Effects of HPV Pseudotype Virus in Cutting E6 Gene Selectively in SiHa Cells

The objectives of this study were to investigate the effects of the CRISPR/Cas9 system mediated by the HPV pseudotype virus on SiHa cytobiology behavior by cutting the HPV16 E6 gene selectively and to explore the role of this system in the treatment of cervical cancer. After designing specific gRNA sequences targeting HPV 16 E6, generating hCas9-EGFP and E6-gRNA-RFP plasmids, and preparing the pseudovirus of HPV16 carrying E6-gRNA and Cas9 plasmids, we determined the titer of the pseudotype virus using the TCID50 method. We obtained the pseudotype virus of HPV16 carrying E6-gRNA and Cas9 plasmids to transfect cervical cancer SiHa cells. Experimental subjects were divided into control group, empty virus group, E6-gRNA transfected group, Cas9 transfected group and Cas9＋E6-gRNA transfected group. The molecular size of the cutting sequence was detected using the T7E1 enzyme digestion method and agarose gel electrophoresis, and the cleavage function of CRISPR/Cas9 on the E6 gene was determined at the same time. RT-PCR and Western blotting were performed to detect the mRNA and protein expression levels of E6 in all the groups; the Transwell cell migration assay was performed to detect the cell migration ability and metastasis in all groups. Heterotopic transplantation tumors were incorporated into mice and were used to investigate the effects of the CRISPR/Cas9 system mediated by the HPV pseudovirus on the tumorigenic ability of SiHa cells by selectively cutting HPV16 E6. The HPV16 pseudotype virus carrying E6-gRNA and Cas9 plasmids could successfully infect SiHa cells, and there were two cutting zones in the Cas9＋E6- gRNA transfected group. However, the empty virus group, E6-gRNA transfected group and Cas9 transfected group had no corresponding zone. Compared with those in the control group, the empty virus group, E6-gRNA transfected group and Cas9 transfected group, the mRNA and protein expression levels of E6 in SiHa cells were downregulated in the Cas9＋E6-gRNA transfected group （P〈0.01）. In addition, the proliferation and migration abilities of SiHa cells were significantly inhibited （P〈0.01）. There were no significant differences among the other groups. In contrast to the control group, the HPV pseudotype virus carrying E6-gRNA and Cas9 plasmids could significantly delay the growth of tumor cells of the ectopic tumor transplantation model （P〈0.01）. The CRISPR/Cas9 system mediated by the HPV pseudotype virus to knockout E6 gene expression exhibited a clear inhibitory effect on the biological function of SiHa cells, which indicated that knocking out the E6 gene using the CRISPR/Cas9 system mediated by the HPV pseudotype virus had a potential effect of eliminating HPV infection and inhibiting the growth of HPV-related tumors. Taken together, these findings provide insight into a new treatment strategy for the prevention and treatment of hr-HPV infected disease, particularly in HPV-related tumors.

Construction of pseudotyped human coronaviruses and detection of pre-existing antibodies in the human population

In order to clarify the pre-exist immunity background of different human coronaviruses(HCoV),this study investigated the positive rate of spike(S)protein antibodies of HCoV,including HCoV-severe acute respiratory syndrome(SARS)-associated coronavirus(SARS-CoV-1),severe acute respiratory syndrome coronavirus 2(SARS-CoV-2),Middle East respiratory syndrome coronavirus(MERS-CoV),HCoV-229E,HCoV-NL63,HCoV-HKU1 and HCoV-OC43,before and after the Coronavirus Disease 2019(COVID-19)outbreak.We utilized pseu-dotyped virus-based neutralization assays(PBNA)or enzyme-linked immunosorbent assays(ELISA)to detect antibody levels against HCoV in serum samples collected in 2009-2010 and 2023.The PBNA results showed that neutralizing antibodies against SARS-CoV-1 and the MERS-CoV were negative.In the serum samples from 2009 to 2010,neutralizing antibodies against SARS-CoV-2(D614G)were negative,whereas in the serum sam-ples from 2023,73 samples(73%)showed neutralizing reactions with the SARS-CoV-2 D614G strain,96 sam-ples(96%)with the BA.5 strain,and 91 samples(91%)with the BF.7 strain.Among pre-COVID-19 samples,33%(33/100)showed neutralizing reactions with HCoV-229E and 63%(63/100)with HCoV-NL63.Among post-COVID-19 samples,50%(50/100)showed neutralizing reactions with HCoV-229E and 49%(49/100)with HCoV-NL63.Due to the different receptors of alpha coronavirus genus compared to other beta coron-avirus genus,neutralizing antibodies against HCoV-OC43 and HCoV-HKU1 virus cannot be detected by con-structing corresponding pseudotyped virus.Binding antibodies against HCoV-OC43 and HCoV-HKU1 virus were detected using ELISA.The results revealed that among pre-COVID-19 samples,83%(83/100)and 45%(45/100)had binding activity with HCoV-OC43 and HCoV-HKU1,respectively.Among post-COVID-19 samples,100%(100/100)and 81%(81/100)had binding activity with HCoV-0C43 and HCoV-HKU1,respectively.

Comparison of lentiviruses pseudotyped with S proteins from coronaviruses and cell tropisms of porcine coronaviruses

The surface glycoproteins of coronaviruses play an important role in receptor binding and cell entry. Different coronaviruses interact with their specific receptors to enter host cells. Lentiviruses pseudotyped with their spike proteins(S) were compared to analyze the entry efficiency of various coronaviruses. Our results indicated that S proteins from different coronaviruses displayed varied abilities to mediate pseudotyped virus infection. Furthermore, the cell tropisms of porcine epidemic diarrhea virus(PEDV) and transmissible gastroenteritis virus(TGEV) have been characterized by live and pseudotyped viruses. Both live and pseudoviruses could infected VeroCCL-81(monkey kidney), Huh-7(human liver), and PK-15(pig kidney) cells efficiently. CCL94(cat kidney) cells could be infected efficiently by TGEV but not PEDV. Overall, our study provides new insights into the mechanisms of viral entry and forms a basis for antiviral drug screening.