.2021 Jul 26;17(7):e1009788.

doi: 10.1371/journal.ppat.1009788. eCollection 2021 Jul.

Neuroinvasiveness of the MR766 strain of Zika virus in IFNAR-/- mice maps to prM residues conserved amongst African genotype viruses

Eri Nakayama^{1 2}, Fumihiro Kato¹, Shigeru Tajima¹, Shinya Ogawa³, Kexin Yan², Kenta Takahashi⁴, Yuko Sato⁴, Tadaki Suzuki⁴, Yasuhiro Kawai⁵, Takuya Inagaki¹, Satoshi Taniguchi¹, Thuy T Le², Bing Tang², Natalie A Prow^{2 6}, Akihiko Uda⁷, Takahiro Maeki¹, Chang-Kweng Lim¹, Alexander A Khromykh^{6 8}, Andreas Suhrbier^{2 6}, Masayuki Saijo¹

Affiliations

¹ Department of Virology I, National Institute of Infectious Diseases, Tokyo, Japan.
² QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.
³ Department of Applied Biological Chemistry, School of Agriculture and Life Sciences, The University of Tokyo, Tokyo, Japan.
⁴ Department of Pathology, National Institute of Infectious Diseases, Tokyo, Japan.
⁵ Management Department of Biosafety and Laboratory Animal, Division of Biosafety Control and Research, National Institute of Infectious Diseases, Tokyo, Japan.
⁶ Australian Infectious Disease Research Centre, GVN Center of Excellence, The University of Queensland and QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.
⁷ Department of Veterinary Science, National Institute of Infectious Diseases, Tokyo, Japan.
⁸ School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia.

PMID:34310650
PMCID: PMC8341709
DOI: 10.1371/journal.ppat.1009788

Neuroinvasiveness of the MR766 strain of Zika virus in IFNAR-/- mice maps to prM residues conserved amongst African genotype viruses

Eri Nakayama et al. PLoS Pathog.2021.

.2021 Jul 26;17(7):e1009788.

doi: 10.1371/journal.ppat.1009788. eCollection 2021 Jul.

Authors

Affiliations

¹ Department of Virology I, National Institute of Infectious Diseases, Tokyo, Japan.
² QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.
³ Department of Applied Biological Chemistry, School of Agriculture and Life Sciences, The University of Tokyo, Tokyo, Japan.
⁴ Department of Pathology, National Institute of Infectious Diseases, Tokyo, Japan.
⁵ Management Department of Biosafety and Laboratory Animal, Division of Biosafety Control and Research, National Institute of Infectious Diseases, Tokyo, Japan.
⁶ Australian Infectious Disease Research Centre, GVN Center of Excellence, The University of Queensland and QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.
⁷ Department of Veterinary Science, National Institute of Infectious Diseases, Tokyo, Japan.
⁸ School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia.

PMID:34310650
PMCID: PMC8341709
DOI: 10.1371/journal.ppat.1009788

Abstract

Zika virus (ZIKV) strains are classified into the African and Asian genotypes. The higher virulence of the African MR766 strain, which has been used extensively in ZIKV research, in adult IFNα/β receptor knockout (IFNAR-/-) mice is widely viewed as an artifact associated with mouse adaptation due to at least 146 passages in wild-type suckling mouse brains. To gain insights into the molecular determinants of MR766's virulence, a series of genes from MR766 were swapped with those from the Asian genotype PRVABC59 isolate, which is less virulent in IFNAR-/- mice. MR766 causes 100% lethal infection in IFNAR-/- mice, but when the prM gene of MR766 was replaced with that of PRVABC59, the chimera MR/PR(prM) showed 0% lethal infection. The reduced virulence was associated with reduced neuroinvasiveness, with MR766 brain titers ≈3 logs higher than those of MR/PR(prM) after subcutaneous infection, but was not significantly different in brain titers of MR766 and MR/PR(prM) after intracranial inoculation. MR/PR(prM) also showed reduced transcytosis when compared with MR766 in vitro. The high neuroinvasiveness of MR766 in IFNAR-/- mice could be linked to the 10 amino acids that differ between the prM proteins of MR766 and PRVABC59, with 5 of these changes affecting positive charge and hydrophobicity on the exposed surface of the prM protein. These 10 amino acids are highly conserved amongst African ZIKV isolates, irrespective of suckling mouse passage, arguing that the high virulence of MR766 in adult IFNAR-/- mice is not the result of mouse adaptation.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

