The effects of host factors ADP-ribosylation factor 4 (ARF4) and ADP-ribosylation factor 5 (ARF5) upon Zika virus (ZIKV) infection in vivo were characterized by construction of gene knockout mice via CRISPR-Cas9. Firstly, ARF5 and ARF4 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 ARF4 or ARF5 knockout (ARF4KO or ARF5KO) mice, and ARF4/5 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 ARF4 by 37.8%-50.0% but not ARF5 in ARF4KO^-/+ compared with the wild-type mice. Meanwhile, complete knockout of ARF5 and no changes in ARF4 were observed in ARF5KO^-/- mice. Additionally, completed knockout of ARF5 and down-regulated mRNA level of ARF4 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 in vivo. This study provided animal models for studying the roles of ARF4 and ARF5 in ZIKV infection and developing antivirals.
Animals ; ADP-Ribosylation Factors/metabolism* ; Zika Virus Infection/genetics* ; Mice ; Mice, Knockout ; Zika Virus/genetics* ; Mice, Inbred C57BL ; CRISPR-Cas Systems ; Viral Load ; Male ; Female

Objective:To test the HIV virus nucleic acid using immunoblot method (Western blotting, WB) and to follow-up with the negative and indeterminate samples in the Dujiangyan area, compare the WB and nucleic acid results before and after followed-up, and try to reduce the WB band′s false-negatives and false-positives.Methods:The 286 suspected HIV infection samples in the Dujiangyan region from January to October 2021 were confirmed by WB, the HIV virus load were tested for the samples that were WB negative and WB indeterminate, those patients were followed-up with epidemiological history and viral load results, and the results before and after tracking were compared.Results:In the 286 samples of suspected HIV infection included in this study, we reported 213 (74.48%) WB positive, 37 WB negative (12.94%), and 36 WB indeterminate (12.58%); 10 of 37 WB negative samples were followed-up; 18 of 36 WB indeterminate samples were followed-up. Among the followed-up WB negative and indeterminate samples, 17 of them had virus nucleic acid detection prior to the follow-up, and all of them turned positive after following-up. The others with no previous virus nucleic acid detection were confirmed to be negative.Conclusions:Among the followed-up samples, 2 samples were false-negative in WB negative results, and 3 were false-positive in WB indeterminate results. The viral nucleic acid must be tested and followed-up in WB negative and indeterminate samples.

OBJECTIVESTo investigate the effects of antisense oligodeoxynucleotide(ASON) on the cyclic nucleotide monophosphates (cNMP) in smooth muscle cells of human corpus cavernosum, and provide experimental groundwork for the gene therapy of erectile dysfunction.

METHODSPDE5 gene ASON(containing exon 1) was transfected into the corpus cavernosum smooth muscle cells with the presence of liposome DOTAP. Another sense oligodeoxynucleotide(SON) and 1% of bovine serum were also transducted into the cells as controls. Two of cNMP, cAMP and cGMP, were probed and measured by ELISA at 1, 2, 4, 6, 10, 24 and 48 h after transfection.

RESULTSAfter transfection, the level of cGMP(1-6 h) in human corpus cavernosum smooth muscle cells was significantly higher than that in controls(P < 0.01).

CONCLUSIONSThe PDE5 gene ASON had been showed to manifest stimulative effect on the cGMP in smooth muscle cells of human corpus cavernosum in vitro, and it provides experimental groundwork for the gene therapy of erectile dysfunction.

3',5'-Cyclic-GMP Phosphodiesterases ; antagonists & inhibitors ; genetics ; Cyclic AMP ; metabolism ; Cyclic GMP ; metabolism ; Cyclic Nucleotide Phosphodiesterases, Type 5 ; Humans ; Male ; Muscle, Smooth ; drug effects ; metabolism ; Oligodeoxyribonucleotides, Antisense ; pharmacology ; Penis ; cytology