Two newly introduced Wolbachia endosymbionts induce cell host differences in competitiveness and metabolic responses

Wolbachia endosymbionts can induce multiple reproductive manipulations in their hosts, with cytoplasmic incompatibility (CI) being one of the most common manipulations. The important agricultural pests, white-backed planthopper ( Sogatella furcifera ) and brown planthopper ( Nilaparvata lugens ), are usually infected with CI-inducing Wolbachia w Fur and non-CI-inducing Wolbachia w Lug, respectively. The biological effects of these infections when present in a host cell are unknown. Here, we introduced the two Wolbachia strains into an Aedes albopictus cell line to stably establish a w Fur-infected cell line (WFI) and a w Lug-infected cell line (WLI). In a mixed culture, WFI cells were completely replaced by WLI cells, pointing to a stronger competitiveness of the WLI cell line. We found that infection by both Wolbachia strains reduced cell growth rates, but WLI had a faster cell growth rate than WFI, and this difference in cell growth rate combined with possible Wolbachia differences in diffusivity may have affected cell competitiveness. By examining gene expression and metabolites in the two lines, we found that some genes and key metabolites responded to differences in cell competitiveness. These results point to potential mechanisms that could contribute to the relative performance of hosts infected by these strains and also highlight the substantial impact of a non-CI Wolbachia on metabolism, which may in turn influence fitness of its native host. IMPORTANCE Wolbachia transinfection in insects can be used to suppress pests and block virus transmission. We stably introduced two Wolbachia strains from rice planthoppers into cell lines of an important arbovirus mosquito vector, Aedes albopictus . The competitiveness of host cells from the lines infected by the two Wolbachia strains was different, as were metabolic responses of the cell lines. These results suggest potential metabolic effects of Wolbachia on native hosts which could be exploited when they are transinfected into novel hosts for pest control.

Development and validation of a targeted sequencing panel for application to treatment-refractory solid tumor.

245 Background: Next generation sequencing has revolutionised genomic studies of cancer, having facilitated the development of precision oncology treatments based on a tumour’s molecular profile. We aimed to develop a targeted sequencing panel for application to treatment-refractory solid tumor types with particular focus on tumours of the stomach cancer, plus test for utility in FFPE, fluid sample and cancer cell lines. Methods: “CancerMaster” is custom RNA probes for target enrichment sequencing. It consists of all unions (7,811 regions, 2.5Mb) of reported exons of 524 tumor and immune related genes. The panel contains special RNA probes, which enables detection of microsatellite instability, Epstein-Barr virus and Human papillomavirus. Also, the CancerMaster panel analyzes 524 genes as well as genomic signatures including microsatellite instability (MSI), tumor mutational burden (TMB) and HLA allele. FFPE samples and 49 gastric cancer (GC) cell lines were collected for performance and application testing. Panel sensitivity and precision were measured using well-characterised DNA controls (n = 117), and specificity by Sanger sequencing, pyrosequencing, FoundationOne CDx. Results: We achieved a mean coverage of 1,032x, with sensitivity and specificity of >99% and precision of >97%. In this study, we evaluated 26 Yonsei Cancer Center (YCC) GC cell lines and 23 GC cell lines from other sources (ATCC, KCLB, and JCRB). Application to 49 gastric cancers cell lines resulted in detection of copy number variant (CNV), single number variant (SNV) and small InDel aligned to whole exome sequencing data. In addition to previous novel EBV infected cell line (YCCEL1/YCC-10, J Gen Virol. 2013), we observed amplification and overexpression of receptor tyrosine kinase (RTK) including HER2 (YCC-19, 32, 33, 38), EGFR (YCC-11, 21), Met (YCC-31, 34), and FGFR2 (YCC-28, 30) in YCC GC cell lines; confirming that protein was overexpression by Western blot or Flow cytometry in 19/49 (38.8%). Interestingly, amplification of RTKs was detected mutually exclusive pattern with other RTK amplification. Conclusions: We have developed and validated an analytically-validated panel for application to cancers of treatment-refractory types.

