key: cord-0291449-sdy4dtf1
authors: Bohm, Ellie K.; Vangorder-Braid, Jennifer T.; Jaeger, Anna S.; Moriarty, Ryan V.; Baczenas, John J.; Bennett, Natalie C.; O’Connor, Shelby L.; Fritsch, Michael K.; Fuhler, Nicole A.; Noguchi, Kevin K.; Aliota, Matthew T.
title: Zika virus infection of pregnant Ifnar1−/− mice triggers strain-specific differences in fetal outcomes
date: 2021-05-15
journal: bioRxiv
DOI: 10.1101/2021.05.14.444269
sha: a5ad22659564f1023da3c58f19a9ffc1fea3ab09
doc_id: 291449
cord_uid: sdy4dtf1

Zika virus (ZIKV) is a flavivirus that causes a constellation of adverse fetal outcomes collectively termed Congenital Zika Syndrome (CZS). However, not all pregnancies exposed to ZIKV result in an infant with apparent defects. During the 2015-2016 American outbreak of ZIKV, CZS rates varied by geographic location. The underlying mechanisms responsible for this heterogeneity in outcomes have not been well defined. Therefore, we sought to characterize and compare the pathogenic potential of multiple Asian/American-lineage ZIKV strains in an established Ifnar1−/− pregnant mouse model. Here, we show significant differences in the rate of fetal demise following maternal inoculation with ZIKV strains from Puerto Rico, Panama, Mexico, Brazil, and Cambodia. Rates of fetal demise broadly correlated with maternal viremia but were independent of fetus and placenta virus titer, indicating that additional underlying factors contribute to fetus outcome. Our results, in concert with those from other studies, suggest that subtle differences in ZIKV strains may have important phenotypic impacts. With ZIKV now endemic in the Americas, greater emphasis needs to be placed on elucidating and understanding the underlying mechanisms that contribute to fetal outcome. IMPORTANCE Zika virus (ZIKV) actively circulates in 89 countries and territories around the globe. ZIKV infection during pregnancy is associated with adverse fetal outcomes including birth defects, microcephaly, neurological complications, and even spontaneous abortion. Rates of adverse fetal outcomes vary between regions, and not every pregnancy exposed to ZIKV results in birth defects. Not much is known about how or if the infecting ZIKV strain is linked to fetal outcomes. Our research provides evidence of phenotypic heterogeneity between Asian/American-lineage ZIKV strains and provides insight into the underlying causes of adverse fetal outcomes. Understanding ZIKV strain-dependent pathogenic potential during pregnancy and elucidating underlying causes of diverse clinical sequelae observed during human infections is critical to understanding ZIKV on a global scale.

Zika virus (ZIKV) exposure during pregnancy can cause a constellation of adverse fetal outcomes, 26 collectively termed congenital Zika syndrome (CZS). However, a substantial proportion of pregnancies 27 with in-utero ZIKV exposure result in babies without apparent defects. Only an estimated 5-15% of 28

infants have ZIKV-related birth defects (1-3). Importantly, infants who are born apparently healthy can 29 manifest developmental and neurocognitive deficits months to years after birth (4-7), even if maternal 30 exposure resulted in asymptomatic infection (8) . Furthermore, there was an unequal distribution of 31 ZIKV cases and severe outcomes in all areas where ZIKV emerged in the Americas, demonstrating that 32 risk of CZS varied over time and with geographic location (reviewed in (9)). For example, the rate of 33 microcephaly differed between French Polynesia (1%) (10), the U.S. Territories and Freely Associated 34

States (5-6%) (11) , and the Dominican Republic (11%) (7) . Within Brazil, the rate of microcephaly 35 varied between São Paulo (0%) (12) , Pernambuco (2.9%) (13) , Rio de Janeiro (3.5%) (14) , Southeast 36 Brazil (1.5%) and Northeast Brazil (13%) (15) . However, it should be noted that accurate diagnosis of 37 microcephaly requires multiple measures after birth and the use of inconsistent definitions of cases and 38 complications can bias reporting (9) . For example, initial microcephaly rates were overestimated in 39

