Spondweni virus infection in pregnant rhesus macaques causes placental pathology without apparent fetal harm

Hunter J Ries; Livia Romanov; Maria C Charles; Chelsea M Crooks; Carson DePagter; Alex Richardson; Grace A VanSleet; Andrea M Weiler; Jens C Eickhoff; Katharina S Stewart; Leandro B C Teixeira; Eric Peterson; Michele Schotzko; Heather A Simmons; Jenna R Rosinski; Lauren E Raasch; Anna S Jaeger; Elaina R Razo; Emma L Mohr; David H O’Connor; Christina M Newman; Matthew T Aliota; Thomas C Friedrich

doi:10.64898/2026.08.10.743875

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[Preprint]. 2026 Aug 10:2026.08.10.743875. [Version 1] doi: 10.64898/2026.08.10.743875

Spondweni virus infection in pregnant rhesus macaques causes placental pathology without apparent fetal harm

Hunter J Ries ¹, Livia Romanov ¹, Maria C Charles ¹, Chelsea M Crooks ¹, Carson DePagter ¹, Alex Richardson ², Grace A VanSleet ², Andrea M Weiler ², Jens C Eickhoff ³, Katharina S Stewart ⁴, Leandro B C Teixeira ¹, Eric Peterson ², Michele Schotzko ², Heather A Simmons ², Jenna R Rosinski ⁵, Lauren E Raasch ⁵, Anna S Jaeger ⁶, Elaina R Razo ⁷, Emma L Mohr ^7,⁸, David H O’Connor ⁵, Christina M Newman ⁵, Matthew T Aliota ⁶, Thomas C Friedrich ^1,^2,^#

¹Department of Pathobiological Sciences, University of Wisconsin–Madison, Madison, Wisconsin, USA

²Wisconsin National Primate Research Center, University of Wisconsin–Madison, Madison, Wisconsin, USA

³Department of Biostatistics and Medical Informatics, University of Wisconsin–Madison, Madison, Wisconsin, USA

⁴Department of Obstetrics and Gynecology, University of Wisconsin–Madison, Madison, Wisconsin, USA

⁵Department of Pathology and Laboratory Medicine, University of Wisconsin–Madison, Madison, Wisconsin, USA

⁶Department of Veterinary and Biomedical Sciences, University of Minnesota, Twin Cities, St. Paul, Minnesota, USA

⁷Department of Pediatrics, University of Wisconsin–Madison, Madison, Wisconsin, USA

⁸Department of Medical Microbiology and Immunology, University of Wisconsin–Madison, Madison, Wisconsin, USA

Author contributions:

H.J.R.—Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Methodology, Project Administration, Software, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

L.R.—Data Curation, Investigation, Methodology, Validation, Writing – Review & Editing

M.C.C.—Data Curation, Investigation, Methodology, Validation, Writing – Review & Editing

C.M.C.—Conceptualization, Data Curation, Funding Acquisition, Investigation, Methodology, Validation, Writing – Review & Editing

C.D.—Data Curation, Investigation, Methodology, Validation, Writing – Review & Editing

A. R.—Data Curation, Investigation, Methodology, Validation, Writing – Review & Editing

G.V.S.—Data Curation, Investigation, Methodology, Validation, Writing – Review & Editing

A.M.W.—Data Curation, Investigation, Methodology, Validation, Writing – Review & Editing

J.C.E.—Data Curation, Formal Analysis, Investigation, Methodology, Software, Validation, Writing – Review & Editing

K.S.S.—Data Curation, Formal Analysis, Investigation, Writing – Review & Editing

L.B.C.T.—Data Curation, Investigation, Writing – Review & Editing

E.P.—Investigation, Project Administration, Resources, Writing – Review & Editing

M. S.—Investigation, Project Administration, Resources, Writing – Review & Editing

H.A.S.—Data Curation, Formal Analysis, Investigation, Methodology, Resources, Validation, Writing – Review & Editing

J.R.R.—Investigation, Writing – Review & Editing

L.E.R.—Investigation, Writing – Review & Editing

A.S.J.—Conceptualization, Investigation, Methodology, Resources, Validation, Visualization, Writing – Review & Editing

E.R.R.—Investigation, Resources, Writing – Review & Editing

E.L.M.—Investigation, Resources, Writing – Review & Editing

D.H.O.—Investigation, Resources, Writing – Review & Editing

C.M.N.—Investigation, Resources, Writing – Review & Editing

M.T.A.—Conceptualization, Funding Acquisition, Project Administration, Resources, Supervision, Writing – Review & Editing

T.C.F.—Conceptualization, Funding Acquisition, Project Administration, Resources, Supervision, Validation, Writing – Original Draft Preparation, Writing – Review & Editing

Correspondence should be addressed to T.C.F. (tfriedri@wisc.edu).

PMCID: PMC13640649 PMID: 42845459

Abstract

The 2015–2016 Zika virus (ZIKV) epidemic revealed the potential of flaviviruses to emerge rapidly, cause severe disease, and affect pregnancy outcomes. In 2016, Spondweni virus (SPOV), the closest known relative of ZIKV, was detected in mosquitoes in Haiti, suggesting it may also have the potential to emerge in the Western Hemisphere. The risks that close relatives of ZIKV pose to pregnant individuals are not well understood. Previously, we showed that SPOV can cause fetal demise, placental pathology, and vertical transmission in a mouse model. Here we report SPOV’s pathogenic potential in pregnant rhesus macaques. We inoculated four macaques with SPOV at gestational day 30 (early first trimester) and compared their viral loads and fetal outcomes with those of macaques infected in the first trimester with either African-lineage ZIKV (ZIKV-DAK) or an Asian-lineage ZIKV isolate from Puerto Rico (ZIKV-PR) in previous studies. Plasma viremia persisted 10–31 days in SPOV-inoculated dams, whereas viremia resolved within 10–17 days for ZIKV-DAK and 5–52 days for ZIKV-PR. Cesarean deliveries near term (gestational day 152–157) revealed no demise, premature birth, or gross abnormalities in fetuses of dams inoculated with SPOV or ZIKV-PR. In contrast, under near-identical conditions, all ZIKV-DAK-inoculated dams experienced fetal demise between 12 and 20 days post-inoculation. At cesarean section, we did not detect SPOV RNA above the limit of detection in maternal (e.g., spleen, liver), placental, or fetal tissues, in contrast to previous findings with ZIKV-PR. Histological analysis revealed mononuclear/lymphohistiocytic villitis in all placentas of SPOV-exposed macaques, along with other pathological changes in individual placentas. Our findings suggest that SPOV infection of macaques in early pregnancy may result in placental pathology without overt fetal harm. Our results suggest that flaviviruses in the Spondweni serocomplex, which includes ZIKV and SPOV, may vary in their pathogenic potential during pregnancy.

