Objective:We genetically modified our non-replicating vaccinia virus NTV to improve its immunogenicity.Methods:We constructed NTV-modified strain NTV-ΔF1L-C7L by homologous recombination of vaccinia virus based on CRISPR-Cas9 technology by inserting the C7L gene while deleting the F1L gene. The recombinant virus NTV-ΔF1L-C7L was then immunized with 10 7 PFU in BALB/c mice, and the levels of humoral and cellular immunity induced by NTV-ΔF1L-C7L were detected by ELISA and ELISpot method, respectively, and the levels of neutralizing antibodies were determined by the phage-reduced neutralization assay. Results:The PCR and western- blot identification proved that the F1L gene of the constructed NTV-modified strain NTV-ΔF1L-C7L was missing, while the C7L gene was inserted back in the region, and the C7L gene could be expressed normally, indicating that the recombinant virus was constructed correctly. After immunization of mice with NTV-ΔF1L-C7L, ELISA result showed that the recombinant virus NTV-ΔF1L-C7L induced a higher level of IgG antibody than NTV; ELISpot result also showed that the recombinant virus was able to induce a higher level of IFN-γ; and the result of plaque reduction neutralization test showed that the recombinant virus was able to induce a higher level of IFN-γ antibody than that of NTV.Conclusions:We correctly constructed the NTV gene-modified strain NTV-ΔF1L-C7L, which induced stronger humoral and cellular immunity compared with NTV, and provided reference data for the research and development of replacement products for smallpox or monkeypox vaccines.

Objective:To accurately ascertain the whole genome sequencing and the composition of open reading frames (ORFs) of non-replicating Tiantan strain of vaccinia virus (NTV) using next-generation long-read sequencing technology.Methods:NTV, obtained from our laboratory stock, was amplified and purified on chicken embryo fibroblast cells(CEFs), and the full-length genomic nucleic acid of NTV was extracted. The PacBio HiFi sequencing platform was utilized for de novo assembly to obtain the complete genomic sequence of NTV. Using a homology annotation strategy, we identified its ORF composition and compared it with known non-replicating vaccinia virus strains. Results:The total length of NTV′s genome was 171 729 bp, with a GC content of 33%. Its unique inverted terminal repeat (ITR) region comprised hairpin structures, two tandem repeat regions, and three non-repeat regions. NTV contained 166 ORFs, with major differences observed in the ITR and its surrounding regions when compared to MVA-BN and NYVAC. These three strains shared a common set of 138 ORFs. NTV encoded six unique ORFs related to virus evasion of host antiviral response.Conclusions:This study accurately determines the whole genome sequencing and ORFs composition of NTV, and reveals its similarities and differences with other replication-deficient vaccinia virus strains, which pave a way for the development and application of the next generation of monkeypox vaccines and novel viral vectors.

Objective:To develop a recombinase-aided amplification (RAA)-clustered regularly interspaced short palindromic repeats(CRISPR)/Cas12a-based nucleic acid assay for monkeypox virus with high specificity and sensitivity.Methods:RAA primers and CRISPR RNA (crRNA) were designed based on the known conserved regions of the monkeypox virus gene and synthesized, and specific crRNAs were screened using fluorescence detection. The sensitivity and specificity of the detection system were evaluated.Results:An RAA-CRISPR/Cas12a-based nucleic acid assay for monkeypox virus was developed with a sensitivity of 2.5 copies/reaction and high specificity without cross-reactivity with ectromelia virus and vaccinia virus.Conclusions:An RAA-CRISPR/Cas12a-based nucleic acid assay for monkeypox virus was established, which would provide a powerful tool for efficient, rapid and specific detection of monkeypox virus.