Brazil before INTERGROWTH-21st reference-based standards were implemented (16) . 40 Microcephaly is not the only adverse birth outcome that results from gestational ZIKV infection (17) , 41 and these rates varied as well. The U.S. Territories and Freely Associated States reported birth defects 42 in 14% of ZIKV-exposed pregnancies (11) . Pernambuco, Brazil reported adverse outcomes in 20% of 43 exposed pregnancies (13) , whereas São Paulo reported a 28% rate of adverse neurological outcomes 44 (12) . Strikingly, in Rio de Janeiro, 42% of infants born to ZIKV-exposed pregnancies had adverse 45 outcomes; however, this study used a broader definition for ZIKV-associated outcomes (14) . Because 46 current diagnostic testing remains suboptimal and inconsistent for the detection of congenital ZIKV 47 infection (18) , the relative risk of CZS in infants from ZIKV-exposed pregnancies remains unknown, and 48 it remains unknown whether the risk is equal in different geographic areas. Was the unequal 49 distribution in CZS incidence over time and region stochastic or were there other factors that 50 influenced these regional differences? A provocative explanation for the appearance of CZS in the 51

Americas is that contemporary ZIKV strains evolved from strains that cause fetal lethality to those that 52 cause birth defects and this may have facilitated recognition of ZIKV's ability to harm the developing 53 fetus (19) . Whether ongoing virus evolution during geographic spread in the Americas gave rise to 54 phenotypic variants that differ in their capacity to harm the developing fetus remains an open question. 55

Large case-control studies of pregnant women may prove useful for determining whether infecting 56 ZIKV genotype affects overall pathogenesis during pregnancy. However, these types of studies are 57 observational and are complicated by participant heterogeneity, including history of infection with 58 other flaviviruses, and the precise time, dose, and genetic makeup of the infecting virus. We therefore 59 aimed to better understand heterogeneity in ZIKV-associated pregnancy outcomes by investigating 60 whether there are Asian/American-lineage strain-specific phenotypic differences by using mice lacking 61 type I interferon signaling (Ifnar1 -/-). Although there are limitations regarding the translational relevance 62 of this model, transplacental ZIKV infection and fetal damage have been demonstrated (20) (21) (22) (23) , and it 63 has been used to compare maternal infection parameters, placental pathology, fetal infection, and 64 outcomes between ZIKV strains and the closely-related Spondweni virus (20) (21) (22) . Congenital ZIKV 65 studies in pregnant mouse models have used a variety of virus strains, as well as timing, route, and dose 66 of inoculation (22) (23) (24) (25) . This heterogeneity in design has made it difficult to compare results across 67 mouse studies because both inoculation dose and time of ZIKV exposure during pregnancy play a role 68 in determining fetal outcomes (26) . Therefore, we assessed fetal outcomes following infection by a 69 panel of five geographically distinct, low-passage Asian/American-lineage ZIKV strains at embryonic 70 day 7.5 (E7.5). Here, we found that all ZIKV strains infected the placenta but varied in their capacity to 71 cause overt fetal harm, suggesting that there is phenotypic heterogeneity in pregnancy outcomes that 72 is dependent on the infecting ZIKV genotype. PR, ZIKV-PAN, ZIKV-MEX, and ZIKV-BRA share >99.5% genome-wide nucleotide identity, and ZIKV-82 CAM shares over 98% genome-wide nucleotide identity, resulting in only 4-18 amino acid differences 83 between strains (Tables 1 and 2 measurements in mm of morphologically normal fetuses at E14.5 using ImageJ software. Significance 119 was determined by one-way ANOVA. (F) Tissue titer was measured by plaque assay for individual 120 homogenized placentas, fetuses, and concepti (when the fetus and placenta were indistinguishable due 121

to severe resorption). Symbols represent individual placenta, fetus, or conceptus from 4-6 independent 122 experiments for each treatment group. Bars represent the mean viral titer of each treatment group and 123

significance was determined by one-way ANOVA. Significance annotations for all figures: 124 ****p≤0.0001; ***p≤0.001; **p≤0.01; *p≤0.05. 125

Adverse fetal outcomes are dependent on the infecting ZIKV strain 126

Next, to assess fetal outcomes, dams were necropsied on E14.5. Gross examination of each conceptus 127 revealed overt differences among fetuses within pregnancies and with uninfected counterparts. 128

Fetuses appeared as either morphologically normal or undergoing embryo resorption, as defined in 129 (20) . At time of necropsy, we observed high rates of resorption from ZIKV-BRA-and ZIKV-CAM-130 infected pregnancies (ZIKV-BRA: 51% and ZIKV-CAM: 46%), which were significantly higher than the 131 other virus-inoculated groups and PBS-inoculated controls (Fisher's Exact test, p < 0.0001) ( Figure 1B) . 