Keywords: Spondweni virus, Zika virus, rhesus macaques, non-human primates, pregnancy, placental pathology, fetal growth

Author Summary

Zika virus (ZIKV) can cause fetal harm. Does this risk extend to its closest known relative, Spondweni virus (SPOV)? Should SPOV circulate in humans, what risks would it pose in pregnancy? SPOV can injure fetuses in immunocompromised mice, but the physiology of pregnancy in mice differs greatly from that of humans. We therefore infected pregnant rhesus macaques with SPOV during early gestation and compared maternal viremia, placental pathology, and fetal outcomes with macaques infected with African- or Asian-lineage ZIKVs at the same gestational age. All fetuses survived to near-term pregnancy, fetal tissues were negative for SPOV RNA, and fetal growth tracked within expected ranges. Nonetheless, all SPOV-exposed pregnancies showed placental injury, including mononuclear/lymphohistiocytic villitis and maternal vascular malperfusion. Despite the absence of detectable SPOV RNA in fetal tissues, SPOV RNA persisted at term in maternal-fetal interface tissues in two of four animals. These data indicate placental injury without detectable vertical transmission in this translational model. Our results suggest that SPOV and ZIKV display a wide range of risks to the developing fetus. Identifying viral and host factors that increase the potential for fetal harm will be important for assessing risks posed by emerging viruses in this family.

Introduction

Zika virus (ZIKV), dengue virus (DENV), and yellow fever virus (YFV) are closely related orthoflaviviruses that cause hundreds of millions of infections annually and place half of the world’s population at risk of infection [1–4]. As climate change expands the geographic range of their primary mosquito vectors, Aedes aegypti and Aedes albopictus, these flaviviruses pose a growing global health threat [1,5]. Despite their genetic similarity, these viruses exhibit markedly different pathogenic profiles: ~5% of symptomatic DENV infections progress to severe dengue that can be life-threatening [6–8], ~12% of YFV infections enter a severe “toxic” phase in which approximately half of cases are fatal [9,10], and ZIKV infection during the first trimester of pregnancy carries up to 15% risk of ZIKV-associated birth defects [11–14]. This teratogenic capacity distinguishes ZIKV from other flaviviruses, yet the determinants underlying this unique pathology remain poorly understood. Determining whether this capacity extends to ZIKV’s closest relatives is critical for assessing emerging threats and developing targeted interventions.

Following its identification in 1947, ZIKV was perceived to have low pathogenic potential and little capacity to cause major outbreaks [15,16]. This perception began to shift following explosive outbreaks on Yap Island (2007) and French Polynesia (2013), both characterized by high attack rates, although pregnancy complications were not recognized at the time [17,18]. The 2015–16 Americas epidemic revealed ZIKV’s capacity to cause congenital Zika syndrome (CZS), a newly identified spectrum of fetal harm including microcephaly, neurological impairment, and fetal loss [19–23]. Given that ZIKV’s potential for fetal harm went unrecognized for decades, we posited that close genetic relatives of ZIKV might also possess this potential.

Spondweni virus (SPOV) is ZIKV’s closest known relative, sharing ~68% nucleotide similarity and ~75% amino acid similarity [24]. Both viruses have documented co-circulation in sub-Saharan Africa and antibodies against either virus exhibit extensive cross-reactivity with the other, complicating surveillance and diagnostic efforts [24–28]. Due to their extensive serological cross-reactivity, SPOV and ZIKV are grouped together taxonomically in the Spondweni virus serocomplex. Despite these genetic and geographic similarities, SPOV has caused only six documented human infections, most presenting as mild-to-moderate, self-limiting febrile illness [27,29–33]. These cases span from SPOV’s first identification in Nigeria in 1952 [29] to its most recent documented human case in Burkina Faso in 1979 [33]. While the limited number of reported cases presented with relatively mild symptoms, some cases have included vascular or neurological symptoms, such as epistaxis (nosebleed) [31] and photophobia [33], suggesting the possibility of more severe clinical manifestations [27]. Historically, SPOV has been detected only in sub-Saharan Africa, where it has been isolated from humans and mosquitoes [24,31,34,35]. In 2016, however, SPOV was unexpectedly detected in Culex quinquefasciatus mosquitoes in Haiti [36], raising the possibility that SPOV, similar to ZIKV, may have the potential to expand its geographic range beyond Africa. Given these concerns and SPOV’s close genetic relatedness to ZIKV, we sought to evaluate whether SPOV shares ZIKV’s capacity to cause fetal harm. We previously demonstrated that SPOV can cause fetal demise and placental pathology and can be vertically transmitted in a mouse pregnancy model [37]. However, mouse placental structure is labyrinthine rather than villous, as seen in humans, and human flaviviruses like DENV, ZIKV, and SPOV can only replicate in mice lacking type-I interferon responses, so it is important to evaluate their pathogenic potential during pregnancy in additional models [37–39].

To overcome these limitations, our group has used rhesus macaques to model flavivirus pathogenesis during pregnancy [40–46]. Rhesus macaques recapitulate core features of human pregnancy, including gestational timing, villous placental architecture, and immune function [47–49]. Using this model, our group has demonstrated that viral lineage and timing of infection are crucial determinants of ZIKV-associated fetal harm. In previous studies with Asian-lineage ZIKV (PRVABC59 strain; hereafter ZIKV-PR) we found infrequent fetal demise and no cases of microcephaly in pregnant rhesus macaques, though infection was associated with neurodevelopmental deficits and presence of vRNA in the maternal-fetal interface (MFI) [42,50,51]. In contrast, African-lineage ZIKV (Dak Ar 41524 strain; hereafter ZIKV-DAK) consistently caused frequent fetal demise between 12 and 20 days post-inoculation when inoculated during mid-first trimester (gestational day 30) [41]. Motivated by these observations and prior evidence that SPOV replicates in non-pregnant rhesus macaques [52], we designed an experiment to assess the comparative pathogenic potential of SPOV, ZIKV-DAK, and ZIKV-PR in pregnancy. We inoculated four pregnant rhesus macaques with SPOV and compared maternal viremia, fetal growth, and placental pathology at delivery to a previously characterized cohort of five macaques infected with ZIKV-DAK and eight macaques infected with ZIKV-PR at the same approximate gestational age [41,53]. This study tests whether SPOV, under matched conditions, produces placental infection and fetal injury comparable to ZIKV-DAK or ZIKV-PR.

Results

Study design

We inoculated four pregnant rhesus macaques with SPOV at approximately 30 days after the estimated date of conception (range GD 31–34; Fig 1). All four macaques were inoculated with 1×10^4 plaque-forming units (PFU) of SPOV (SAAr94 strain), a dose within the range thought to be delivered by mosquitoes [54]. In a previous study [41], we inoculated five pregnant rhesus macaques with 1×10^4 PFU of ZIKV (ZIKV-DAK; Dak Ar 41524 strain) at approximately GD 30. Recently [53], we also inoculated eight pregnant rhesus macaques with 1×10^4 PFU of ZIKV (ZIKV-PR; PRVABC59 strain) at approximately GD 30. In each study, we assessed maternal plasma viral load throughout the acute phase of infection and fetal growth throughout gestation until the study endpoint. In the SPOV and ZIKV-DAK studies, we also assessed fetal and placental tissue viral loads following necropsy. In the ZIKV-PR study, we assessed placental but not fetal tissues, as infants remained with their dams for developmental studies following cesarean delivery at approximately GD 160 (term in rhesus macaques is approximately 165 days) [53].

