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. 2018 Dec 17;9(1):5350.
doi: 10.1038/s41467-018-07782-x.

Routes of Zika virus dissemination in the testis and epididymis of immunodeficient mice

Konstantin A Tsetsarkin  1 , Olga A Maximova  1 , Guangping Liu  1 , Heather Kenney  1 , Natalia Teterina  1 , Marshall E Bloom  2 , Jeffrey M Grabowski  2 , Luwanika Mlera  2 , Bianca M Nagata  3 , Ian Moore  3 , Craig Martens  4 , Emerito Amaro-Carambot  5 , Elaine W Lamirande  5 , Stephen S Whitehead  5 , Alexander G Pletnev  6
Affiliations

Routes of Zika virus dissemination in the testis and epididymis of immunodeficient mice

Konstantin A Tsetsarkin et al. Nat Commun. .

Abstract

Sexual transmission and persistence of Zika virus (ZIKV) in the male reproductive tract (MRT) poses new challenges for controlling virus outbreaks and developing live-attenuated vaccines. To elucidate routes of ZIKV dissemination in the MRT, we here generate microRNA-targeted ZIKV clones that lose the infectivity for (1) the cells inside seminiferous tubules of the testis, or (2) epithelial cells of the epididymis. We trace ZIKV dissemination in the MRT using an established mouse model of ZIKV pathogenesis. Our results support a model in which ZIKV infects the testis via a hematogenous route, while infection of the epididymis can occur via two routes: (1) hematogenous/lymphogenous and (2) excurrent testicular. Co-targeting of the ZIKV genome with brain-, testis-, and epididymis-specific microRNAs restricts virus infection of these organs, but does not affect virus-induced protective immunity in mice and monkeys. These defined alterations of ZIKV tropism represent a rational design of a safe live-attenuated ZIKV vaccine.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Model to study the effects of miRNA targeting on ZIKV tissue tropism and pathogenesis. a Schematics of ZIKV-NS3m and 2×scr viral genomes. Green boxes indicate position of scr sequence insertions. b Survival of AG129 mice inoculated ip with 106 pfu of ZIKV-NS3m, 2×scr virus or with a diluent (mock). c–g Growth kinetics of ZIKV-NS3m and 2×scr viruses in the serum c, spleen d, brain e, testis f, or epididymis g of adult AG129 male mice inoculated ip with 106 pfu of virus. Mean viral load in the serum or organ homogenates (n = 3–7 per time point) ± standard deviation (SD; shown as error bars) was determined by titration in Vero cells. The dashed lines indicate the limit of virus detection: 1.5 log10(pfu/mL) for serum c, 1.7 log10(pfu/g) for spleen, brain, or testis d–f, and 0.7 log10(pfu/mouse) for epididymis g
Fig. 2
Fig. 2
Effect of miRNA target insertions on ZIKV replication in the serum and organs of AG129 mice. a Relative expression of selected miRNAs in the testis compared with their expression in the brain. Data indicate the ratio (folds change [FC]) of normalized counts for miRNAs isolated from the testis to those isolated from the brain of individual AG129 mice (n = 3). Horizontal line and error bar are geometric mean and geometric SD, respectively. b List of viruses with inserted miRNA targets. c Growth kinetics of recombinant viruses in Vero cells. Results are presented as an average titer of two biological replicates ± SD (shown as error bars). d–h Adult AG129 mice were infected ip with 106 pfu of 2×scr or miRNA-targeted viruses and were killed at 3 or 12 dpi. Mean viral titer ± SD in the serum at 1 dpi d, spleen at 3 dpi e, testis at 3 dpi f, and 12 dpi g, and brain at 12 dpi h was determined by titration in Vero cells. Mice infected with 2×202(T) and 2×202/141(T) viruses were divided into two groups (stable, stb and mutant, mut) based on the miRNA target sequence stability in the testis at 12 dpi. The titers of 2×202(T) or 2×202/141(T) viruses in the brain and testis in these two groups of mice are presented separately. The dashed lines indicate the limit of virus detection: 0.7 log10(pfu/mL) for Vero cells (c), 1.5 log10(pfu/mL) for serum d and 1.7 log10(pfu/g) for spleen, brain, or testis d–h. Differences between the titer of 2×scr and the titer of each of miRNA-targeted virus in mouse serum or organs were compared using one-way ANOVA (***p < 0.001, **p < 0.01, *p < 0.05). Differences between the replication of 2×scr and other viruses in Vero cells were compared using two-way ANOVA, and were not statistically significant (p > 0.05)
Fig. 3
Fig. 3