To further characterize pathogenic outcomes during pregnancy, we measured crown-to-rump length 143 (CRL) to assess overall fetal growth (20, 22) . Only fetuses that appeared morphologically normal were 144 included for measurement of CRL to provide evidence for intrauterine growth restriction (IUGR). There 145 was a statistically significant reduction in CRL in ZIKV-BRA fetuses compared to fetuses from PBS-146 inoculated dams (One-way ANOVA with Tukey's multiple comparisons, p < 0.0001) ( Figure 1E) . In 147 contrast, mean CRL did not differ significantly between fetuses from ZIKV-MEX-, ZIKV-CAM-, ZIKV-148 PAN-, ZIKV-PR-, and PBS-inoculated dams (p > 0.1561). The lack of apparent IUGR for the other ZIKV 149 strains is contrary to other studies using Asian-lineage ZIKVs (French Polynesia and Cambodia) in which 150 fetuses developed severe IUGR (22, 23) . However, the discrepancy in outcomes may be the result of 151 differences in timing of challenge and necropsy, dose and/or route of inoculation, dam age, litter size, 152 or metrics for defining grossly normal fetuses compared to those undergoing resorption at a later 153 embryonic age. 154

Next, to determine the potential of each ZIKV strain to be vertically transmitted, a subset of placentas 156 and fetuses were collected for plaque assay at time of necropsy from each litter in all treatment groups. 157

No infectious virus was detected by plaque assay in any fetus sample from any treatment group ( Figure  158 1F) and the absence of ZIKV fetal infection was confirmed by RNA In Situ Hybridization (ISH). In 159 contrast, virus was detected in placentas from all virus-inoculated groups at time of necropsy at E14.5 (7 160 dpi). ZIKV-PR placenta titers were significantly higher than ZIKV-PAN and ZIKV-MEX titers (One-way 161 ANOVA with Tukey's multiple comparisons, p <0.0001 and p = 0.0006), but only modestly higher than 162 ZIKV-CAM and ZIKV-BRA titers (p = 0.5591 and p = 0.5693) ( Figure 1F ). In ZIKV-PR, ZIKV-PAN, ZIKV-163 MEX, and ZIKV-BRA groups, placenta titer was not a predictor of partner fetus outcome (One-way 164 ANOVA, p > 0.0970). Although limited by the number of data points, ZIKV-CAM placentas with 165 resorbed fetuses had significantly higher titers than those from normal fetuses (p = 0.0182). Therefore, 166

we hypothesized that additional factors, outside of fetus and placenta virus levels, contribute to poor 167 fetal outcomes. 168 thrombi, inflammation, and apoptosis (20, 21) . Interestingly, the overall severity observed within virus 192 groups was relatively subtle compared to our previous studies (20, 21) , which may account for the 193 higher background scores noted in the PBS control group (Figure 2A-C) . There also were clear strain-194 specific differences in the amount of placental pathology observed, with ZIKV-CAM displaying the most 195 severe histologic phenotype (Figure 2 ). Similar to placenta titer, pathology severity score was not a 196 predictor of adverse fetal outcome for any treatment group. 197

Due to the lack of vertical transmission and an association between fetal outcome and placenta 199 infection and pathology, we hypothesized that IFN induction in the placenta was responsible for 200 determining fetal outcome. Indeed, it has previously been shown that type I IFN signaling, not the 201 levels of virus, mediated pathology following intravaginal ZIKV infection in Ifnar1 +/fetuses and 202 placentas (23) . Accordingly, we examined the transcriptional changes of the interferon-stimulated 203 genes (ISGs) Oasl2, Mx1, and Ifit1 in the placenta to determine if IFN induction (or the lack thereof) may 204 be contributing to fetal demise. We observed that Oasl2, Mx1, and Ifit1 were induced regardless of 205 infecting ZIKV genotype in our model (Figure 3A-C) . Interestingly, ZIKV-MEX placentas had 206 significantly lower Mx1, Oasl2, and Ifit1 transcript abundance compared to the other virus groups (One-207 way ANOVA, p < 0.0357). Across virus groups, pregnancies with better outcomes (i.e., lower rates of 208 We then examined if infection outcomes were due to differential expression of IFN-λ or its 226 heterodimeric receptor Ifnλr1/Il10rβ. We measured the relative transcript abundance of Ifnλr1, Ifnλ2, 227

and Ifnλ3 in the placenta at time of necropsy at E14.5. Ifnλ1 is a pseudogene and the genomic region of 228