Objective:Through the study of the cell biological characteristics and virulence in mice in vivo of three non-replicating vaccinia virus modified strains to provide reference for the development of smallpox/monkeypox vaccine replacement products.Methods:Replicating vaccinia virus Tiantan strain VTT and non-replicating Tiantan Vaccinia Strain NTV were studied in BHK-21/CEF and its modified strains NTV-C7L, NTV-△F1L-C7L, NTV-K1L were amplified, purified, and identified by Western blotting. The virulence and diffusivity of each strain in cells were evaluated by immune-plaque assay. The replication dynamics curves were used to compare the replication differences between the strains, and the weight loss was observed by intranasal route in the mouse model to compare the virulence levels of the viruses.Results:In this study, Western blotting result proved that the amplified and purified vaccinia virus strains were correct. Immunophagocytosis and replication kinetics showed that the replication capacity of the three NTV modified strains in CEF was similar to that of NTV. The diffusion ability and replication ability between Vero cells were improved, but the replication multiple was less than 100 times. The replication level of MRC-5 was significantly enhanced compared with that of NTV, and the replication ratio of NTV-C7L was more than 20000 times. The virulence in mice showed that the body weight of the three NTV modified strains had no statistical significance compared with that of NTV.Conclusions:The three NTV modified strains recovered their replication ability in human MRC-5 cells, but their virulence in mice was similar to that of NTV, which provided the preliminary conditions for being candidate strains of smallpox/monkeypox vaccine replacement products.

Objective:To study the cell morphological changes and related molecular mechanisms of non-replicating vaccinia virus NTV infection with human cells and to provide a scientific basis for the further optimization, transformation and application of NTV vectors.Methods:HeLa cells were infected with vaccinia virus Tiantan strain VTT and non-replicating vaccinia virus NTV, and the morphological changes of cells were observed. Then cells were harvested, and rRNA break levels were detected by electrophoresis and the molecular signals associated with apoptosis were detected by Western blotting, and the pathways and mechanisms of NTV-induced early apoptosis were preliminarily determined.Results:In this study, in terms of cell morphology, it was observed that cells infected with NTV were able to have cell rounding, wrinkles and other lesions at an earlier time compared with VTT. DAPI staining showed that NTV-infected nuclei exhibited high chromatin aggregation, marginalization, and disintegration over time. The rRNA fracture level test result indicate that rRNA has been broken and degraded after 16 hours of NTV infection. The Western blotting test result showed that the molecular signals of PARP, caspase-3 and caspase-9 that were stronger than in normal cells could be detected in NTV-infected HeLa cells, but there was no significant increase in caspase-8, while the result of VTT were the opposite of those in NTV.Conclusions:NTV can induce apoptosis in the early stage and provide a theoretical basis for the modification of vaccinia vectors.

Objective:Using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) technology to edit Vaccinia virus (VACV) thymidine kinase (TK) Region for targeted recombination to establish an efficient vaccinia virus insertion recombination technology.Methods:We designed and synthesized guide RNAs(gRNA)targeting the TK region and then cloned individual gRNA into the PX458 vector that removes nuclear localization signals, and modified the original TK region recombinant plasmid. The gRNA and Cas9 co-expression plasmids were transfected into 293T cells separately to mediate the homologous recombination of vaccinia virus (VACV) and TK region recombination plasmid, and then the rate of viral recombination was evaluated by the appearance of blue and white spots.Results:The recombination efficiency mediated by the gRNA sequence designed and synthesized in this study is more than 1%, which is more than 10 times higher than the classical homologous recombination method .Conclusions:This study used CRISPR/Cas9 technology to establish a highly efficient recombinant vaccinia virus system, which provides technical support for pre-clinical research in vaccines or multivalent research of emerging infectious diseases, as well as tumor treatment.

