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

Total nucleic acid (TNA) was extracted from the blood and necropsied tissues using EZ1 Virus Mini Kit v2.0 and QIAsymphony DSP Virus/Pathogen Mini Kit (QIAGEN, Hilden, Germany), respectively, as we previously described (Zheng et al., 2008; Chan et al., 2016b; Chan et al., 2015a) . ZIKV envelope gene was measured by using QuantiNova Probe RT-PCR Kit (QIAGEN) in LightCycler 96 Real-Time PCR System (Roche Diagnostics, Basel, Switzerland). 5 μl of purified TNA was amplified in a 20 μl-reaction containing 10 μl of 2× QuantiNova Probe RT-PCR Master Mix, 0.2 μl QN Probe RT-mix, 0.8 μM forward primer, 0.8 μM reverse primer, and 200 nM probe. Forward primer (5′-CGYTGCCCAACACAAGG-3′), reverse primer (5′-CCACYAAYGTTCTTTTGCABACA-3′), and probe (5′-HEX-AGCCTACCTTGAYAAGCARTCAGACACTC-IABkFQ-3′) targeting the ZIKV envelope gene as we previously described were used (Chan et al., 2016b) . Reactions were incubated at 45°C for 10 min, followed by 95°C for 5 min, and then thermal cycled for 50 cycles (95°C for 5 s, 55°C for 30 s). Internal control β-actin gene was measured by using QuantiNova SYBR Green RT-PCR Kit (QIAGEN) in LightCycler 96 Real-Time PCR System. 5 μl of purified TNA was amplified in a 20 μl-reaction containing 10 μl of 2× QuantiNova SYBR Green RT-PCR Master Mix, 0.2 μl QN SYBR Green RT-mix, 0.5 μM forward primer (5′-ACGGCCAGGTCATCACTATTG-3′) and 0.5 μM reverse primer (5′-CAAGAAGGAAGGCTGGAAAAG-3′) for the β-actin gene. Reactions were incubated at 50°C for 10 min, followed by 95°C for 2 min, and then thermal cycled for 50 cycles (95°C for 5 s, 60°C for 10 s). A series of 10-fold dilutions equivalent to 1 × 10 2 to 1 × 10 6 copies/reaction mixture were prepared to generate standard curves and run in parallel with the test samples.