Pregnant macaques support SPOV infection with occasional prolonged shedding

All SPOV-inoculated macaques became productively infected, reaching a median peak maternal plasma viral loads of 2.51×10^4 copies/mL (range: 1.38×10^4 – 3.85×10^5 copies/mL) approximately six days post-inoculation (Fig 2A). Plasma viremia persisted for a median duration of 16.5 days (range: 10–31 days). We define prolonged viremia as ≥21 days of detectable plasma vRNA on serial sampling, because in non-pregnant rhesus macaques challenged with 1×10^4 PFU of SPOV, ZIKV-DAK, or ZIKV-PR, plasma viremia typically resolves within ten days [52]. By this definition, macaques 102 and 104 exhibited prolonged viremia (31 and 23 days, respectively), whereas macaques 101 and 103 were viremic for ten days each. All macaques were serologically naïve to SPOV prior to inoculation and seroconverted by 30 days post-inoculation (S1 Fig).

Fig 2. — **(A)** Longitudinal plasma viral RNA loads in pregnant macaques inoculated with Spondweni virus (SPOV; orange), Zika virus Dak Ar 41524 strain (ZIKV-DAK; blue), or Zika virus PRVABC59 strain (ZIKV-PR; green). Viral loads are shown as log10 RNA copies per mL of plasma over days post-inoculation. **(B–E)** Group comparisons of **(B)** peak viral load, **(C)** time to peak viremia (days), **(D)** duration of viremia (days above the assay lower limit of detection, LLOD), and **(E)** area under the curve (AUC) defined as area above the LLOD (units: log10·days). Points represent individual macaques (SPOV n = 4; ZIKV-DAK n = 5; ZIKV-PR n = 8). Horizontal brackets in B and C mark pairwise two-tailed Wilcoxon rank-sum tests. Stars denote Benjamini-Hochberg-adjusted p-values within each metric (* < 0.05, ** < 0.01, *** < 0.001); non-significant p-values are not shown. ZIKV-DAK-inoculated macaques were previously reported by Rosinski and Raasch *et al.* [41]. ZIKV-PR-inoculated macaques were previously reported by Krabbe *et al*. [53].

In comparison, ZIKV-DAK-infected macaques had higher peak maternal plasma viral loads than those infected with SPOV (9.12×10^5 copies/mL; BH-adjusted p = 0.0238), whereas peak viremia for SPOV was not significantly different from ZIKV-PR (p = 0.570; Fig 2B). In the SPOV group, viral loads peaked approximately six days post-inoculation (Fig 2C), significantly later than both ZIKV-DAK (five days; p = 0.0397) and ZIKV-PR (three days; p = 0.0242). The duration of viremia (Fig 2D) did not differ significantly between SPOV and ZIKV-DAK (ten days; p = 0.524) or SPOV and ZIKV-PR (seven days; p = 0.345). To quantify the overall burden of viral replication, we calculated the area under the curve (AUC) for each animal, excluding values at or below the lower limit of detection. AUC did not differ significantly across pairs, although the ZIKV-DAK group had an approximately two-fold greater AUC compared to the SPOV group (p = 0.333; Fig 2E). Collectively, these findings demonstrate that SPOV-inoculated pregnant rhesus macaques exhibited later peak viremia than animals infected with either of the ZIKV strains. While the total burden of virus replication trended lower for SPOV compared to ZIKV-DAK, this difference was not significant.

Previously, we detected ZIKV RNA in urine from all five ZIKV-DAK-inoculated macaques [41], with one animal reaching a peak urine viral load of nearly 48,000 copies/mL. In this study, we detected SPOV RNA in urine from two SPOV-inoculated macaques (101 and 104; S2 Fig). Macaque 101 had detectable viral RNA (vRNA) at 118 copies/mL five days post-inoculation, increasing to 1,016 copies/mL by day ten. Macaque 104 had detectable vRNA at 159 copies/mL nine days post-inoculation. These results suggest that SPOV, similar to ZIKV-DAK, can be detected in urine but appears to exhibit more limited urinary shedding in this rhesus macaque model.

Prenatal ultrasound reveals no consistent evidence of SPOV-associated intrauterine growth restriction

All pregnancies in SPOV-inoculated macaques progressed to the study endpoint (GD 152–157; term = 165 ± 10 days) without fetal demise, premature birth, or gross anatomical abnormalities. This finding contrasts with our prior study of ZIKV-DAK-inoculated macaques inoculated at the same point in the first trimester; each of those pregnancies ended in fetal demise by ~20 days post-inoculation [41]. This suggests that SPOV has reduced pathogenic potential compared to ZIKV-DAK, but the absence of overt adverse outcomes does not preclude more subtle effects on fetal development. To monitor fetal growth, we conducted biweekly ultrasounds from early gestation through to near-term (gestational day 155), measuring fetal biparietal diameter (BPD), head circumference (HC), abdominal circumference (AC), and femur length (FL). Growth trajectories appeared broadly consistent and uninterrupted across gestation (Fig 3A). To contextualize these trajectories, we compared our measurements to age-matched normative data from a large California National Primate Research Center (CaNPRC) cohort of rhesus macaques (n = 55) [55,56]. We then calculated Z-scores to quantify the extent to which each measurement deviated from the CaNPRC mean (Fig 3B). SPOV-exposed fetuses showed statistically significant deviations from expected mean Z-score values for all measurements (p < 0.05), but their growth trajectories (slope of the Z-score curves) were not significantly different from the normative data (Fig 3B). All SPOV-exposed fetuses had HC that measured below the CaNPRC mean throughout gestation (p < 0.001; Fig 3A), but differences were small. In human fetal medicine, the threshold for abnormal size is defined as a Z-score ≥2, and Z-scores for SPOV-exposed fetuses never reached this threshold [57,58]. The deviation in HC was most pronounced after 15 weeks of gestation, where fetus 003 (from macaque 103) had an HC Z-score of −1.98 at 20 weeks of gestation, just under the abnormality threshold (S3A Fig). BPD was not as markedly reduced (Fig 3A) and remained closer to the normative mean, suggesting that the observed reduction in HC may reflect differences in cranial shape or growth pattern rather than a global restriction in fetal head size. AC was also statistically significantly smaller in SPOV-exposed fetuses than in the normative data (p < .01), but FL was significantly elevated (p < 0.05; Fig 3B). Together our fetal growth data suggest that SPOV-exposed fetuses in our study were somewhat smaller than fetuses in the normative dataset, but that their growth followed a similar trajectory. It appears likely that size differences relative to the normative cohort were statistically, but not biologically, significant. Comparison of our SPOV-exposed fetuses with the growth trajectories of other rhesus macaques studied at the Wisconsin National Primate Research Center (WNPRC), including mock-infected (n = 4), ZIKV-DAK (GD 30; n = 4), ZIKV-PR (GD 30; n = 8) and ZIKV-PR cohorts (GD 45; n = 4), supports this conclusion: we observed no apparent differences in growth trajectories between our SPOV-exposed animals and these groups (S3B Fig) [42]. Thus, the HC and AC values of WNPRC fetuses may consistently fall below CaNPRC normative curves, regardless of viral exposure. Together, our observations indicate that SPOV does not cause an apparent phenotype of fetal growth restriction in rhesus macaques.

