Image_3_Zika Virus Induced More Severe Inflammatory Response Than Dengue Virus in Chicken Embryonic Livers.TIF

Dengue (DENV) and Zika virus (ZIKV) are important flaviviruses in tropical and subtropical regions, causing severe Dengue Hemorrhagic Fever (DHF)/Dengue Shock Syndrome (DSS) and microcephaly, respectively. The infection of both viruses during pregnancy were reported with adverse fetal outcomes. To investigate the effects of ZIKV and DENV infections on fetal development, we established an infection model in chicken embryos. Compared with DENV-2, the infection of ZIKV significantly retarded the development of chicken embryos. High viral loads of both DENV-2 and ZIKV was detected in brain, eye and heart 7 and 11 days post-infection, respectively. Interestingly, only ZIKV but not DENV-2 was detected in the liver. Even both of them induced apparent liver inflammation, ZIKV infection showed a more severe inflammatory response than DENV-2 infection based on the inflammation scores and the gene expression levels of IL-1β, TNF, IL-6, and TGFβ-2 in liver. Our results demonstrated that ZIKV induced more severe inflammatory response in chicken embryo liver compared to DENV-2, which might partially attribute to viral replication in liver cells. Clinicians should be aware of the potential liver injury associated with ZIKV infection in patients, especially in perinatal fetuses.</p

Image_4_Zika Virus Induced More Severe Inflammatory Response Than Dengue Virus in Chicken Embryonic Livers.TIF

Dengue (DENV) and Zika virus (ZIKV) are important flaviviruses in tropical and subtropical regions, causing severe Dengue Hemorrhagic Fever (DHF)/Dengue Shock Syndrome (DSS) and microcephaly, respectively. The infection of both viruses during pregnancy were reported with adverse fetal outcomes. To investigate the effects of ZIKV and DENV infections on fetal development, we established an infection model in chicken embryos. Compared with DENV-2, the infection of ZIKV significantly retarded the development of chicken embryos. High viral loads of both DENV-2 and ZIKV was detected in brain, eye and heart 7 and 11 days post-infection, respectively. Interestingly, only ZIKV but not DENV-2 was detected in the liver. Even both of them induced apparent liver inflammation, ZIKV infection showed a more severe inflammatory response than DENV-2 infection based on the inflammation scores and the gene expression levels of IL-1β, TNF, IL-6, and TGFβ-2 in liver. Our results demonstrated that ZIKV induced more severe inflammatory response in chicken embryo liver compared to DENV-2, which might partially attribute to viral replication in liver cells. Clinicians should be aware of the potential liver injury associated with ZIKV infection in patients, especially in perinatal fetuses.</p

Image_2_Zika Virus Induced More Severe Inflammatory Response Than Dengue Virus in Chicken Embryonic Livers.TIF

Dengue (DENV) and Zika virus (ZIKV) are important flaviviruses in tropical and subtropical regions, causing severe Dengue Hemorrhagic Fever (DHF)/Dengue Shock Syndrome (DSS) and microcephaly, respectively. The infection of both viruses during pregnancy were reported with adverse fetal outcomes. To investigate the effects of ZIKV and DENV infections on fetal development, we established an infection model in chicken embryos. Compared with DENV-2, the infection of ZIKV significantly retarded the development of chicken embryos. High viral loads of both DENV-2 and ZIKV was detected in brain, eye and heart 7 and 11 days post-infection, respectively. Interestingly, only ZIKV but not DENV-2 was detected in the liver. Even both of them induced apparent liver inflammation, ZIKV infection showed a more severe inflammatory response than DENV-2 infection based on the inflammation scores and the gene expression levels of IL-1β, TNF, IL-6, and TGFβ-2 in liver. Our results demonstrated that ZIKV induced more severe inflammatory response in chicken embryo liver compared to DENV-2, which might partially attribute to viral replication in liver cells. Clinicians should be aware of the potential liver injury associated with ZIKV infection in patients, especially in perinatal fetuses.</p

Image_1_Zika Virus Induced More Severe Inflammatory Response Than Dengue Virus in Chicken Embryonic Livers.TIF

Dengue (DENV) and Zika virus (ZIKV) are important flaviviruses in tropical and subtropical regions, causing severe Dengue Hemorrhagic Fever (DHF)/Dengue Shock Syndrome (DSS) and microcephaly, respectively. The infection of both viruses during pregnancy were reported with adverse fetal outcomes. To investigate the effects of ZIKV and DENV infections on fetal development, we established an infection model in chicken embryos. Compared with DENV-2, the infection of ZIKV significantly retarded the development of chicken embryos. High viral loads of both DENV-2 and ZIKV was detected in brain, eye and heart 7 and 11 days post-infection, respectively. Interestingly, only ZIKV but not DENV-2 was detected in the liver. Even both of them induced apparent liver inflammation, ZIKV infection showed a more severe inflammatory response than DENV-2 infection based on the inflammation scores and the gene expression levels of IL-1β, TNF, IL-6, and TGFβ-2 in liver. Our results demonstrated that ZIKV induced more severe inflammatory response in chicken embryo liver compared to DENV-2, which might partially attribute to viral replication in liver cells. Clinicians should be aware of the potential liver injury associated with ZIKV infection in patients, especially in perinatal fetuses.</p

EDIII -Fc induction the production of anti-zika antibodies in rhesus macaques A.

