Zika Virus Infection Damages the Testes in Pubertal Common Squirrel Monkeys (Saimiri collinsi)

Abstract

During the Zika virus (ZIKV) outbreak and after evidence of its sexual transmission was obtained, concerns arose about the impact of the adverse effects of ZIKV infection on human fertility. In this study, we evaluated the clinical-laboratory aspects and testicular histopathological patterns of pubertal squirrel monkeys (Saimiri collinsi) infected with ZIKV, analyzing the effects at different stages of infection. The susceptibility of S. collinsi to ZIKV infection was confirmed by laboratory tests, which detected viremia (mean 1.63 × 10⁶ RNA copies/µL) and IgM antibody induction. Reduced fecal testosterone levels, severe testicular atrophy and prolonged orchitis were observed throughout the experiment by ultrasound. At 21 dpi, testicular damage associated with ZIKV was confirmed by histopathological and immunohistochemical (IHC) analyses. Tubular retraction, the degeneration and necrosis of somatic and germ cells in the seminiferous tubules, the proliferation of interstitial cells and an inflammatory infiltrate were observed. ZIKV antigen was identified in the same cells where tissue injuries were observed. In conclusion, squirrel monkeys were found to be susceptible to the Asian variant of ZIKV, and this model enabled the identification of multifocal lesions in the seminiferous tubules of the infected group evaluated. These findings may suggest an impact of ZIKV infection on male fertility.

Keywords: Zika virus; squirrel monkeys; testes damage.

Figure 1

Experimental design of the study. The upper portion of the figure shows the timeline of sample collection and biometric data from the animals evaluated; the periods were divided into (1) Pre-inoculation, (2) Acute and (3) Convalescence phases of infection, each with its corresponding color according to the legend on the right of the figure; on the ruler we can see the days marked in red on which biological material was collected, the day of inoculation and euthanasia of the animals, as well as what was collected and evaluated, and on which days they were evaluated. In the lower portion of the figure, we have a flowchart showing to which methodology each of the samples was forwarded and the culmination of the entire study.

Figure 2

Noninvasive evaluation of experimental ZIKA infection in male S. collinsi. (A) Measurement of rectal temperature, showing feverish period of infected group between 3 to 10 dpi. The dashed line represents the temperature cut-off based in control group data. The (B) RNA viral load in blood by RT-qPCR and (C) IgM antibody profile of the infected group detected from 14 dpi. The dashed line represents the cut-off of ELISA assay. Group evaluation, mean and min-max values: (D) body weight, (E) testicular volume, (F) gonadosomatic index, and (G) testosterone by stages of infection, highlighting the decrease of testicular volume, GSI and testosterone values during acute phase of infection in G1.

Figure 3

Ultrasonographic analysis at 21 dpi. The left testicle of animal AT-003 (G1) showed normal homogeneity (A) and the left testicle of animal AT-163 (G2) presented hypoechoicity in all its extensions (B). The testicular echogenicity of G1 and G2 was evaluated at the indicated day (C) and by stages of ZIKV infection (D). Mean and min-max values.

Figure 4

Postmortem evaluation of experimental ZIKA infection in male S. collinsi. (A) Control animal AT-001, macroscopy of the right and left testes. (B) Infected animal AT-156, macroscopy showing a slight difference in size between the right and left testes. (C) Postmortem GSI graph at 21 dpi of each animal. (D) Graph of the detection of ZIKV RNA copies/mg in a pool of both testes of each infected animal. (E) Graph of Pearson’s correlation: demonstrating a significant inverse and very strong correlation between GSI X ZIKV tissue variables, R-value = −0.9986 and p = 0.033.

Figure 5

Microscopic evaluation of S. collinsi. (A) Integrity of testicular tissue from control animal AT-001 (100×). (B) Seminiferous tubule of the control animal, in a higher magnification lens, showing sperm (arrow), score 10 (400×). (C) AT-005, infected with ZIKV, showing diffuse tubular edema and tubular retraction (star). Highlighted (circle) interstitial cell proliferation associated with inflammatory infiltrate (100×). (D) Perivascular inflammatory infiltrate (circle), with the presence of lymphocytes and neutrophils. The seminiferous tubule of an infected animal, showing a decrease in the number of germ cells, which are degenerated (arrowhead) (400×). (E) AT-163 infected animal, showing proliferation of Leydig cells and degenerated germ cells. (F) Infected animal, showing seminiferous tubule containing degenerating spermatogenic cells (hollow arrows), presence of pyknotic nuclei and cytoplasmic macrovacuolization (arrowhead), score 4. In the extra tubular area, there is an increase in the proliferation of Leydig cells (black arrows) (400×). (G) Light micrograph showing IHC staining with peroxidase using anti-ZIKV polyclonal antibodies. Seminiferous tubule from infected animal AT-005, showing ZIKV antigen labeling on both the germ cells of the seminiferous tubule (arrow) and the Leydig cells (circle). (H) Graph showing the quantification of viral antigen per group in the testicular tissue of the control and infected animals. ** p < 0.03. (I) Graph showing the quantification of ZIKV antigen per animal based on the IHC test. In blue is the animal AT-163, in green the AT-156 and in red the AT-005; the filled columns represent the right testis and the empty columns the left testis.