Fig 3. — **(A)** Fetal growth measurements from the macaques inoculated with Spondweni virus (SPOV): biparietal diameter (BPD), head circumference (HC), abdominal circumference (AC), and femur length (FL). Points denote individual macaques (n = 4), with lines connecting repeated measurements over gestational age (weeks). Normative reference curves are shown as a black line (mean) with flanking grey lines representing ±1, ±2, and ±3 standard deviations at each gestational age, derived from rhesus macaque reference cohorts (n = 55) [55,56]. **(B)** Z-scores for SPOV-inoculated macaques at each time point for the same four metrics. Values reflect the number of standard deviations each measurement differed from the gestational-age-matched normative mean. A linear regression line (dark grey) is overlaid in each panel. p-values indicate one-sample t-tests (vs 0) and a linear mixed-effects model (slope). Horizontal grey lines indicate ±1, ±2, and ±3 Z-score thresholds, corresponding to the mean-flanking grey lines in A.

SPOV RNA is present at low levels in maternal and fetal tissues upon delivery

Despite the lack of overt fetal growth restriction, we next asked whether SPOV RNA was detectable in maternal, fetal, or maternal-fetal interface (MFI) tissues at delivery. SPOV RNA was sporadically detected at very low levels in placental tissues and the maternal spleen at the time of fetectomy (Fig 4). Specifically, vRNA was identified in the spleens of SPOV-infected macaques 101 and 102, with macaque 101 testing positive in one of two technical replicates (0 and 7 copies/mg) and macaque 102 testing positive in both wells (34 and 40 copies/mg) (Fig 4; Table 1). Additionally, we detected 10 copies/mg in the decidua basalis of macaque 101. In macaque 104, we detected SPOV RNA in various samples of the decidua basalis (1–4 copies/mg across samples), placental parenchyma tissue (4–8 copies/mg), and chorionic plate (5–42 copies/mg; Fig 4). This pattern of low-level tissue vRNA burden resembles our findings with ZIKV-PR (mesenteric lymph node and MFI: decidua basalis and placental parenchyma). In contrast, we previously detected ZIKV-DAK RNA in maternal organs (liver, spleen, mesenteric lymph node) and multiple MFI compartments, frequently at higher levels. Because tissues from animals infected with ZIKV-DAK were sampled at the time of fetal demise (within 20 days of inoculation), whereas tissues from SPOV- and ZIKV-PR-infected pregnancies were sampled much later, over 100 days post-inoculation, we cannot determine from these data whether higher ZIKV-DAK tissue burdens reflect inherently greater replication in the MFI or earlier sampling for ZIKV-DAK during gestation. Per-animal tissue vRNA loads are summarized in Table 1 for SPOV-inoculated macaques. SPOV RNA was undetectable across 60 fetal anatomical compartments, including CNS, ocular, cardiopulmonary, lymphoid, gastrointestinal, reproductive, and endocrine (S1 Table). Because we previously found evidence of ocular pathology in fetuses from ZIKV-infected dams [59], eyes of four SPOV-exposed fetuses were analyzed histopathologically via light microscopy, but no abnormalities (developmental, inflammatory or otherwise) were identified (data not shown).

Fig 4. — Viral RNA levels (log10 copies per mg tissue) measured at delivery from pregnant macaques inoculated with Spondweni virus (SPOV; orange), Zika virus Dak Ar 41524 strain (ZIKV-DAK; blue), or Zika virus PRVABC59 strain (ZIKV-PR; green) earlier in gestation. Tissues are grouped into maternal (Dam) and maternal-fetal interface (MFI) compartments. Each point is a single sample from one animal (SPOV n = 4; ZIKV-DAK n = 5; ZIKV-PR n = 8). Only samples with detectable viral RNA are shown; dam 103 and her fetus had no detectable vRNA. The horizontal dashed line indicates the assay’s lower limit of detection (LLOD = 108 copies/mg for SPOV and 81 copies/mg for ZIKV), defined as the concentration detected ≥95% of the time in validation experiments (see Methods). Values plotted below this threshold reflect occasional true-positive detections but are not reliably quantifiable. ZIKV-DAK-inoculated macaques were previously reported by Rosinski and Raasch *et al.* [41]. ZIKV-PR-inoculated macaques were previously reported by Krabbe *et al*. [53].

Table 1.

Virological and pathological findings

ID	Maternal spleen vRNA¹	MFI vRNA¹	Fetal vRNA¹	Chronic chorionic villitis²	Maternal vascular malperfusion	Villous ischemic injury²	Trans-mural ischemia²
101	±³	+	−	+	−	+	+
102	+	−	−	+	+	+	+
103	−	−	−	+	+	+	+
104	−	+⁴	−	+	+	+	+

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Detection of Spondweni vRNA by qRT-PCR (duplicate wells per tissue). In this table + = both wells amplified (2/2); ± = equivocal (1/2); − = neither well amplified (0/2). All detections were below the assay lower limit of detection (LLOD = 108 copies/mg tissue) and are not reliably quantifiable. All fetal tissues (60 compartments) were negative (see S1 Table for the full list of tissues tested).

Abbreviations: vRNA, viral RNA; MFI, maternal-fetal interface.

Pathology scoring on H&E. (+) present; (−) not observed. Chronic chorionic villitis refers to mononuclear/lymphohistiocytic chorionic villitis. Transmural ischemia was defined as any plate-to-plate area of ischemic necrosis present on any full-thickness section from either placental disc. See S2 Table for a summary of placental histopathological findings.

101 maternal spleen: one of two wells amplified (0 and 7 copies/mg).

⁴

104 MFI: one placental parenchyma sample was equivocal, and a second parenchyma sample tested positive in both wells (7 and 8 copies/mg). Decidua basalis (two samples) and chorionic plate (four samples) tested positive but below the LLOD in both wells.

SPOV exposure induces placental inflammation and vascular pathology

We reasoned that placental histopathology might reveal signs of earlier infection or vascular injury during acute maternal viremia, even in the absence of gross fetal abnormalities or detectable SPOV RNA in fetal tissues at delivery. To investigate this possibility, we examined H&E-stained full-width and full-thickness central sections (“center cuts”) from the midpoint of each placental disc together with representative sections from each cotyledon collected at cesarean section. Macaques typically have bidiscoid placentas, and both placental discs were sampled per pregnancy. In the placentas from 3 of 4 SPOV-inoculated dams (102, 103, and 104), we observed persistently muscularized arteries in the decidua basalis (Fig 5A), suggestive of impaired maternal spiral-artery remodeling, a hallmark of maternal vascular malperfusion (MVM; for a normal, remodeled maternal spiral artery, see the bottom of S4A Fig, showing a large basal-plate artery with a wide lumen and thin vessel wall). These findings were accompanied by dense clusters of cells with segmented (multilobed) nuclei, morphologically consistent with neutrophilic infiltration, in the decidua and small proximal vessels (Fig 5B), indicating acute (neutrophilic) deciduitis and vasculitis. Larger decidual vessels, in contrast, exhibited predominantly mononuclear infiltrates composed of small round lymphocyte-like cells and evidence of prior hemorrhage, including interstitial hemosiderin deposition and hemosiderophages. and fibroblast disintegration (Fig 5C–D). Per-animal summaries are provided in S2 Table. These findings are consistent with mononuclear inflammation of the larger decidual vessels. In aggregate, these findings suggest that SPOV infection can induce placental inflammation and vascular pathology that may impair maternal-fetal blood exchange, even in the absence of overt fetal harm.