**Fig 1. Virulence of MR766 and PRVABC59 after subcutaneous infection in IFNAR^-/- mice.**
(A) Survival of 7 to 32-week-old IFNAR^-/- mice after s.c. infection with MR766 or PRVABC59. Mice were infected with the indicated doses of MR766 or PRVABC59 and monitored until 14 dpi. Each group consisted of n = 6–12 mice, with similar numbers of male and female mice per group. Comparison of Kaplan-Meier survival curves between groups was performed by log-rank analysis. (B) Mean viral titers in the brain of mice s.c. infected with 1 × 10³ PFU of MR766 or PRVABC59 (n = 4 mice per group). Brains were harvested at 2, 4 and 6 dpi, with tissue titers determined by CCID₅₀ assays. Four uninfected mouse brains were used as controls (Cont.). Kolmogorov-Smirnov test was used for statistical analysis. Limit of detection was 0.83 log₁₀CCID₅₀/g indicated by the horizontal dashed line. (C) ZIKV RNA levels in brains of mice s.c. infected with 1 × 10³ PFU of MR766 or PRVABC59 were determined by qRT-PCR with the E gene-specific primers (n = 4 mice per group). ZIKV RNA levels were normalized to RPL13 mRNA levels. Four uninfected mouse brains were used as controls (Cont.). Kolmogorov-Smirnov test was used for statistical analysis. Limit of detection is indicated by the horizontal dashed line. (D) Mean viremia titers of mice s.c. infected with 1 × 10³ PFU of MR766 or PRVABC59 (n = 4 mice per group). Sera were collected at 2, 4 and 6 dpi, and titers were determined by CCID₅₀ assays. Four uninfected mouse sera were used as controls (Cont.). Kolmogorov-Smirnov test was used for statistical analysis. Limit of detection was 2 log₁₀CCID₅₀/ml indicated by the horizontal dashed line. (E) Blood-brain barrier permeability. IFNAR^-/- mice were infected s.c. with 1 × 10³ PFU of MR766 or PRVABC59 (n = 4 mice per group). At 2, 4 and 6 dpi, mice received 10 mg sodium fluorescein intraperitoneally. Sera and brains were harvested after 30 min. The ratio of brain to serum fluorescence was determined for each animal, and the mean values are shown. Four uninfected mice were used as controls (Cont.). Kolmogorov-Smirnov test was used for statistical analysis. (F) TNFα mRNA levels in brains of mice infected s.c. with 1 × 10³ PFU of MR766 or PRVABC59 (n = 4 mice per group). Four uninfected mice were used as controls (Cont.). Cytokine mRNA levels were normalized to RPL13 mRNA levels. Kolmogorov-Smirnov test was used for statistical analysis. (G) As in (F) for IL-1β mRNA. (H) As in (F) for IL-6 mRNA. (I) H&E staining of meninx and brain parenchyma at 5 dpi with MR766. Inserts show cellular infiltrates. (J) As in (I) with higher magnification showing neuronophagia (arrowheads). (K) IHC of brain parenchyma at 5 dpi with MR766 using anti-ZIKV NS1 antibody. Positive signal (brown) was seen in cells with neuronal morphology. (L-N) As in (I-K), after infection with PRVABC59. (O) H&E staining of spinal cord at 7 dpi with MR766. Black circles indicate vacuolation of the parenchyma. Inserts with arrowheads show infiltrates in the meninx. (P) As in (O) with higher magnification. Arrowheads indicate infiltration of neutrophils. (Q) IHC of spinal cord at 7 dpi with MR766 using anti-ZIKV NS1 antibody showing positive staining of neural cells. (R-T) As in (O-Q) after infection with PRVABC59.

**Fig 2. Chimeric MR766/PRVABC59 viruses and their virulence in IFNAR^-/- mice.**
(A) Schematic representation of viral genes of wild-type and chimeric ZIKVs. (B) Growth kinetics of wild-type and chimeric virusesin vitro. Vero cells were infected at a MOI of 0.01, and the supernatant was collected at the indicated times. The viral titres were determined by plaque assay on Vero cells [34]. Each data point represents the average of 3 or 4 wells. (C) Survival of 8–14-week-old IFNAR^-/- mice s.c. infected with 1 × 10⁴ PFU of MR766 (n = 10), PRVABC59 (n = 12) or each chimeric virus (n = 6–13). Comparisons of Kaplan-Meier survival curves between different groups were performed by log-rank analysis. The indicated comparisons are between MR766 vs. MR/PR(prM) (p = 0.0002), MR766 vs. MR/PR(NS5) (p = 0.001) and MR766 vs. MR/PR(E) (p = 0.008). (D) Mean percent weight change relative to day 0 after s.c. infection with 1 × 10⁴ PFU of MR766 (n = 12), PRVABC59 (n = 6) or each chimeric virus (n = 6). Seven to nine-week-old IFNAR^-/- mice were s.c. infected with 1 × 10⁴ PFU of each virus. Statistical analysis was performed by repeat measure ANOVA for 2 to 5 dpi.