Ifnλ4 is missing in mice (28) and, therefore, were not measured. Consistent with our ISG data, we 229 observed modest induction of Ifnλr1 for all ZIKV strains ( Figure 4A) . Importantly, pregnancies with 230 lower rates of resorption had higher expression of Ifnλr1 (Spearman, p = 0.0302) (Figure 4B) , and ZIKV-231 = 0.0087; mean ± SEM: -1.148 ± 0.333; n = 10). In contrast, Ifnλ2 and Ifnλ3 were not induced in any 233 placenta sample from ZIKV-infected mice (Figure 4C-D) . This, perhaps, was not surprising since a 234 previous mouse study showed that type III IFNs played little to no role in placental antiviral defenses 235 before placentation (25) . In our model, dams were infected on E7.5 and placental development is not 236 complete until E8.5-10.5 in mice (29, 30) . Still, it remains unknown whether the mouse placenta 237 constitutively releases type III IFNs in a manner similar to the human placenta, or whether these IFNs 238 are induced systemically or in response to placental infection (31) . Here, we did not detect robust 239 evidence for induction of type III IFNs despite detection of infectious virus in the placenta at time of 240 necropsy at E14.5. 241 without vertical transmission is sufficient to cause adverse outcomes. Placental insufficiency is now 260 being recognized as a potential contributor to some of these adverse outcomes (34, 35) , and our data 261 suggest that pregnancy loss is not solely driven by fetal infection. 262

One possible explanation for differences in fetal outcomes observed between treatment groups could 263 be due to differences in activation of and/or susceptibility to antiviral signaling at the MFI. It is 264

becoming increasingly apparent that IFN responses can have protective and/or pathogenic effects in 265 pregnancy (reviewed in (28)). Protection associated with IFN production prevents uncontrolled virus 266 replication, fetal infection, and maternal mortality (36) (37) (38) ; however, overproduction of type I IFNs are 267 known to be an underlying cause of pregnancy complications, including developmental defects similar 268 to those that result from infections with teratogenic pathogens (23, 39, 40) . As a result, there likely is a 269 critical balance that must occur between the beneficial antiviral effects of the IFN response to virus and general involvement in the success of human pregnancies (28, 36, 52) . Our data suggest that IFN 275 activation did not contribute to fetal demise and, in some cases, may have played a protective role. 276

Maternal viremia appeared to drive ISG induction in the placenta, which may not be surprising given 277 that the mouse labyrinth is perfused with maternal blood (53) . Higher maternal viremia also positively 278 correlated with increased resorption rate across virus groups. Therefore, maternal viremia may also 279 contribute to an increased risk of adverse fetal outcomes, alone or in combination with IFN-dependent 280 causes, direct pathogenic effects of the virus, or as a bystander effect associated with immune 281 responses unrelated to IFN induction. Importantly, we only assessed ISG expression at a single 282 timepoint, at time of necropsy (E14.5, 7 dpi), and expression profiles may differ depending on the 283 timing of collection. More studies are needed to better understand antiviral signaling at the MFI and the 284 mechanisms these virus strains exploit to harm the feto-placental unit. 285

Differences observed in fetal outcomes and histopathology across ZIKV strains may also be due to virus 286 genetic determinants of virulence and pathogenesis during congenital infection. Because 287 contemporary ZIKV isolates are so closely related, they are oftentimes used interchangeably in 288 laboratory research. But even though there is high genetic similarity between ZIKV strains, it is possible 289 that subtle genotypic differences could result in small, but biologically important, phenotypic 290 differences between strains. For example, evidence suggests that ZIKV virulence can be governed both 291 by viral nucleotide sequence and/or amino acid sequence (41, (54) (55) (56) (57) (58) , but the impact of a single amino 292 acid substitution may vary in the different strains chosen for analyses (20, 59) . ZIKV strains used here 293 share >98% genome-wide nucleotide identity and while it is unclear whether the differences in amino 294 acid and/or nucleotide sequence are responsible for the differences in the observed phenotypes, it is 295 possible that there is not a single determinant of ZIKV fetal pathogenicity. Future reverse genetic 296 studies will be needed to fully understand if there is a link between viral genotype and phenotype. 297

Our findings highlight that phenotypic heterogeneity exists between closely related ZIKV strains that 298 are commonly used for pathogenesis studies. To more rigorously assess the relative capacity of 299