Fig 5. — **(A)** Persistently muscularized artery (black arrow) in the decidua basalis from macaque 103. **(B)** Neutrophilic deciduitis (top arrow) and vasculitis (bottom arrows) of a small vessel in the decidua parietalis from macaque 101. The bottom arrows show neutrophils in the lumen (right-most arrow) and epithelium (left-most arrow) of the vessel. **(C)** Mononuclear/lymphohistiocytic vasculitis of a large artery within the basal plate of macaque 102. **(D)** Mononuclear/lymphohistiocytic vasculitis of a large basal plate vessel from macaque 102, with the left-most arrow showing a hemosiderin deposit and the right-most arrow showing a fibroblast with a disintegrating nucleus.

We next evaluated the histopathology of the chorionic villi, which mediate the oxygenation of fetal blood. All four SPOV-exposed macaques exhibited evidence of villous injury, although the severity and histological features varied. A representative image of normal villous architecture (S4B Fig) shows branching villi without evident inflammation. Macaques 102, 103, and 104 showed multifocal mononuclear/lymphohistiocytic villitis with associated perivillous fibrin deposition, mineralization, and ischemic necrosis. Among these, macaque 103 exhibited the most severe villous pathology, with widespread necrosis and collapse of villous architecture (Fig 6A). Macaque 102 displayed moderate villitis and hemorrhage, along with diffuse perivillous fibrin and mineral accumulation within the basal plate and adjacent villi (Fig 6B). Macaque 104 showed mononuclear/lymphohistiocytic villitis accompanied by small plasma cell aggregates consistent with chronic deciduitis (Fig 6C). Most strikingly, macaque 104 also had extensive transmural placental ischemic necrosis and loss of parenchymal villi that caused the chorionic plate to collapse into a distinctive “V” configuration (S5 Fig). In contrast, macaque 101 had relatively mild mononuclear/lymphohistiocytic chorionic villitis with focal villous ischemic injury, including small areas of necrosis, thrombosis, and mineralization (Fig 6D). Across animals, MVM was present in 3/4 and villous ischemic injury in 4/4, and transmural ischemia was present in all four placentas by our definition (any plate-to-plate region of ischemic coagulative necrosis), with the most striking example observed in macaque 104 (Table 1). The spectrum of villous injury by animals is summarized in S2 Table. Together, these findings demonstrate that SPOV exposure can result in diverse villous pathology ranging from focal inflammation to extensive multifocal ischemia, highlighting that significant placental pathology may occur without translating to overt fetal harm. Two of four infants did have minimal to mild multifocal neutrophilic inflammation involving the liver (fetuses 002 and 003 from macaques 102 and 103, respectively), spleen (fetus 003), salivary gland (fetus 002), and axillary lymph node (fetus 002). No other organs, including the eyes and brain, had significant histologic changes in any of the infants.

Fig 6. — **(A)** Mononuclear/lymphohistiocytic chorionic villitis in Macaques 103, **(B)** 102, **(C)** 104, and **(D)** 101. In each image, representative clusters of lymphohistiocytic infiltration are denoted by black arrows, and the loss of villous architecture is circumscribed by a dashed black line.

Discussion

ZIKV and SPOV are close relatives and the two known members of the Spondweni virus serocomplex. It is currently unknown whether SPOV has the potential to cause fetal harm. This study provides the first assessment of SPOV infection during pregnancy in a translational rhesus macaque model. We demonstrate that SPOV induces placental pathology, yet does not cause fetal demise or gross fetal abnormalities when fetuses were collected just prior to term. Nevertheless, SPOV was associated with placental pathology in all macaques and is likely able to infect the placenta.

We found very low levels of SPOV RNA, below the assay’s lower limit of detection, in several tissue specimens from 3 of 4 dams and/or fetuses. We believe these findings represent bona fide detection of SPOV genomic material for several reasons. RT-PCR assays can detect vRNA at lower concentrations, but below this limit we expect detection of true positives to become increasingly sporadic. Values below the LLOD are therefore detectable but not reliably quantifiable, limiting our ability to draw conclusions about prolonged viral replication at these sites. Extraction-negative and no-template controls were uniformly negative, and positive controls were uniformly positive on every instrument run. Presence of SPOV RNA is also consistent with findings of pathological changes in the placenta of some SPOV-infected dams. Together, our data therefore argue against cross-contamination and suggest true SPOV RNA presence at very low levels in maternal and fetal tissues ~120 days after inoculation.

Placental pathology in all SPOV-inoculated macaques and low, but detectable, levels of vRNA at term (~120 days post-inoculation) in maternal-fetal interface tissues of n = 2/4 macaques suggest that SPOV is able to induce placental injury that can persist even in the absence of detectable fetal infection (Table 1). This is similar to what we observed with the Asian-lineage ZIKV strain PRVABC59 but direct comparisons of placental viral burdens with ZIKV-DAK are complicated by sampling at different timepoints: ZIKV-DAK tissues were collected at the time of fetal demise (within 20 days of inoculation), when some dams remained viremic; SPOV and ZIKV-PR tissues were collected more than 100 days after inoculation, long after maternal viremia had resolved. Findings of mononuclear/lymphohistiocytic chorionic villitis and maternal vascular malperfusion suggest that SPOV induced placental damage early in pregnancy, triggering lasting inflammatory damage and impaired spiral artery remodeling. Further, SPOV induced neutrophil-predominant inflammation in the decidua parietalis of macaque 101, which also contained low levels of vRNA in the decidua basalis, together suggesting that SPOV is capable of prolonged viral persistence and sustained inflammatory responses in both the placenta and the fetal membranes. Last, SPOV can induce severe placental pathology: macaque 104, which contained low vRNA in the decidua, parenchyma, and chorionic plate, developed significant regions of ischemia and villous necrosis S5 Fig). These observations indicate that, despite considerable pathology and vRNA presence upon delivery, placental structure and function remained resilient enough to prevent any detectable vertical transmission of SPOV or notable injury to the fetus.

Gestational timing of viral inoculation profoundly affects ZIKV congenital outcomes in non-human primate models. In previous studies, inoculation later in the first trimester (around GD 45) with African-lineage ZIKV-DAK or Puerto Rican ZIKV-PRVABC59 was not consistently associated with fetal demise or marked fetal growth restriction, and in some cohorts no ZIKV RNA was detected in infants at delivery. [42,51,60,61]. Vertical transmission can occur at this gestational age: inoculation in the mid-first trimester (GD 31–38) with a French Polynesian strain of ZIKV (ZIKV-FP) caused congenital infection with fetal ocular injury [46]. In contrast, when inoculated at GD ~30, ZIKV-DAK caused uniform fetal demise and vertical transmission 12–20 days after inoculation [41]. Notably, infectious ZIKV-DAK has been recovered from amniotic fluid within ~2 weeks of inoculation at GD ~45 in at least one pregnancy, indicating that African-lineage ZIKV can also cause detectable fetal infection, particularly when the fetal compartment is sampled soon after infection [62]. Similarly, the absence of severe fetal outcomes in our SPOV inoculations at GD ~30 does not exclude the possibility of SPOV posing a congenital infection risk in humans.

