[Characterization of host factors <i>ARF4</i> and <i>ARF5</i> upon Zika virus infection <i>in vivo</i> by construction of gene knockout mice].

The effects of host factors ADP-ribosylation factor 4 (ARF4) and ADP-ribosylation factor 5 (ARF5) upon Zika virus (ZIKV) infection <i>in vivo</i> were characterized by construction of gene knockout mice via CRISPR-Cas9. Firstly, <i>ARF5</i> and <i>ARF4</i> genes were modified by the CRISPR-Cas9 system and then microinjected into the fertilized eggs of C57BL/6JGpt mice. Fertilized eggs were transplanted to obtain <i>ARF4</i> or <i>ARF5</i> knockout (ARF4KO or ARF5KO) mice, and <i>ARF4</i>/<i>5</i> double knockout mice were achieved by the mating between ARF4KO and ARF5KO mice (ARF4KO/ARF5KO). Then, the mouse genotypes were identified by PCR to identify the positive knockout mice, and RT-qPCR was employed to examine the knockout efficiency. The mice were then infected with ZIKV and the blood and tissue samples were collected after 2, 4, and 6 days. RT-qPCR was then employed to determine the virus load, and hematoxylin-eosin staining was employed to observe the pathological changes in the tissue. The results showed that expected PCR bands were detected from ARF4KO^-/+, ARF5KO^-/-, and ARF4KO^-/+/ARF5KO^-/- mice, respectively. The results of mRNA transcription measurement indicated the significant knockdown of <i>ARF4</i> by 37.8%-50.0% but not <i>ARF5</i> in ARF4KO^-/+ compared with the wild-type mice. Meanwhile, complete knockout of <i>ARF5</i> and no changes in <i>ARF4</i> were observed in ARF5KO^-/- mice. Additionally, completed knockout of <i>ARF5</i> and down-regulated mRNA level of <i>ARF4</i> in the lung, kidney, and testis were detected in ARF4KO^-/+/ARF5KO^-/-mice in comparison with the wild-type mice. The virus load in the serum decreased in ARF4KO^-/+ mice, while it showed no significant change in ARF5KO^-/- or ARF4KO^-/+/ARF5KO^-/- mice compared with that in the wild type. Meanwhile, ARF4KO^-/+ mice showcased no significant difference in virus load in various tissues but attenuated pathological changes in the brain and testis compared with the wild-type mice. We successfully constructed ARF4KO and ARF5KO mice by CRISPR-Cas9 in this study. ARF4 rather than ARF5 is essential for ZIKV infection <i>in vivo</i>. This study provided animal models for studying the roles of ARF4 and ARF5 in ZIKV infection and developing antivirals.