Asian/American-lineage ZIKVs to cause adverse fetal outcome, future studies should carefully consider 300 the specific characteristics of the virus strains being used and consider them in the specific context of 301 the questions being asked. One important limitation to our study is that it is unclear whether the same 302 phenotypes would be recapitulated during human infection. Further, we do not argue that the 303 phenotypic differences we observe between strains indicate diminished risk of adverse outcomes 304 following infection during pregnancy with a certain ZIKV genotype (60) . On the contrary, the presence 305 of infectious ZIKV in the placenta for all strains tested is concerning and suggests that all ZIKV strains 306 have the capacity to harm the developing fetus depending on the specific pathophysiological context of 307 infection at the MFI. Here, our results provide a comparative framework to further investigate 308 underlying factors that determine fetal outcome during in-utero ZIKV exposure. 309

Ethical Approval 311 

A vial of all viral stocks used for challenges were each deep sequenced by preparing libraries of 337 fragmented double-stranded cDNA using methods similar to those previously described (20, 21, 61) . 338

Briefly, the sample was centrifuged at 5000 rcf for five minutes. The supernatant was then filtered 339 through a 0.45-μm filter. Viral RNA was isolated using the QIAamp MinElute Virus Spin Kit (Qiagen, Zealand). Briefly, using the viral-ngs workflow, host-derived reads that map to a human sequence 352 database and putative PCR duplicates were removed. The remaining reads were loaded into Geneious 353

Pro and mapped to NCBI Genbank Zika (GenBank:KX601166) reference sequences using bbmap local 354 alignment. Mapped reads were aligned using Geneious global alignment and the consensus sequence 355 was used for intra sample variant calling. Variants were called that fit the following conditions: have a 356 minimum p-value of 10e-60, a minimum strand bias of 10e-5 when exceeding 65% bias, and were 357 nonsynonymous. Consensus-level nucleotide substitutions and minor nucleotide variants are reported 358

in Table 3 

Quantification of virus titer in maternal serum, placenta, and fetuses were completed by plaque assay 363 on Vero cells. Duplicate wells were infected with 0.1 mL aliquots from serial 10-fold dilutions in growth 364 medium and virus was adsorbed for 1 hour. After incubation, the monolayers were overlaid with 3 mL 365 containing a 1:1 mixture of 1.2% oxoid agar and 2X DMEM (Gibco, Carlsbad, CA) with 10% (vol/vol) FBS 366 and 2% (vol/vol) Antibiotic Antimycotic. Cells were incubated at 37°C in 5% CO 2 for three days (ZIKV-367 PR, ZIKV-BRA, ZIKV-CAM), four days (ZIKV-PAN), or five days (ZIKV-MEX) for plaque development. 368

Cell monolayers were then stained with 3 mL of overlay containing a 1:1 mixture of 1.2% oxoid agar 369 with 4% neutral red (Gibco) and 2X DMEM with 2% (vol/vol) FBS, and 2% (vol/vol) Antibiotic 370

Antimycotic. Cells were incubated overnight at 37°C in 5% CO 2 and plaques were counted. 371

Female Ifnar1 -/mice on the C57BL/6 background were bred in the specific pathogen-free animal 373 facilities of the University of Minnesota within the College of Veterinary Medicine. Male C57BL/6 were 374 purchased from Jackson Laboratories. Timed matings between female Ifnar1 -/mice and male C57BL/6 375 mice resulted in Ifnar1 +/progeny. 376

All pregnant dams were between six and ten weeks of age and were randomly assigned to infected or 378 control groups. Matings between Ifnar1 -/dams and wildtype sires were timed by checking for the 379 presence of a vaginal plug, indicating gestational age E0.5. At embryonic day 7.5 (E7.5) dams were 380 inoculated in the right hind footpad with 1x10 3 PFU of the selected ZIKV strain in sterile PBS or with 381 sterile PBS alone to serve as experimental controls. All animals were closely monitored by laboratory 382 staff for adverse reactions and/ or clinical signs of disease. A submandibular blood draw was performed 383 at 2, 4, and 7 days post inoculation (dpi), and serum was collected to verify viremia. Mice were 384 humanely euthanized and necropsied at E14.5. 385

