Wolbachia wStri Blocks Zika Virus Growth at Two Independent Stages of Viral Replication.

Mosquito-transmitted viruses are spread globally and present a great risk to human health. Among the many approaches investigated to limit the diseases caused by these viruses are attempts to make mosquitos resistant to virus infection. Coinfection of mosquitos with the bacterium Wolbachia pipientis from supergroup A is a recent strategy employed to reduce the capacity for major vectors in the Aedes mosquito genus to transmit viruses, including dengue virus (DENV), Chikungunya virus (CHIKV), and Zika virus (ZIKV). Recently, a supergroup B Wolbachia wStri, isolated from Laodelphax striatellus, was shown to inhibit multiple lineages of ZIKV in Aedes albopictus cells. Here, we show that wStri blocks the growth of positive-sense RNA viruses DENV, CHIKV, ZIKV, and yellow fever virus by greater than 99.9%. wStri presence did not affect the growth of the negative-sense RNA viruses LaCrosse virus or vesicular stomatitis virus. Investigation of the stages of the ZIKV life cycle inhibited by wStri identified two distinct blocks in viral replication. We found a reduction of ZIKV entry into wStri-infected cells. This was partially rescued by the addition of a cholesterol-lipid supplement. Independent of entry, transfected viral genome was unable to replicate in Wolbachia-infected cells. RNA transfection and metabolic labeling studies suggested that this replication defect is at the level of RNA translation, where we saw a 66% reduction in mosquito protein synthesis in wStri-infected cells. This study's findings increase the potential for application of wStri to block additional arboviruses and also identify specific blocks in viral infection caused by Wolbachia coinfection.IMPORTANCE Dengue, Zika, and yellow fever viruses are mosquito-transmitted diseases that have spread throughout the world, causing millions of infections and thousands of deaths each year. Existing programs that seek to contain these diseases through elimination of the mosquito population have so far failed, making it crucial to explore new ways of limiting the spread of these viruses. Here, we show that introduction of an insect symbiont, Wolbachia wStri, into mosquito cells is highly effective at reducing yellow fever virus, dengue virus, Zika virus, and Chikungunya virus production. Reduction of virus replication was attributable to decreases in entry and a strong block of virus gene expression at the translational level. These findings expand the potential use of Wolbachia wStri to block viruses and identify two separate steps for limiting virus replication in mosquitos that could be targeted via microbes or other means as an antiviral strategy.

Viral E6-E7 transcription in the basal layer of organotypic cultures without apparent p21cip1 protein precedes immortalization of human papillomavirus type 16- and 18-transfected human keratinocytes.

Organotypic cultures of human keratinocytes provide a useful model system to study human papillomavirus (HPV)-host cell interactions. In this study, we analyzed organotypic cultures of two HPV type 16 (HPV16) (FK16A and FK16B)- and two HPV18 (FK18A and FK18B)-transfected keratinocyte cell lines through the process of immortalization in vitro. For FK16A and FK18B cells, passages of both mortal cells in their extended life span and subsequent immortal stages were studied. Mortal cells of FK16A and FK18B showed a morphology reminiscent of mild to moderate dysplasia, whereas in their immortal descendants, severely dysplastic features were observed. Immortal FK18A cells were mildly to moderately dysplastic, while FK16B cells were severely dysplastic. The increasing degrees of dysplasia were associated with a decreasing expression of differentiation markers cytokeratin 10 and profilaggrin. All raft cultures expressed E6-E7 mRNAs in the basal layer, while the amount of viral transcripts in the suprabasal cells was in general proportional to the degree of dysplasia. In all cases, E6-E7 transcription and dysplastic features were highly correlated with cellular proliferation, as assessed by Ki-67 (MIB-1) antigen expression. Moreover, high levels of E6-E7 transcription and expression of p21cip1 protein in the basal layer seemed to be mutually exclusive. We conclude that expression of E6-E7 in the basal cells associated with increased proliferation in the absence of detectable p21cip1 protein is apparently necessary but not sufficient for immortalization, or for the loss of terminal differentiation, for which yet to be discovered additional events are required. The model system described in this study provides a valuable tool to analyze alterations in viral transcription regulation during HPV-mediated cell transformation.

Differences in magnitude and duration of opioid-induced respiratory depression and analgesia with fentanyl and sufentanil.

The magnitude and duration of analgesia and respiratory depression induced by fentanyl (1.0, 2.0, and 4.0 micrograms/kg) and sufentanil (0.1, 0.2, and 0.4 microgram/kg) after intravenous administration over 30 s were measured in 30 healthy young adult male volunteers divided into three groups and studied in a double-blind, randomized fashion. Each volunteer received one dose of fentanyl or sufentanil and no sooner than 48 h later, the corresponding equipotent dose of the other opioid. End-tidal CO2 and ventilatory and occlusion pressure responses to CO2 rebreathing were used to measure drug-induced respiratory effects. Analgesic effects were assessed by changes in the pain threshold to electric shock applied to the forearm. Plasma levels of fentanyl and sufentanil were measured by radioimmunoassay. Testing and sampling intervals were 5, 30, 60, 90, 120, 240, 300, and 360 min after drug administration. The magnitude and duration of depression of the ventilatory and occlusion pressure response were significantly less with sufentanil compared with fentanyl, irrespective of dose. Ventilatory and occlusion pressure responses returned to control values by 30 and 30 min, respectively, after sufentanil and by 240 and 120 min, respectively, after fentanyl. Statistically significant elevations of the pain threshold were, however, greater and longer lasting after sufentanil compared with fentanyl. Pain threshold returned to control values 180 min after sufentanil but only 90 min after fentanyl. These results suggest that sufentanil may provide better patient comfort with less respiratory depression than does fentanyl.