**Fig 3. prM cleavage and pH stability.**
(A) Sequence alignment of prM from MR766 and MR/PR(prM) (or PRVABC59). The N-linked-glycosylation site (black arrowhead) and motif (underlined) are shown. The furin cleavage site (red arrowhead) and motif (red underline) are indicated. (B) Structure prediction of MR766 prME monomer in the immature virus. The homology model based on dengue virus structure (PDB accession code: 4B03) was generated by MOE homology modeler. The pr, M and E proteins are shown in light pink, yellow and green, respectively. Different amino acid residues in prM protein between MR766 and MR/PR(prM) are shown as atomic spheres. The furin cleavage site (red arrowhead) and motif (red) are indicated. (C) Representative example of a Western blot of purified MR766, PRVABC59 and MR/PR(prM). (D) Densitometry analyses of prM and E proteins from Western blots of purified MR766, PRVABC59 and MR/PR(prM). The intensities of the prM and E bands were determined using ImageJ software, with the mean (7 purified preparations of MR766, 6 purified preparations of PRVABC59 and MR/PR(prM)) prM/E ratio shown. Statistical analysis was performed byt-test. (E) pH stability of MR766, PRVABC59 and MR/PR(prM). MR766 (n = 6 replicates), PRVABC59 (n = 8) and MR/PR(prM) (n = 6) were treated at the indicated pH for 30 min, and virus titers were determined by CCID₅₀ assays. Kolmogorov-Smirnov test ort-test was used for statistical analysis.

**Fig 4. Viral titers in tissues and brain cytokine levels in IFNAR^-/- mice infected with wild-type or chimeric viruses.**
(A) Virus titers in tissues after s.c. infections with 1 × 10⁴ PFU of MR766, PRVABC59, MR/PR(C), MR/PR(prM) or MR/PR(E) (n = 5–10 mice per group). Organs were harvested at 6 dpi, and viral titers were determined by CCID₅₀ assays. Limit of detection was 0.83 log₁₀CCID₅₀/g indicated by the horizontal dashed line. Kolmogorov-Smirnov test ort-test were used for statistical analyses. (B) Inverse correlation between percent survival and mean virus titer in the brain and spinal cord. Significance was determined by Spearman’s correlation test. (C) Virus titers in tissues after i.c. infections with 1 × 10⁴ PFU of MR766, PRVABC59, MR/PR(C), MR/PR(prM) or MR/PR(E) (n = 5–12 mice per group). Organs were harvested at 4 dpi, and viral titers were determined by CCID₅₀ assays. Limit of detection was 0.83 log₁₀CCID₅₀/g indicated by the horizontal dashed line. Statistical analysis was performed byt-test. (D) Survival of 10–11-week-old IFNAR^-/- mice infected with 1 × 10⁴ PFU of MR766 (n = 12), PRVABC59 (n = 12), each chimeric virus (n = 6) or uninfected (n = 4). Comparisons of Kaplan-Meier survival curves between different groups were performed by log-rank analysis. (E) mRNA levels of TNFα, IL-1β and IL-6 in brains at 6 days after s.c. infections (n = 6 mice per group). Three uninfected mice were used as controls. Cytokine mRNA levels in brains were normalized to RPL13 mRNA levels. Statistical analysis was performed byt-test. (F) As in (E) at 4 days after i.c. infection.

**Fig 5. Fetal brain infections.**
(A) ZIKV RNA levels in fetal brains. Individual dams are indicated on the x axis; each square representing one fetus. Vertical dashed grey lines separate litters from each dam. qRT-PCR was performed using the prM gene-specific primers. ZIKV RNA levels were normalized to RPL13 mRNA levels. Five fetal brains from uninfected dam were used as controls. The percentage of fetuses that were infected for each dam at E18.5 are indicated (%). Statistics shown on the figure were performed by Kolmogorov-Smirnov tests. Limit of detection is indicated by the horizontal dashed line. ND, not detected. (B) Viral titers in the placentas. Viral titers were determined by CCID₅₀ assays. + indicates the titers are from placentas of fetuses with detectable virus in the brain;—indicates the titers are from placentas of fetuses with no detectable virus in the brain. Kolmogorov-Smirnov test was used for statistical analysis. Limit of detection was 0.83 log₁₀CCID₅₀/g. (C) No correlation between placental viral titers and ZIKV RNA levels in fetal brains were found. Significance was determined by Spearman’s correlation test. (D) Viral titers in maternal tissues. Indicated tissues of pregnant mice were harvested at 3 dpi, and viral titers were determined by CCID₅₀ assays (n = 4–8 mice per group). Limit of detection was 0.83 log₁₀CCID₅₀/g indicated by the horizontal dashed line.T-test was used for statistical analysis.