Following inoculation with ZIKV or PBS, mice were sacrificed at E14.5. Tissues were carefully dissected 387 using sterile instruments that were changed between each mouse to minimize possible cross 388 contamination. Each organ and neonate were morphologically evaluated in situ prior to removal. Using 389 sterile instruments, the uterus was removed and dissected to remove individual concepti. Each 390 conceptus was placed in a sterile culture dish and dissected to separate the fetus and the placenta, 391 when possible, for gross evaluation. Fetuses were characterized as "normal" or "resorbed", with the 392 latter being defined as having significant growth retardation and reduced physiological structure 393 compared to littermates and controls, accompanied by clearly evident developmental delay or 394 visualization of a macroscopic plaque in the uterus. A subset of fetuses and placentas from each litter 395 were reserved for viral titer analysis (preserved in PBS supplemented with 20% FBS and 1% Antibiotic 396

Antimycotic) or fixed in 10% neutral buffered formalin for imaging and histology. 397

Crown-to-rump length (CRL) was measured by tracing the distance from the crown of the head to the 399 base of the tail, using ImageJ. Infection-induced resorbed fetuses were excluded from measurement 400 analyses because they would not survive if the pregnancy was allowed to progress to term (20) . 401

Placenta tissues were fixed in 10% neutral buffered formalin at room temperature for 36-48 hours and 403 then transferred to 70% ethanol until alcohol-processed and embedded in paraffin. Paraffin sections (5 404 μm) were stained with hematoxylin and eosin (H&E) and the degree of pathology was scored by a 405 blinded pathologist, as described in (20) . The degree of placental pathology was rated on a relative 406 scale of 0-4: zero represents normal histologic features and 4 represents the most severe features 407 observed. Each zone of the placenta was scored individually for general overall pathology, amount of 408 inflammation, and amount of vascular injury. Only 'General' scores are shown because they were 409 representative of 'inflammation' and 'vascular injury' scores. 410

Immediately following necropsy, fetuses were fixed in 10% neutral buffered formalin at room 412 temperature for 36-48 hours and then transferred to 70% ethanol until alcohol-processed and 413 embedded in paraffin. Paraffin sections (5 μm) were deparaffinized and a hydrogen peroxide quench 414 was performed, followed by boiling in target retrieval reagent (catalog #322000). Tissue was then 415 incubated in Protease Plus solution (catalog #322330) in a HybEZ II Oven at 40˚C before hybridization 416 with the ZIKV probe (catalog #468361) and chromogen labeling using the RNAscope 2.5 HD Red Assay 417 (catalog #322360). In Situ Hybridization (ISH) was performed using the RNAscope Assay using products 418 and instructions (62) provided by the manufacturer (Advanced Cell Diagnostics. Inc., Newark, CA). Each 419 ISH run included ZIKV-infected positive control tissue to confirm the protocol was run as properly. After 420 labeling, tissue was counterstained using hematoxylin before cover-slipping for evaluation. 421

An Omni TH115 Homogenizer (Omni International, Omni Tissue Homogenizer (TH) -115V) was used to 423 homogenize fetus and placenta samples following necropsy. Samples were submerged in chilled PBS 424 supplemented with 20% FBS and 1% Antibiotic Antimycotic in 15mL Omni sealed plastic tubes (Omni 425

International, Catalog # 00-2015-25). Omni soft tissue probes (Omni International, Catalog # 30750) 426 were used to homogenize samples at the highest speed for 15 seconds (placentas) or 30 seconds 427 (fetuses). Homogenized samples were clarified by centrifugation at 10,000 x g for 2 minutes. The 428 supernatant was removed and 0.1mL was immediately plated for plaque assay. The remainder was 429 stored at -80°C. Gapdh and then 2-delta delta CT was calculated relative to PBS-inoculated controls. 439

All analyses were performed using GraphPad Prism. Unpaired Student's t-test was used to determine 441 significant differences in crown-rump lengths. Fisher's exact test was used to determine differences in 442 rates of normal versus resorbed concepti. One-way ANOVA with Tukey's multiple comparison test was 443 conducted to compare virus titers in maternal serum, placentas, fetuses, and concepti. Nonparametric 444

Spearman correlation was used to evaluate the relationship between variables. 445

Virus stock sequence data have been deposited in the Sequence Read Archive (SRA) with accession 447 codes SRX4510825, SRR14467422, and SRR14467421. The authors declare that all other data 448

supporting the findings of this study are available within the article. 

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Table 1: Total number of amino acid differences between strains and

CAM (FSS13025; GenBank:AFD30972), BRA (Paraiba_01