuV, whereas RusV FL2 residues differ from those of RuV FL2 to a greater extent. The color scale indicates normalized QMEAN local score.

Extended Data Table 5 |

Conservation of B- and T-cell epitopes in the E1 fusion protein of the rubella wild-type virus 1B, ruhugu virus, and rustrela virus.

	Epitope	Rubella virus (JN635282)	Ruhugu virus (MN547623)	Rustrela virus (MN552442)
Linear, neutralizing B-cell epitopes	NT1:E11221–239	LGSPNCHGPDWASPVCQRHS	VGLPNCHGPDWASPVCQQHS	VPAPDCFGPAWASPVCARHM
	NT2: E1245–251	LVGATPE	LTGVPPE	LTGATPG
	NT3 :E1260–266	ADDPLLR	ADDPRLT	ADDLGWH
	NT4 :E1274–285	VWVTPVIGSQAR	VWAVAVKGTQPK	VWYQPVIGRQPR
CD8+ T-cell epitopes	C9–22	MEDLQKALEAQSRA	LADLQRLLEKQSAE	Deleted
	C11–29	DLQKALEAQSRALRAELAA	DLQRLLEKQSAELRAEMAR	Deleted
	C264–272	RIETRSARH	KQDVKSDKV	RKEOLGATSGAA

Amino acid differences are in bold, insertions are underlined, and GenBank accession numbers are in parentheses.

The RuhV E1 fusion loops (FL1: residues 87–92; FL2: residues 130–136) are predicted to support the unusual metal ion complex necessary for E1-mediated RuV membrane fusion due to the presence of amino acids N87 and D135 (homologous to RuV N88 and N136, respectively; Fig. 4b)[5]. By contrast, FL2 of RusV is predicted to be less similar to RuV due to two amino acid residue replacements, P134A and T135A, the latter being a change from a polar to a non-polar residue (Fig. 4c). Across the RuV, RuhV, and RusV genomes, regions of marked conservation and stabilizing selection are evident immediately upstream of the putative methyltransferase domain of P150, in the RdRp domain of P90, and proximal to the aforementioned NT1 epitope of E1 (Extended Data Fig. 2).

The similarity or near identity of certain RuV, RuhV, and RusV B-cell epitopes (Extended Data Table 5) suggests that existing serologic assays for anti-rubella antibodies might detect RuhV, RusV, and other as-yet undiscovered rubella-like viruses. Future studies evaluating the performance of existing serologic tests for RuV infection in animals would be useful, as would the development of novel assays that can detect and differentiate among rubella-like viral infections in animals and humans. Implicating RuhV or RusV as zoonotic agents is currently speculative, but bats and rodents possess biological attributes that predispose them to hosting many zoonotic viruses[35-37], so this scenario ought not to be dismissed. The ability of RusV to infect both placental and marsupial mammals and to cause clinical disease resembling severe encephalitic forms of rubella in humans[38,39] reinforces such a precautionary stance.

The World Health Organization’s (WHO’s) Global Measles and Rubella Strategic Plan aims to control or eliminate rubella and congenital rubella syndrome in 5/6 WHO regions by the end of 2020[40]. Our discoveries of relatives of RuV infecting asymptomatic bats and rodents suggests that rubella may have arisen as a zoonosis. Furthermore, the ability of RusV to infect mammals across wide taxonomic distances and to cause severe encephalitis in spillover hosts raises concern about the potential for zoonotic transmission of RuhV, RusV, or other as-yet undiscovered rubella-like viruses. Despite these concerns, our findings clearly augur well for comparative studies of RuV that were heretofore not possible, including the potential development of novel animal models for rubella and congenital rubella syndrome.

Extended Data Table 6 |

Immunohistochemical markers and applications.

Marker	Antibody	Antigen Retrieval	Secondary reagents
Active caspase 3	Anti-Active Caspase 3 (Promega, Walldorf, Germany), 1:200, overnight	n/a	ABC Kit Vectastain Elite PK 6100, 30 min (Dako)
CD79a	Mouse anti-CD79A (clone HM57) monoclonal, (LifeSpan BioSciences, Seattle, WA, USA), 1:50, overnight	HIER, 10 mM Tris/1mM EDTA buffer pH 9.0, 20 min	Dako EnVision+ System-HRP Labelled Polymer Anti-mouse, 30 min
CD3	Rabbit anti-CD3 polyclonal (Dako), 1:100, overnight	HIER, 10 mM Tris/1mM EDTA buffer pH 9.0, 20 min	Dako EnVision+ System- HRP Labelled Polymer Anti-rabbit, 30 min
Iba-1	Iba1 (Wako), 1:800, overnight	HIER, Citrate buffer pH 6.0, for 20 min	Dako EnVision+ System- HRP Labelled Polymer Anti-rabbit, 30 min

HIER: Heat-induced epitope retrieval; HRP: horse-radish peroxidase. n/a = not applicable.