**Fig 6. Viral transcytosis and uptake in bEnd.3 cells.**
(A)*In vitro* Transwell setup. Mouse endothelial cells (bEnd.3) were seeded onto the luminal side of the Transwell insert. After the bEnd.3 monolayers reached confluence, MR766, PRVABC59 or MR/PR(prM) viruses were added to the top well at a MOI of 1. After 2, 4 and 6 hrs, viral RNA levels in the cells were determined by qRT-PCR, and after 24 hrs, virus titers in the upper and lower chambers were determined by CCID₅₀ assays. After collecting the culture medium from the upper and lower chambers at 24 hrs, NaF was added to the top wells and PBS to the bottom chambers; after 30 min, samples from the bottom chambers were analyzed by fluorometer. (B) ZIKV levels in the lower chambers. The medium in the lower chamber was collected and virus titers were determined. Kolmogorov-Smirnov tests were used for statistics. Data were obtained from two independent experiments. The horizontal line indicates the mean. (C) The percent of virus-induced permeability. The percent virus-induced permeability was calculated relative to a linear standard curve where “no virus control” was 0% and “no cell control” was 100%.T-test was used for statistical analysis. (D) ZIKV levels in the upper chambers. The medium in the upper chambers was collected and virus titers were determined. Kolmogorov-Smirnov tests were used for statistical analyses. (E) Viral uptake by bEnd.3 cells. bEnd.3 cells were inoculated at a MOI of 1 and incubated for 2, 4 and 6 hrs. The cells were washed three times with PBS, trypsinized, washed three times with PBS, and dissolved in TRIzol (n = 4–9 replicates per virus per time point). Uninfected bEnd.3 cells were used as controls (n = 3). ZIKV RNA levels in the cells were determined by qRT-PCR and normalized to RPL13 mRNA levels and graphed relative to MR766 for each time point. Data were obtained from three independent experiments.

**Fig 7. Surface hydrophobicity and positively charge on the prM proteins of MR766 and MR/PR(prM).**
(A) Molecular model showing the top surface of the prME trimer in the immature virus. The homology models based on the pr structure (PDB accession code: 5U4W) was generated by MOE homology modeler. The pr, M and E proteins are shown in light pink, yellow and green, respectively. Different amino acids in the pr protein between MR766 and MR/PR(prM) are colored red on the trimetric molecular structure. Amino acids (in text) are indicated for one of the pr proteins in the trimer. Bold text indicates amino acids that have side chains exposed on the surface (position 17, 21, 26, 31 and 35; see S2 Table), with font color corresponding to the amino acids highlighted in (B) and (C) (green–hydrophobic patch, red–positively charged). (B) Patch analysis shows that the pr protein of PRVABC59 has an additional hydrophobic patch due to the A26P and V31M substitutions (Patch 1, 50Å²) and an enlarged hydrophobic patch (50 to 90Å²) due to the H35Y substitution (Patch 2) (dashed circles). The total increase in hydrophobic surface area was 90Å². A further increase is evident for Patch 3 (60 to 110Å²), although this patch sits on the side of the trimeric spike. The threshold of the patch area was 50Å². (C) Analyses of positively charged residues on the surface of the pr protein show that the K21E and H35Y substitutions in MR/PR(prM) result in the loss of two positively charged residues exposed (see S2 Table) on the surface of the pr protein (dashed circles). Light pink–histidine (H). Red–Lysine (K). Dark red–Arginine (R). (D) Surface positively charged patches on the M dimer. The patch analysis shows the M protein of MR/PR(prM) (or PRVABC59) decreased a positively charged patch due to the K31R substitution (150 to 90Å²) (dashed circles). The M structure of MR766 was homology-modelled based on the structure (PDB accession code: 5IZ7). Top view of M dimer relative to the viral surface is shown. The threshold of the patch area was 50Å². The default MOE settings were used for protein surface patch analysis.

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This research was supported by the Research Program on Emerging and Re-emerging Infectious Diseases of the Japan Agency for Medical Research and Development (AMED, Grant Number JP19fk0108035.https://www.amed.go.jp/en/program/list/11/02/002.html. S.T. and C.K.L.). E.N. was supported in part by the Daiichi Sankyo Foundation of Life Science, Japan (http://www.ds-fdn.or.jp/support/studying_abroad.html). A.S. holds an Investigator grant (APP1173880) from the National Health and Medical Research Council (NHMRC) of Australia (https://www.nhmrc.gov.au/). The project was also funded in part by an NHMRC Project grant APP1144950 (A.S.), JSPS KAKENHI Grant Number JP20K07530 (https://www.jsps.go.jp/english/index.html. E.N.) and AMED Grant Number JP19fk0108067 (C.K.L.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Neuroinvasiveness of the MR766 strain of Zika virus in IFNAR-/- mice maps to prM residues conserved amongst African genotype viruses

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Neuroinvasiveness of the MR766 strain of Zika virus in IFNAR-/- mice maps to prM residues conserved amongst African genotype viruses

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