ZIKV genome targeting for mir-202–5p restricts virus ability to infect cells of the seminiferous tubules. Adult AG129 mice were infected ip with 106 pfu of 2×scr, various miRNA-targeted viruses, or mock-inoculated and killed at 3 dpi a–c or 12 dpi d–i. Representative images of ZIKV antigen distribution in the testes of mice that were mock-inoculated or infected with indicated viruses are shown on the indicated dpi (2–6 mice per group). ZIKV antigen in a whole testis are shown in the top panel. Red boxes indicate the areas which are presented at higher magnification images on the bottom of each panel. Testicular structural elements are indicated in a–c. The cellular elements of seminiferous tubules are indicated in d. Labeling used within panels is indicated at the bottom of the figure. Scale bars: 10 µm
Fig. 4
Fig. 4
Replication of the miRNA-targeted ZIKVs in the epididymis of AG129 mice. a Relative expression of selected miRNAs in the mouse epididymis compared with their expression in the testis. Data show the ratio (FC) of normalized counts for miRNAs isolated from the epididymis to those isolated from the testis of AG129 mice (n = 3). Horizontal lines and error bars are geometric means and geometric SDs, respectively. b–k AG129 mice were infected ip with 106 pfu of 2×scr or miRNA-targeted viruses and were killed at 3 or 12 dpi. b and c Mean viral titers ± SD (shown as error bars) in the epididymis of mice at 3 dpi b and 12 dpi c. Epididymides dissected from mice infected with 2×202(T) or 2×202/141(T) at 12 dpi were divided into two groups based on the miRNA target sequence stability (see Fig. 2 for details). The dashed line indicates the limit of virus detection (0.7 log10(pfu/epididymis)). Differences between the titers of 2×scr and each of miRNA-targeted viruses in the epididymis were compared using one-way ANOVA (***p < 0.001). d–l Representative images of ZIKV antigen distribution in the epididymides of mice that were mock-inoculated or infected with indicated viruses are shown on the indicated dpi (2–6 mice per group). The epididymal interstitium is indicated. Labeling used within panels is indicated at the bottom of the figure. Scale bars: 10 µm
Fig. 5
Fig. 5
Replication and immunogenicity of C/3’NCR-mir(T) virus in adult AG129 mice. a Schematic representation of viral genomes used in the study. dCGR—duplicated capsid gene region; ΔC—truncated C gene; C-opt—a full-length copy of C gene containing synonymous mutations introduced in each AA codon (except AUG and UGG); colored boxes indicate miRNA targets for mir-9–5p (cherry), mir-141–3p (purple), mir-202–5p (blue), mir-124–3p (red); 2 A—autoprotease 2 A from foot-and-mouth disease virus (FMDV); the curved arrow indicates position of 2 A protease cleavage; striped cherry and red boxes indicate mutated targets for mir-9–5p and mir-124–3p, respectively; striped green box indicates scr sequence; striped gray box indicates random sequence of 21 nt. b–h mice were infected ip with 106 pfu of the indicated viruses. Mean virus titer ± SD (shown as error bars) in the serum at 1 and 3 dpi b, spleen at 3 dpi c, testis at 3 dpi d, and 12 dpi f, epididymis at 3 dpi e and 12 dpi g, and brain at 12 dpi h was determined by titration in Vero cells. The dashed lines indicate the limit of virus detection. Differences between viral titers in the mouse serum or organs at 1 or 3 dpi were compared using unpaired two-tailed t test (ns denotes not statistically significant; p > 0.05). Differences between viral titers in the organs at 12 dpi were compared using one-way ANOVA (ns p > 0.05; *p < 0.05; **p < 0.01; ****p < 0.0001). i–j Mice were infected ip with 105 pfu of C/3’NCR-mir(T) and C/3’NCR-scr. At 29 days post immunization, animals were challenged with 105 pfu of wt ZIKV (strain Paraiba_01/2015). Neutralizing antibody titer in the serum of immunized mice at 28 dpi i and 56 dpi (27 days post challenge) j were compared using unpaired two-tailed t test (ns denotes not statistically significant (p > 0.05]). Horizontal lines denote geometric mean ± geometric SD (shown as error bars)
Fig. 6
Fig. 6
C/3′NCR-mir(T) virus is immunogenic to the rhesus macaques. a Experimental design of the study. b Mean viremia ± SD (shown as upper error bars) post immunization with ZIKV-NS3m or C/3′NCR-mir(T) viruses. c Neutralizing antibody response in immunized monkeys before and after the challenge with 105 pfu of wt ZIKV. Horizontal lines denote geometric mean ± geometric SD (shown as error bars). d Mean viremia + SD (shown as upper error bars) after the challenge with wt ZIKV. Differences between viral titer and between ZIKV-specific NA titer in serum were compared using two-way ANOVA and one-way ANOVA, respectively (**p < 0.01, ***p < 0.001; ns denotes not statistically significant (p > 0.05). The dashed lines indicate the limit of virus detection (0.7 log10(pfu/mL)) for serum b and d, or the limit of NA titer detection c
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