A variety of viral and host factors likely contribute to these differences in infection outcomes. The stark contrast in fetal survival rates for dams inoculated with ZIKV-DAK at GD 30 vs. GD 45 suggests that there is a critical window during the mid-first trimester when the fetus is particularly susceptible to infection, perhaps because the MFI is still undergoing rapid trophoblast invasion and spiral-artery remodeling at GD 30, while these processes are more advanced by GD 45 [41]. Inoculum dose likely also plays a role. All infected dams whose viremia data are summarized in this manuscript were inoculated with 1×10^4 PFU virus, an amount in the middle of the range thought to be delivered by mosquitoes [54]. Notably, we previously showed that 1×10^8 PFU ZIKV-DAK inoculated at GD ~45 led to fetal infection, indicating that high doses can overcome barriers to vertical transmission that develop over the course of the first trimester [40]. However, strain differences are prominent: inoculation with 1×10^4 PFU of ZIKV-FP at GD 31–38 resulted in vertical transmission with fetal ocular injury [46]. Across experiments that maintained the same inoculum dose (1×10^4 PFU), inoculation route, gestational timing (GD ~30), and animal care conditions, outcomes diverged by virus: ZIKV-DAK produced uniform fetal demise with vertical transmission [41], whereas in the present study, SPOV produced neither fetal demise nor detectable vertical transmission despite consistent placental pathologies. Indeed, SPOV infection more closely resembled our previous observations of ZIKV-PR infection at GD 30, in which very low levels of vRNA were detected in MFI tissues near the time of delivery. Interestingly, the duration of infection during pregnancy appeared more variable for ZIKV-PR—with viremia lasting over 50 days in one dam—than we observed for SPOV, which was cleared from plasma within 30 days by all four dams in this study (Fig 2). Taken together, our studies therefore indicate that viruses within the Spondweni virus serocomplex likely possess a wide range of pathogenic potential during gestational infection. Improving risk assessments for these viruses will require further studies to identify specific viral and host determinants of pathogenic outcomes.

Several limitations constrain our findings. Our data, from a small cohort (n = 4) with one SPOV strain, dose, and inoculation at a single gestational time point, suggest that SPOV can damage the placenta without demonstrable fetal infection or gross abnormalities observable at term. Because fetal tissues were uniformly negative for SPOV vRNA while placentas showed consistent pathology (and occasional low-level MFI vRNA), our findings are most consistent with placenta-focused infection and injury rather than direct infection of the fetus. Whether such injury manifests in postpartum impacts such as neurodevelopmental effects is unknown and would require long-term follow-up. Further, our study design, which terminated pregnancies at near-term rather than during acute infection, may have reduced our ability to detect vertical transmission or characterize acute placental responses. To understand the placental response and vRNA distribution during acute maternal infection, future studies should include necropsies at earlier post-inoculation timepoints [63]. Additionally, longer-term studies should assess comprehensive developmental outcomes, which are critical to detect the subtle sequelae that we know ZIKV is capable of causing. The clinical challenge lies in recognizing that the absence of gross fetal abnormalities does not indicate the absence of risk; moreover, while cohort data exist for Asian-lineage ZIKV, comparable human data are lacking for SPOV and for African-lineage ZIKV, so we are unable to determine whether SPOV poses a different risk to human pregnancies.

The contrast between the consistent placental pathologies of SPOV and the direct fetal effects of ZIKV-DAK suggests that distinct or sequential pathways may underlie placental and fetal outcomes. This positions SPOV as a valuable comparative model for identifying the viral and host factors governing adverse pregnancy outcomes. Beyond its utility as a comparator, SPOV’s impact on the placenta raises important clinical concerns. While we observed no evidence of direct fetal harm in this cohort, the placental pathologies suggest the potential for indirect developmental effects through placental dysfunction [64]. These outcomes were not assessed in this study design but remain biologically plausible, given similar findings in ZIKV-exposed infants with developmental impairment [65,66]. These insights underscore the need for mechanistic studies of flavivirus-induced placental and fetal injury, as well as potential long-term sequelae. The severity of placental pathology we observed here warrants sustained surveillance and research in the context of expanding mosquito vector ranges and regions with ecological conditions favorable for flavivirus emergence.

Materials and Methods

Ethical approval

This study was approved by the University of Wisconsin College of Letters and Sciences and the Vice Chancellor for Research and Graduate Education Centers Institutional Animal Care and Use Committee (protocol numbers G006139 and G006256). The University of Wisconsin–Madison Institutional Biosafety Committee approved this work under protocol numbers B00000117 and B00000182.

Care and use of rhesus macaques

All rhesus macaques involved in this study were housed and cared for by the Wisconsin National Primate Research Center (WNPRC) staff, as previously described by our team [40–44,46,52,67]. Care practices strictly adhered to the guidelines outlined in the Animal Welfare Act, the National Research Council’s Guide for the Care and Use of Laboratory Animals, and the Weatherall report [68].

As previously described [40–44,46,52,67], the macaques’ diets included a variety of fruits, vegetables, nuts, cereals, seed mixtures, yogurt, peanut butter, popcorn, and marshmallows. In addition to dietary enrichment, structural enrichment (e.g., climbing structures) and manipulanda (e.g., toys) were provided to promote psychological well-being. Trained animal care staff evaluated all study animals twice daily for signs of pain, distress, or illness by observing appetite, stool quality, activity level, and overall physical condition. Macaques displaying abnormal clinical signs received appropriate care from attending veterinarians. Prior to all experimental procedures, including virus inoculation, blood collection, and physical examinations, animals were anesthetized with an intramuscular dose of ketamine (10 mg/kg) and closely monitored until fully recovered.

Study design

In this study, we infected four pregnant rhesus macaques (Macaca mulatta) with SPOV and compared their viral loads and fetal development with five macaques infected with African-lineage ZIKV and eight macaques infected with Asian-lineage ZIKV. Animals that met study criteria (no prior flavivirus exposure history or major surgery) were confirmed pregnant by ultrasound and subcutaneously inoculated during the first trimester (approximately gestational day 30) with 1×10^4 plaque-forming units of either SPOV (SAAr94), African-lineage ZIKV (Dak Ar 41524), or Asian-lineage ZIKV (PRVABC59). All macaques used in the study were screened to confirm that they were not infected with the following viruses at the time of inoculation: Macacine herpesvirus 1, simian retrovirus type D (SRV), simian T-lymphotropic virus type 1 (STLV), and simian immunodeficiency virus (SIV). Pan collection of urine via removable cage bottoms was performed at all sampling time points. Blood samples were obtained from the femoral or saphenous vein using a Vacutainer system or needle and syringe. Blood was collected on all dams prior to virus exposure (0 days post-inoculation; 0 dpi), daily from 1–10 dpi, twice weekly until two sequentially negative maternal plasma vRNA loads, then once weekly until study completion. Plasma and urine samples were collected at all time points; serum samples were collected at 0, 2, 4, 7, 10, 14, 21, and 30 dpi; peripheral blood mononuclear cells (PBMCs) were collected on 0, 7, 14, 21, and 30 dpi. Pregnancies were monitored throughout gestation using ultrasound (described below) to assess fetal viability and ensure the well-being of both the dam and the fetus. Fetal outcomes were assessed upon cesarean-section delivery near GD 155, approximately 10 days prior to full term.