Objective To evaluate the virulence of NTV modified strain NTV-C7L in vitro and in vivo.Methods The non-replicative vaccinia virus modified strain NTV-C7L was constructed by homologous recombination,in order to evaluate the virulence of NTV-C7L by cell diffusion ability and virus replication,intranasal challenge,and scratch of tail of mice.Results The homologous recombination non-replicating vaccinia virus modified strain NTV-C7L was selected by blue-white screening,the purity of NTV-C7L was identified by PCR and Western blot,the C7L gene was successfully inserted and expressed;In human embryonic lung fibroblasts (MRC-5) as well as mouse embryonic fibroblasts (BALB/C3T3),the replication ability of NTV-C7L was recovered compared with that of NTV,but was lower than that of VTT;the result of the intranasal route with 106 PFU vaccinia virus showed that the animals infected with TTV had slightly greater weight loss than NTV or NTV-C7L(t =6.56,P＜0.001;t =5.73,P＜0.001).Compared with the NTV-C7L group,the weight loss of the NTV group was not statistically significant (t =0.597,P =0.081);the intranasal route with 107pFU vaccinia virus showed that the weight loss of the NTV group and NTV-C7L group was statistically significant compared with the TTV group (t =12.86,P＜0.001;t =10.71,P＜ 0.001).Compared with the NTV-C7L group,the weight loss of the NTV group was statistically significant(t =4.616,P＜0.001);the result of skin scratch indicated that mice scarified with TTV (106 PFU) formed more obvious skin redness,swelling and injury on the post infected 5 days,whereas only redness was detected after scarification with a much higher dose (107) of NTV or NTV-C7L.Conclusions The intracellular and mouse virulence of NTV modified strain NTV-C7L was recovered compared with NTV,but was significantly lower than that of TTV,which provided reference for optimization and application of vaccinia virus vector.

Objective: To detect the expression level of early and late protein of vaccinia virus and to preliminarily explore replication-defective mechanism of highly attenuated NTV strain of vaccinia virus Tiantan. Methods: We constructed prokaryotic expression vector, expressed and purified homologous early protein E3 and late protein A27 closely related to replication and prepared mouse polyclonal antiserum by immunizing mice with homologous proteins. Early and late protein expression levels of NTV were detected. Results: We have expressed and purified vaccinia virus proteins respectively in E. coli expression system and prepared homologous mouse polyclonal antiserum. Early protein E3 and late protein A27 could be highly efficient expression in NTV infected non-permissive Hela cells, while expression of late protein F17 was blocked detected by Western blot. Conclusions The expression limitation of late protein F17 may be an explanation for the replication-defective mechanism of NTV.

Objective: To investigate the cellular and humoral immune responses induced by combined immunization with the fusion protein of human papillomavirus type 18 (HPV18) and the recombinant vaccinia virus. Methods: Purified HPV18L2_31-600E7E6 fusion protein, expressed by prokaryotic expression system, were immunized in combination with the recombinant vaccinia virus vaccine expressing HPV18E7E6 fusion protein (rVV18E7E6) by using various prime-boost regiments in C57BL/6 mice. Cellular and humoral immune responses were analyzed by enzyme-linked immunospot assay (ELISPOT), enzyme-linked immunosorbent assay (ELISA), and pseudovirus neutralization assay. Results: Higher levels of cellular immune responses were induced in mice primed with the HPV18L2_31-600E7E6 fusion protein/adjuvant CpG and boosted with the recombinant vaccinia virus rVV18E7E6, than in other immunized mice. Higher binding antibody level was induced, and low level neutralizing antibody against pseudovirus was detected simultaneously. Conclusions Priming with HPV18L2_31-600E7E6 fusion protein/CpG and boosting with the recombinant vaccinia virus rVV18E7E6 could induce higher cellular and humoral immune response in immunized mice, which might be taken as vaccine candidate for treatment of HPV18 chronic infection and postoperative adjuvant treatment for cervical cancer.

Objective: To establish a method for detection of human antibodies against monkeypox virus. Mothds: The enzyme linked immunosorbent assay (ELISA) plates were coasted with two monkeypox virus peptides from B21R protein, to establish an indirect ELISA for detecting monkeypox virus IgG antibody. The healthy individuals serum samples, monkeypox virus infected patient serum samples and other virus infected patient sera samples were applied to evaluate specificity of the peptides antigen. The reaction conditions were optimized. Results: Synthesized two peptides from monkeypox virus BR21R protein did not cross react obviously with healthy person serum and other virus infected serum. It was shown that the reaction condition was best with sera dilution at 1∶50 when two combined peptides were coated at 100 ng /well, and second-antibody was diluted at 1∶20 000. At this condition the cut off value of IgG antibody in serum samples for ELISA were A450 reading of 0.393. The detected results of two serum samples collected from the monkeypox patient in Sierra Leone were strongly positive, the titers of IgG antibody in two sera were both 1∶6 400. Conclusions The indirect ELISA for detection of monkeypox virus infection was established preliminarily which provided useful tools for epidemiological study and diagnosis.