Organ-Specific Treatment for COVID-19: Rationale, Evidence, and Potential Candidates

<div><b>Background and Purpose:</b> COVID-19 has become the ongoing public health crisis of our time. Although it was first presented as a respiratory infection, extrapulmonary manifestations are increasingly reported. However, no effective therapeutic strategy for COVID-19 extrapulmonary involvement is currently available. The current study aims to analyze the pathogenesis of COVID-19 extrapulmonary complications to evaluate the rationale for proposing organ-specific treatment as a novel therapeutic strategy to manage these multisystemic complications.</div><div><b>Experimental Approach:</b> In this study, differentially expressed genes (DEGs) of SARS-CoV-2 infected extrapulmonary organs including human pluripotent stem cells (hPSCs)-derived liver organoids, hPSCs-derived pancreatic endocrine cells, and human-induced pluripotent stem cells (hiPSCs)-derived choroid plexus organoids were analyzed. First, pathway enrichment analysis is done based on the identified DEGs to compare the underlying biological pathways enriched upon SARS-CoV-2 infection in different organs to confirm the need for developing organ-specific treatment strategies. Then, these lists of DEGs are used in a connectivity map-based drug repurposing experiment to propose novel organ-specific therapeutic options.</div><div><b>Key Results:</b> The results reveal different biological pathways and networks responsible for SARS-CoV-2 multisystemic pathogenesis based on the organ involved that highlight the need for considering organ-specific treatments. Besides, some FDA-approved drugs are proposed as the potential therapeutic candidates for each infected cell line.</div><div><b>Conclusion and Implications:</b> Although COVID-19 extrapulmonary manifestations are increasing, management of these complications is still challenging. Traditional therapeutic strategies and already repurposed antiviral agents are not effective. In this situation, organ-specific treatment, or in other words personalized therapy might be a promising solution.</div>

Organ-Specific Treatment for COVID-19: Rationale, Evidence, and Potential Candidates

<div><b>Background and Purpose:</b> COVID-19 has become the ongoing public health crisis of our time. Although it was first presented as a respiratory infection, extrapulmonary manifestations are increasingly reported. However, no effective therapeutic strategy for COVID-19 extrapulmonary involvement is currently available. The current study aims to analyze the pathogenesis of COVID-19 extrapulmonary complications to evaluate the rationale for proposing organ-specific treatment as a novel therapeutic strategy to manage these multisystemic complications.</div><div><b>Experimental Approach:</b> In this study, differentially expressed genes (DEGs) of SARS-CoV-2 infected extrapulmonary organs including human pluripotent stem cells (hPSCs)-derived liver organoids, hPSCs-derived pancreatic endocrine cells, and human-induced pluripotent stem cells (hiPSCs)-derived choroid plexus organoids were analyzed. First, pathway enrichment analysis is done based on the identified DEGs to compare the underlying biological pathways enriched upon SARS-CoV-2 infection in different organs to confirm the need for developing organ-specific treatment strategies. Then, these lists of DEGs are used in a connectivity map-based drug repurposing experiment to propose novel organ-specific therapeutic options.</div><div><b>Key Results:</b> The results reveal different biological pathways and networks responsible for SARS-CoV-2 multisystemic pathogenesis based on the organ involved that highlight the need for considering organ-specific treatments. Besides, some FDA-approved drugs are proposed as the potential therapeutic candidates for each infected cell line.</div><div><b>Conclusion and Implications:</b> Although COVID-19 extrapulmonary manifestations are increasing, management of these complications is still challenging. Traditional therapeutic strategies and already repurposed antiviral agents are not effective. In this situation, organ-specific treatment, or in other words personalized therapy might be a promising solution.</div>

Serendipitous Discovery of a Novel Murine Astrovirus Contaminating a Murine Helper T-cell Line and Incapable of Infecting Highly Immunodeficient Mice