Viral inoculations

The SPOV isolate (SAAr94) was derived from a Mansonia uniformis mosquito before five passages in unknown conditions, one passage in Vero cells, and two passages in C6/36 (Aedes albopictus) cells. Contemporary isolates of SPONV do not exist; thus, we used the only available low-passage isolate. Our SPOV challenge stock is 98.8% nucleotide identical to the SPOV genome recovered from mosquitoes in Haiti (GenBank: MG182017; SRA experiment accession: SRX7395146), but we acknowledge that although the sequences are almost identical, the slight difference could result in important phenotypic impacts. The ZIKV-DAK isolate (Zika virus/Aedes africanus-tc/SEN/1984/41524-DAK) was derived from Ae. africanus mosquitoes before two passages in Ae. pseudocutellaris, two passages in Vero cells, and two passages in C6/36 cells (BEI Resources; Manassas, Virginia; NR-50338). The ZIKV-PR isolate (Zika virus/H.sapiens-tc/PUR/2015/PRVABC59_v3c2) was provided by Brandy Russell (CDC, Fort Collins, CO, USA). On the day of the viral challenge, virus stocks were thawed, diluted to 1 mL with phosphate-buffered saline, and loaded into 1-mL syringes, which were kept on ice in a cooler until inoculation. Each dam was inoculated subcutaneously in the upper back, between the shoulder blades (cranial dorsum).

Ultrasonographic analysis of fetal development during gestation

Ultrasounds were conducted every two weeks according to the procedures described previously [46] to monitor the heart rate and growth of the fetus. All dams were anesthetized with an intramuscular dose of ketamine (10 mg/kg) prior to all ultrasounds. Fetal measurements included fetal femur length (FL), biparietal diameter (BPD), head circumference (HC), and abdominal circumference (AC). All measurements were plotted against normative data collected by the California National Primate Research Center [55,56], containing mean measurements and standard deviations for specific gestational days of rhesus macaque pregnancy.

Z-scores represent the number of standard deviations a given fetal measurement deviates from the normative mean for that gestational age. For each metric (FL, BPD, HC, AC), we calculated Z-scores by comparing individual values to gestational age-matched reference data from the California National Primate Research Center [55,56]. To smooth variation in reference values across gestation, we fit quadratic regression models to the normative mean and standard deviation as functions of gestational age and used the resulting estimates to calculate predicted Z-scores. A one-sample t-test was used to determine whether the mean Z-score for each metric differed significantly from zero; in human fetal medicine, Z-scores less than 2 are generally not considered to be clinically meaningful [57,58]. To assess whether Z-scores changed over time, we used a linear mixed-effects model with animal-specific random effects and an autoregressive correlation structure, regressing Z-scores on gestational age. In this model, a significant slope indicated a systematic change in growth trajectory over gestation. Model assumptions were verified by examining residuals. All analyses were performed using SAS software (version 9.4; SAS Institute, Cary, NC), and two-sided p-values < 0.05 were considered statistically significant.

Cesarean-section delivery and tissue collection

At ~GD 155, all SPOV-inoculated pregnancies were surgically delivered via cesarean section, as previously described [41,46]. These were non-terminal survival procedures for the dams. We performed a comprehensive necropsy on the entire conceptus, including fetus, placental discs, fetal membranes, and umbilical cord. Fetuses were euthanized with an intravenous dose of at least 50 mg/kg sodium pentobarbital.

Tissue collection was performed under sterile conditions, as previously described [41,46]. Maternal biopsies were collected during surgery and included the spleen, liver, mesenteric lymph node, and uterine placental bed. Placental and fetal collections were performed immediately following cesarean section, as previously described [41,46]. Briefly, we collected a full-thickness center-cut of each placental disc and cotyledon. Individual cotyledons were then separated into decidua, chorionic plate, and parenchymal components. A full list of fetal, maternal, and maternal-fetal tissues collected can be found in Supplemental Table 1. Each tissue was visually examined and subsequently divided into sections for viral RNA quantification, histology, or long-term storage. When tissue sizes were limited, viral RNA quantification was prioritized.

Processing of blood and urine samples

For larger blood draws, where we wanted to collect plasma and PBMC samples, we used the following protocol: EDTA-treated blood was layered over Ficoll in a 1:1 ratio using sterile transfer pipettes, centrifuged at 1860 relative centrifugal force (rcf; g-force) for 30 minutes, and brake set at 1. Sterile transfer pipettes were used to remove the plasma top layer into a sterile 15-mL conical tube. The “buffy coat” containing peripheral blood mononuclear cells (PBMCs) was then carefully transferred into a 15-mL conical tube with five milliliters of room-temperature R10 media. Both plasma and buffy coat tubes were spun at 670 rcf for 8 minutes. Plasma was stored in two 325-uL aliquots, with the remainder stored in larger aliquots. Media was removed from the buffy coat tubes, and the PBMC pellet was treated with 5 mL of ACK for 5 minutes, then quenched with 5 mL of R10. PBMCs were spun down at 670 rcf for 5 minutes, the media was removed, and the cells were resuspended in R10 before counting in the Beckman Coulter Counter. PBMCs were stored in 1 mL of CryoStor CS5 freezing media (STEMCELL Technologies, Cat #07930) in aliquots of 5e6–10e6 cells per tube. Samples were stored in Mr. Frosty containers (Thermo Fisher Scientific, Cat. No. 5100–0001) at −80°C overnight before moving to liquid nitrogen storage the next day.

For smaller blood draws, where we wanted to only collect plasma or serum, we used the following protocols: smaller, EDTA-treated blood samples were spun at 1400 rcf for 15 minutes. Plasma was transferred to a sterile tube with sterile transfer pipettes, then spun again at 670 rcf for 8 minutes. Plasma was stored in two 325-uL aliquots, with the remainder stored in larger aliquots. Blood in clot activator or serum-separating tubes was spun at 1400 rcf for 20 minutes, transferred to sterile tubes, and spun again at 670 rcf for 8 minutes. The serum was stored in 100 uL aliquots, with the remainder stored in larger aliquots. All plasma and serum samples were stored at −80°C.

Urine samples were collected via pan and were spun down at 500 rcf for 5 minutes to remove debris. 270 uL of urine, to be used for viral loads, was stored in 30 uL of DMSO and stored at 4°C before immediate testing. If the urine was not immediately tested, it was frozen in Mr. Frosty containers at −80°C. The remaining urine was stored in 1 mL aliquots at −80°C.

Viral RNA isolation from fluids

Viral RNA was isolated using the Maxwell 48 RSC instrument (Promega, Madison WI) with the Maxwell Viral TNA kit (Promega, Madison, WI). Samples were vortexed and briefly spun to remove any sample from the tube lid. 330 uL of the provided lysis solution (from the provided lysis and proteinase K) was added to empty microcentrifuge tubes, then 300 uL of each sample was added. Each tube was vortexed and then briefly spun on a benchtop centrifuge before incubation on a 56°C heat block for 10 minutes. New gloves were donned, and the sample tray was prepared per the manufacturer’s instructions during the 10-minute incubation step. Following incubation, all samples were briefly spun down and then run in the Maxwell 48 RSC instrument. After vRNA isolation, samples were transferred to new tubes and stored at −80°C if RT-PCR was performed later.

Viral RNA isolation from tissues

Tissue samples were stored in RNAlater. RNA was recovered from tissue samples using a modification of the method described by Hansen et al. (2013) [69]. Briefly, up to 200 mg of tissue was disrupted in TRIzol (Lifetechnologies) with 2 × 5 mm stainless steel beads using the TissueLyser (Qiagen) for 3 minutes at 25 r/s twice. Following homogenization, samples in TRIzol were separated using Bromo-chloro-propane (Sigma). The aqueous phase was collected and glycogen was added as a carrier. The samples were washed in isopropanol and ethanol precipitated. RNA was fully re-suspended in 5 mM tris pH 8.0.

