Zika Virus Infection in Dexamethasone-immunosuppressed Mice Demonstrating Disseminated Infection with Multi-organ Involvement Including Orchitis Effectively Treated by Recombinant Type I Interferons

Chan, Jasper Fuk-Woo; Zhang, Anna Jinxia; Chan, Chris Chung-Sing; Yip, Cyril Chik-Yan; Mak, Winger Wing-Nga; Zhu, Houshun; Poon, Vincent Kwok-Man; Tee, Kah-Meng; Zhu, Zheng; Cai, Jian-Piao; Tsang, Jessica Oi-Ling; Chik, Kenn Ka-Heng; Yin, Feifei; Chan, Kwok-Hung; Kok, Kin-Hang; Jin, Dong-Yan; Au-Yeung, Rex Kwok-Him; Yuen, Kwok-Yung

Approval was obtained from the Committee on the Use of Live Animals in Teaching and Research of The University of Hong Kong. Male and female BALB/c mice, 6-8 weeks old, were obtained from the Laboratory Animal Unit of The University of Hong Kong. The mice were kept in biosafety level-2 housing and given access to standard pellet feed and water ad libitum. Virus inoculation experiments were performed in a biosafety level-2 animal facility according to the standard operating procedures approved by the Committee on the Use of Live Animals in Teaching and Research of The University of Hong Kong as we described previously (Zhang et al., 2014) . The mice were randomly divided into 11 groups and given different regimens of virus inoculation, dexamethasone, and recombinant interferon treatment (Table 1) . Phosphate-buffered saline (PBS) was used to dilute the virus stocks to the desired concentration, and inocula were back-titrated to verify the dose given. On the day of virus inoculation, a dose of the virus equivalent to 6 × 10 6 TCID 50 (3.24 × 10 6 plaque forming units) in 200 μl of PBS was inoculated via the intraperitoneal route into mice under ketamine (100 mg/kg) and xylazine (10 mg/kg) anesthesia. Mice in the negative-control groups (groups 5 to 8) were injected with the same volume of PBS. Mice were monitored three times each day for clinical signs of disease and a numerical score was assigned at each observation as previously described (Dowall et al., 2016; Graham et al., 2015) . Their body weight and survival were monitored for 14 days post-inoculation (dpi) or until euthanasia. Three mice in each group (except groups 7 and 8 which included mock-infected control mice without dexamethasone immunosuppression and group 9 which included ZIKV-inoculated, dexamethasone-immunosuppressed mice without dexamethasone withdrawal) were sacrificed at 5 dpi for virological, histological, and immunohistochemistry analyses. The remaining mice were sacrificed at 14 dpi or euthanized when there was a 20% weight loss or 10% weight loss with ≥1 clinical sign (Dowall et al., 2016) . Samples of brain, testis/ epididymis (male), prostate (male), ovary/uterus (female), kidney, urinary bladder, spleen, liver, pancreas, intestine, heart, lung, and salivary gland were collected at necropsy. The specimens were separated into two parts, one immediately fixed in 10% PBS-buffered formalin, the other immediately frozen at −80°C until further experiments. Blood samples were also collected for RNA extraction and real-time PCR analysis.