The unexpected seroconversion of sentinel mice in our facility to murine T lymphotrophic virus (MTLV) positivity led to our identification of a novel murine astrovirus that we designated murine astrovirus 2 (MuAstV-2). During our investigation, MuAstV-2 was found to be a contaminant of the T helper cell line (D10. G4.1) that was used to generate the MTLV antigen that we included in the multiplex fluorometric immunoassay (MFIA) that we used for sentinel screening. We eventually determined that cross-reactivity with the astrovirus generated a positive result in the MTLV assay. A confirmatory immunofluorometric assay (IFA) using the same MTLV-infected cell line yielded a similar result. However, the use of antigen prepared from MTLV-infected neonatal mouse thymus did not reproduce a positive result, leading us to suspect that the seroreactivity we had observed was not due to infection with MTLV. A mouse antibody production test showed that mice inoculated with naïve D10. G4.1 cells and their contact sentinels tested positive for MTLV using cell-line generated antigen, but tested negative in assays using MTLV antigen produced in mice. Metagenomic analysis was subsequently used to identify MuAstV-2 in feces from 2 sentinel mice that had recently seroconverted to MTLV. Two closely related astrovirus sequences (99.6% capsid identity) were obtained and shared 95% capsid amino acid identity with the MuAstV-2 virus sequenced from the D10. G4.1 cell line. These viruses are highly divergent from previously identified murine astroviruses, displaying <30% capsid identity, yet were closely related to murine astrovirus 2 (85% capsid identity), which had recently been isolated from feral mice in New York City. A MuAstV-2 specific PCR assay was developed and used to eradicate MuAstV-2 from the infected colony using a test and cull strategy. The newly identified MuAstV2 readily transmits to immunocompetent mouse strains by fecal-oral exposure, but fails to infect NOD-Prkdcem26Cd52Il2rgem26Cd22/NjuCrl (NCG) mice, which have significantly impaired adaptive and innate immune systems. Neither immunocompetent nor immunodeficient mice showed any astrovirus-associated pathology. MuAstV-2 may provide a valuable model for the study of specific aspects of astrovirus pathogenesis and virus-host interactions.

Determination of the Infectivity of Cryopreserved Theileria annu-lata Sporozoites in Tick Derived Stabilates Iran Ak-93 Strain, by In Vivo and In Vitro Methods

Background: The protozoan parasite Theileria annulata is the causative agent of tropical theileriosis in cattle. Vaccination is recommended by administration of attenuated schizont-infected cell lines. The expected protective immunity post-vaccination can be demonstrated by challenge test through inoculation of highly virulent infective sporozoites. The aim of this study was to produce Hyalomma anatolicum anatolicum tick infected with T. annulata (local strain) for preparation of tick-derived sporozoite stabilates for molecular characterization and infectivity test assay. Methods: A local T. annulata strain was used for experimental infection of calves. A field isolate of H. a. anatolicum was isolated, laboratory-reared and infected by blood-feeding on Theileria infected above-mentioned calves. The infectivity of calf, tick and prepared stabilate were confirmed by clinical signs of theileriosis, microscopic inspection, RT-PCR and in vitro cell culture. Results: The tick stabilate was prepared and cryopreserved in liquid nitrogen. The infectivity of the tick stabilate was verified by in vivo bioassay, in vitro cell culture infection, microscopic inspection in salivary glands and RT-PCR assay. The in vitro produced cell line in this study was characterized by T. annulata Cytochrome b gene analyzing. Conclusion: The infectivity of a new prepared tick-derived sporozoite stabilate was confirmed in susceptible calves; by microscopically, post mortem, tick microscopic and molecular assays. Moreover, naïve PBMCs were transformed and proliferated by T. annulata infected tick stabilate to immortal T. annulata schizont infected cell line. The potent infective sporozoite tick derived stabilate could be used for vaccine efficacy and challenge test as well as in vaccine development.