Viral RNA quantification by RT-PCR

Quantitative reverse transcription-PCR (RT-PCR) was performed on a Roche LightCycler 480 (Roche Diagnostics, Indianapolis, IN) with ThermoFisher TaqMan Fast Virus 1-Step Master Mix for qPCR (Cat. No. 4444434) as previously described [52]. Briefly, samples were processed alongside water, where 10 uL of each RNA sample was added to water, master mix, 600 nM forward/reverse primers and random hexamers, and 100 nM probe. The plate containing the samples was sealed, spun for 10 seconds, and then run with the following cycling conditions: 50°C for 5 minutes (ramp rate: 4.4°C/second), 95°C for 20 seconds (ramp rate: 4.4°C/second), 50 cycles of 95°C for 15 seconds (ramp rate: 3.0°C/second) and 60°C for 1 minute (ramp rate: 2.2°C/second) before cooling at 40°C for 30 seconds (ramp rate: 2.2°C/second).

Primer and probe sequences for ZIKV RNA quantification:

Forward primer: 5’-CGYTGCCCAACACAAGG-3’

Reverse primer: 5’-CACYAAYGTTCTTTTGCABACAT-3’

Probe: 5’–6-carboxyfluorescein-AGCCTACCTTGAYAAGCARTCAGACACYCAA-BHQ1–3’

Primer and probe sequences for SPOV RNA quantification:

Forward primer: 5’-GGCATACAGGAGCCACATCAAAC-3’

Reverse primer: 5’-TGCGTGGGCTTCTCTGAA-3’

Probe: 5’–6-carboxyfluorescein-CATCACTGGAACAAYAAGGAGGCGCTGG-BHQ1–3’

The lower limit of detection (LLOD) of this assay in body fluids for ZIKV is 150 copies/ml and 81 copies/mg in tissues. The LLOD of this assay in body fluids for SPOV is 175 copies/ml and 108 copies/mg in tissues. LLOD is defined here as the lowest concentration of viral nucleic acid that can be reliably detected as true-positive with a 95% confidence interval.

Quantification of plaque reduction neutralization by serum

Serum neutralization of SPOV was quantified using a plaque-reduction neutralization test (PRNT) on Vero cells, following the methods outlined by Jaeger et al. [52]. Serum samples were processed in clot activator or serum-separating tubes as described above and screened against the same SPOV stock used for viral inoculation. Neutralization curves were generated in GraphPad Prism 8 through nonlinear regression and evaluated using Prism’s ECanything equation to estimate EC50, which represents the serum dilution titer required to inhibit 50% of viral infectivity. Neutralization curves and EC50 values were generated for serum from each macaque at 0 and approximately 30 days post-inoculation.

Histopathological analysis of placental, neural and ocular tissues

Tissue preparation and histopathological analysis were performed as previously described [46,59]. Briefly, placental tissues were fixed in 4% paraformaldehyde (PFA) overnight (~24 hours) and then transferred to 70% ethanol until routine paraffin embedding. Neural and ocular tissues were fixed separately in 10% neutral buffered formalin (NBF), then routinely processed and paraffin embedded. Paraffin sections (5 μm) were cut and stained with hematoxylin and eosin (H&E) using standard protocols [46,59]. All histological assessments were conducted by veterinary pathologists under blinded conditions with respect to vRNA and fetal-growth findings.

Supplementary Material

Supplement 1

NIHPP2026.08.10.743875v1-supplement-1.pdf^{(15.6MB, pdf)}

Acknowledgments

We thank our colleagues at the Wisconsin National Primate Research Center (Animal Care Services, Veterinary Services, Pathology Services, and Scientific Protocol Implementation) for their invaluable care of the animals and their support throughout the study. We also thank members of the Friedrich and O’Connor laboratories for helpful discussions and technical assistance.

Funding:

This study was funded by AI132563 and AI132132 from the National Institutes of Health (NIH). H.J.R. was supported by the NIH National Institute for General Medical Sciences T32 training grant to the UW-Madison Cellular and Molecular Pathology Training Program (T32GM135119). H.J.R. and C.M.C. were supported by the UW-Madison Parasitology and Vector Biology Training Program, funded by NIH National Institute of Allergy and Infectious Disease (NIAID) award T32AI007414. A.S.J. was supported by the University of Minnesota Institute for Molecular Virology Training Program, funded via NIH-NIAID T32AI083196. The funders had no role in the study design, data analysis, data interpretation, or this report’s writing. All authors had full access to the data in the study and accepted the responsibility to submit it for publication.

Footnotes

Competing interests: The authors declare no competing financial interests.

Additional information: Correspondence and requests for materials should be emailed to T.C.F.

Data availability

Data, code, and figures are available for replication of these results on GitHub (https://github.com/RiesHunter/SPOV).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplement 1

NIHPP2026.08.10.743875v1-supplement-1.pdf^{(15.6MB, pdf)}

Data Availability Statement

Data, code, and figures are available for replication of these results on GitHub (https://github.com/RiesHunter/SPOV).

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PERMALINK

This is a preprint.

Spondweni virus infection in pregnant rhesus macaques causes placental pathology without apparent fetal harm

Hunter J Ries

Livia Romanov

Maria C Charles

Chelsea M Crooks

Carson DePagter

Alex Richardson

Grace A VanSleet

Andrea M Weiler

Jens C Eickhoff

Katharina S Stewart

Leandro B C Teixeira

Eric Peterson

Michele Schotzko

Heather A Simmons

Jenna R Rosinski

Lauren E Raasch

Anna S Jaeger

Elaina R Razo

Emma L Mohr

David H O’Connor

Christina M Newman

Matthew T Aliota

Thomas C Friedrich

Abstract

Author Summary

Introduction

Results

Study design

Fig 1. Study design.

Pregnant macaques support SPOV infection with occasional prolonged shedding

Fig 2. Plasma viremia kinetics in pregnant macaques after Spondweni or Zika virus inoculation.

Prenatal ultrasound reveals no consistent evidence of SPOV-associated intrauterine growth restriction

Fig 3. Fetal growth trajectories in Spondweni virus-inoculated macaque pregnancies.

SPOV RNA is present at low levels in maternal and fetal tissues upon delivery

Fig 4. Tissue viral RNA at delivery in dams and the maternal–fetal interface.

Table 1.

SPOV exposure induces placental inflammation and vascular pathology

Fig 5. Representative images of placental blood vessel histopathology.

Fig 6. Representative images of placental chorionic villi histopathology.

Discussion

Materials and Methods

Ethical approval

Care and use of rhesus macaques

Study design

Viral inoculations

Ultrasonographic analysis of fetal development during gestation

Cesarean-section delivery and tissue collection

Processing of blood and urine samples

Viral RNA isolation from fluids

Viral RNA isolation from tissues

Viral RNA quantification by RT-PCR

Quantification of plaque reduction neutralization by serum

Histopathological analysis of placental, neural and ocular tissues

Supplementary Material

Acknowledgments

Funding:

Footnotes

Data availability

References

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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