Timeline for rhesus macaque immunization with EDIII-Fc and blood collection. Rhesus macaques were immunized with EDIII-Fc or PBS. Pre-immune blood samples were obtained 2 days before immunization, and subsequent samples were collected weekly post-immunization. B. Changes of total antibody level in rhesus macaques after immunization. Serum samples from rhesus macaques immunized with EDIII-Fc (in red) or PBS (in black) were serially diluted at a 1:10 ratio. HRP-labeled mouse anti-monkey monoclonal antibody was used for ELISA detection. C. Detection of neutralizing antibody level in rhesus macaques. Two weeks after the third immunization, the sera to be tested were serially diluted 2-fold in PBS starting from 1:5 for plaque reduction neutralization test. Results are reported as the fold dilution of serum that inhibited 50% of plaque formation. Three replications were done in each experiment, and the experiment was repeated three times. ***: P https://openclipart.org/.</p

Data_Sheet_1_Rapid Construction of an Infectious Clone of the Zika Virus, Strain ZKC2.docx

Zika virus (ZIKV) has had detrimental effects on global public health in recent years. This is because the management of the disease has been limited, in part because its pathogenic mechanisms are not yet completely understood. Infectious clones are an important tool that utilize reverse genetics; these can be used to modify the ZIKV genomic RNA at the DNA level. A homologous recombination clone was used to construct pWSK29, a low copy plasmid that contained sequences for a T7 promoter, the whole genome of ZIKV ZKC2 strain, and a hepatitis delta virus ribozyme. High fidelity PCR was then used to amplify the T7 transcription template. The transcript was then transfected into susceptible cells via lipofection to recover the ZIKV ZKC2 strain. Finally, the virulence of rZKC2 was evaluated both in vitro and in vivo. The rZKC2 was successfully obtained and it showed the same virulence as its parent, the ZIKV ZKC2 strain (pZKC2), both in vitro and in vivo. The 3730 (NS2A-D62G) mutation site was identified as being important, since it had significant impacts on rZKC2 recovery. The 4015 (NS2A, A157V) mutation may reduce virus production by increasing the interferon type I response. In this study, one of the earliest strains of ZIKV that was imported into China was used for infectious clone construction and one possible site for antiviral medication development was discovered. The use of homologous recombination clones, of PCR products as templates for T7 transcription, and of lipofection for large RNA transfection could increase the efficiency of infectious clone construction. Our infectious clone provides an effective tool which can be used to explore the life cycle and medical treatment of ZIKV.</p

The ZIKV EDIII nucleotide sequence alignment.

Sequence alignments were generated using NCBI Multiple Alignment Viewer (https://www.ncbi.nlm.nih.gov/projects/msaviewer/). A total of 1,809 EDIII protein sequences data were obtained from Genbank, and the date was to June 20, 2023). (PDF)</p

Expression of the recombinant EDIII-Fc protein.

A. Positions of EDIII protein in the E protein. The crystal structure of the ZIKV E protein dimer (PDB ID 5LBV) is shown, with EDIII of monomeric subunit A colored in red. B. Sequence Logo visualization of the EDIII alignments. Sequence Logos of the EDIII domain from multiple sequence alignments across all organisms, consisting of 1,809 protein sequences (Sequence data from Genbank, the date to obtain the sequence was to June 20, 2023). C. Schematic map of pLV-ZIKV-EDIII vector construction. D. Western blot detection EDIII expression. Line 1, cell lysate of HEK293T transfected with pLV-eGFP; Line 2, supernatant of HEK293T transfected with pLV-eGFP; Line 3, cell lysate of HEK293T transfected with pLV-ZIKV-EDIII; Line 4, supernatant of HEK293T transfected with pLV-ZIKV-EDIII.</p

Changes in cytokines levels secreted in rhesus macaque after immunization.

Pre-immune PBMCs and those obtained at 4 weeks, 8 weeks, and 12 weeks after immunization from rhesus macaques were incubated with ZIKV E protein for 48 hours. Following incubation, culture supernatants were collected for flow cytometric assays. Three replications were done in each experiment and repeated three times. ****: P < 0.0001.</p

Immunization with EDIII-Fc protects mice from ZIKV challenge.

A. Timeline for mice immunization with EDIII-Fc and blood collection. Balb/c mice were immunized with EDIII-Fc or PBS. Pre-immune blood samples were obtained 2 days before immunization, and subsequent samples were collected weekly post-immunization. B. Changes of total antibody levels in mice after immunization. Serum samples from mice immunized with EDIII-Fc (in red) or PBS (in black) were serially diluted at a 1:10 ratio. HRP-labeled sheep anti-mouse monoclonal antibody was used for ELISA detection. C. Detection of neutralizing antibody levels in mice. Two weeks after the second immunization, the sera to be tested were serially diluted 2-fold in PBS, starting from a 1:5 ratio, for plaque reduction neutralization testing. Results are reported as the fold dilution of serum that inhibited 50% of plaque formation. D. Serum viral load in mice after challenge. One week after the second immunization, seven Balb/c mice were injected subcutaneously with 104 PFU of ZIKV. Daily blood samples were collected and qPCR was carried out for viral load detection. Three replications were done in each experiment, and the experiment was repeated three times. *: P P https://openclipart.org/.</p