Syncytial and Congregative Effects of Dengue and Zika Viruses on the Aedes Albopictus Cell Line Differ among Viral Strains

Dengue viruses (DENV) and Zika virus (ZIKV) are transmitted among humans, or from non-human primates to humans, through mosquito bites. The interaction of the virus with mosquito cells is a key step in the viral life cycle. Therefore, the objective of this study was to determine how DENV and ZIKV interact with mosquito cells. Immunofluorescence assays and a direct visualization system were combined to monitor the syncytial or congregative effects of DENV and ZIKV strains on C6/36 cells. We examined the cytopathic effects of the strains on C6/36 mosquito cells, a widely used laboratory model for studying infection with these viruses. Our results indicated that all strains of DENV-1 and DENV-2, most DENV-4 strains, and some DENV-3 strains caused syncytial effects on C6/36 cells, whereas some DENV-3 and DENV-4 strains, and all tested ZIKV strains, caused cell congregation after infection but no cell fusion. In addition, we detected a range of environmental pH values from 6.0 to 8.0 supporting virus-induced cell fusion. The optimal pH condition was 7.5, at which viral production was also highest. Furthermore, the UV-inactivated virus did not cause cell fusion, thus suggesting that viral replication may be required for DENV’s syncytial effects on C6/36 cells. Syncytial and congregative effects of DENV and ZIKV on Aedes albopictus cells differ among viral strains. Syncytial effects of DENV on C6/36 are important for viral replication

Rapid Neutralization Testing System for Zika Virus Based on an Enzyme-Linked Immunospot Assay.

Zika virus (ZIKV) is a mosquito-borne flavivirus that has been associated with neuropathology in fetuses and adults, imposing a serious health concern. Therefore, the development of a vaccine is a global health priority. Notably, neutralization tests have a significant value for vaccine development and virus diagnosis. The cytopathic effect (CPE)-based neutralization test (Nt-CPE) is a common neutralization method for ZIKV. However, this method has some drawbacks, such as being time-consuming and labor-intensive and having low-throughput, which precludes its application in the detection of large numbers of specimens. To improve this problem, we developed a neutralization test based on an enzyme-linked immunospot assay (Nt-ELISPOT) for ZIKV and performed the assay in a 96-well format. A monoclonal antibody (mAb), 11C11, with high affinity and reactivity to ZIKV was used to detect ZIKV-infected cells. To optimize this method, the infectious dose of ZIKV was set at a multiplicity of infection (MOI) of 0.0625, and a detection experiment was performed after incubating for 24 h. As a result, under these conditions, the Nt-ELISPOT had good consistency with the traditional Nt-CPE to measure neutralizing titers of sera and neutralizing antibodies. Additionally, three neutralizing antibodies against ZIKV were screened by this method. Overall, we successfully developed an efficient neutralization test for ZIKV that is high-throughput and rapid. This Nt-ELISPOT can potentially be applied to detecting neutralizing titers of large numbers of specimens in vaccine evaluation and neutralizing antibody screening for ZIKV

Single-cell multi-omics reveals dyssynchrony of the innate and adaptive immune system in progressive COVID-19.

Dysregulated immune responses against the SARS-CoV-2 virus are instrumental in severe COVID-19. However, the immune signatures associated with immunopathology are poorly understood. Here we use multi-omics single-cell analysis to probe the dynamic immune responses in hospitalized patients with stable or progressive course of COVID-19, explore V(D)J repertoires, and assess the cellular effects of tocilizumab. Coordinated profiling of gene expression and cell lineage protein markers shows that S100

Functional Interaction of Nuclear Domain 10 and Its Components with Cytomegalovirus after Infections: Cross-Species Host Cells versus Native Cells

Species-specificity is one of the major characteristics of cytomegaloviruses (CMVs) and is the primary reason for the lack of a mouse model for the direct infection of human CMV (HCMV). It has been determined that CMV cross-species infections are blocked at the post-entry level by intrinsic cellular defense mechanisms, but few details are known. It is important to explore how CMVs interact with the subnuclear structure of the cross-species host cell. In our present study, we discovered that nuclear domain 10 (ND10) of human cells was not disrupted by murine CMV (MCMV) and that the ND10 of mouse cells was not disrupted by HCMV, although the ND10-disrupting protein, immediate-early protein 1 (IE1), also colocalized with ND10 in cross-species infections. In addition, we found that the UL131-repaired HCMV strain AD169 (vDW215-BADrUL131) can infect mouse cells to produce immediate-early (IE) and early (E) proteins but that neither DNA replication nor viral particles were detectable in mouse cells. Unrepaired AD169 can express IE1 only in mouse cells. In both HCMV-infected mouse cells and MCMV-infected human cells, the knocking-down of ND10 components (PML, Daxx, and SP100) resulted in significantly increased viral-protein production. Our observations provide evidence to support our hypothesis that ND10 and ND10 components might be important defensive factors against the CMV cross-species infection

Western blot assay to detect the effects of ND10 proteins (Daxx, PML, and SP100) on HCMV protein production in mouse cells.

<p><b>A</b>. siRNA to knockdown the ND10 protein. One hundred pmol of siRNA (the sequences are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019187#s2" target="_blank">Materials and Methods</a>) was transfected into NIH3T3 cells for 24 hours, the whole-cell lysates were run on PAGE gels and probed with antibodies to the targeted proteins, as indicated. Tubulin was used as a house-keeping gene for sample-loading control. siRNA-Luc was used as control. <b>B</b>. One hundred pmol of siRNA was used to transfect NIH3T3 cells in a 24-well plate; 12 hours after transfection, the cells were infected with repaired AD169 at an MOI of 1, the cells were harvested at the time indicted, and the whole-cell lysates were used to detect viral-protein production.</p

Immunofluorescent assay to show cytomegalovirus infection and ND10.

<p><b>A–C</b>: After MCMV infection in NIH3T3 cells for 24 hours, cells were stained with anti-PML antibody (rabbit) to show ND10 (in red) (A); anti-IE1 antibody (mouse) was used to show IE1 (in green) (B); the merged picture is shown in C. <b>D–F</b>: After MCMV infection in Mrc-5 cells for 24 hours, cells were stained with anti-PML antibody (rabbit) to show ND10 (in red) (D); anti-IE1 antibody (mouse) was used to show IE1 (in green) (E); the merged picture is shown in F. <b>G–H</b>: After HCMV infection in NIH3T3 cells for 24 hours, cells were stained with anti-PML antibody (rabbit) to show ND10 (in red) (G); anti-IE1 antibody (mouse) was used to show IE1 (in green) (H); the merged picture was shown in I. <b>J–L</b>: After HCMV infection in Mrc-5 cells for 24 hours, cells were stained with anti-PML antibody (rabbit) to show ND10 (in red) (J); anti-IE1 antibody (mouse) was used to show IE1 (in green) (K); the merged picture is shown in L.</p