Situation Report -66 HIGHLIGHTS• Three new countries/territories/areas from the Region of the Americas [1], and African Region [2] have reported cases of COVID-19.• The United Nations launched a US$2 billion COVID-19 Global Humanitarian Response Plan to support the world's most vulnerable countries. More information can be found here.• The WHO Director-General mentioned many key issues and action steps to effectively combat COVID-19, as well as maintaining physical distance but not social distance. More information can be found here.• WHO published the COVID-19: Operational guidance for maintaining essential health services during an outbreak and the Handbook for public health capacity-building at ground crossings and cross-border collaboration on 25 March 2020. All guidance documents can be found here. Western Pacific Region 99 058 confirmed (1292) 3540 deaths (22) European Region 250 287 confirmed (29 771) 13 950 deaths (1964) South-East Asia Region 2536 confirmed (192) 79 deaths (7) Eastern Mediterranean Region 32 442 confirmed (2811) 2162 deaths (154) Region of the Americas 75 712 confirmed (14 878) 1065 deaths (252) African Region 1937 confirmed (275) 31 deaths (2) WHO RISK ASSESSMENTGlobal Level Very High

Vaccinia virus has a wide host range and infects mammalian cells of many different species. This suggests that the cell surface receptors for vaccinia virus are ubiquitously expressed and highly conserved. Alternatively, different receptors are used for vaccinia virus infection of different cell types. Here we report that vaccinia virus binds to heparan sulfate, a glycosaminoglycan (GAG) side chain of cell surface proteoglycans, during virus infection. Soluble heparin specifically inhibits vaccinia virus binding to cells, whereas other GAGs such as condroitin sulfate or dermantan sulfate have no effect. Heparin also blocks infections by cowpox virus, rabbitpox virus, myxoma virus, and Shope fibroma virus, suggesting that cell surface heparan sulfate could be a general mediator of the entry of poxviruses. The biochemical nature of the heparin-blocking effect was investigated. Heparin analogs that have acetyl groups instead of sulfate groups also abolish the inhibitory effect, suggesting that the negative charges on GAGs are important for virus infection. Furthermore, BSC40 cells treated with sodium chlorate to produce undersulfated GAGs are more refractory to vaccinia virus infection. Taken together, the data support the notion that cell surface heparan sulfate is important for vaccinia virus infection. Using heparin-Sepharose beads, we showed that vaccinia virus virions bind to heparin in vitro. In addition, we demonstrated that the recombinant A27L gene product binds to the heparin beads in vitro. This recombinant protein was further shown to bind to cells, and such interaction could be specifically inhibited by soluble heparin. All the data together indicated that A27L protein could be an attachment protein that mediates vaccinia virus binding to cell surface heparan sulfate during viral infection.

Vaccinia virus is a large DNA virus that infects many cell cultures in vitro and animal species in vivo.Although it has been used widely as a vaccine, its cell entry pathway remains unclear. In this study, we showed that vaccinia virus intracellular mature virions bound to the filopodia of HeLa cells and moved toward the cell body and entered the cell through an endocytic route that required a dynamin-mediated pathway but not a clathrin-or caveola-mediated pathway. Moreover, virus penetration required a novel cellular protein, vaccinia virus penetration factor (VPEF). VPEF was detected on cell surface lipid rafts and on vesicle-like structures in the cytoplasm. Both vaccinia virus and dextran transiently colocalized with VPEF, and, importantly, knockdown of VPEF expression blocked vaccinia virus penetration as well as intracellular transport of dextran, suggesting that VPEF mediates vaccinia virus entry through a fluid uptake endocytosis process in HeLa cells. Intracellular VPEF-containing vesicles did not colocalize with Rab5a or caveolin but partially colocalized with Rab11, supporting the idea that VPEF plays a role in vesicle trafficking and recycling in HeLa cells. In summary, this study characterized the mechanism by which vaccinia virus enters HeLa cells and identified a cellular factor, VPEF, that is exploited by vaccinia virus for cell entry through fluid phase endocytosis.The poxviruses form a group of large DNA viruses that includes variola virus, the causative agent of smallpox disease. Though smallpox itself has been eradicated, the fear of biological warfare and the recent occurrence of accidental monkeypox virus transmission between species (22) and of eczema vaccinatum (33) have alerted us to the potential danger of new emerging diseases. In addition, the potential applications of poxviruses as improved vaccines (34) and as oncolytic agents for cancer therapy (57) have raised new interest in poxvirus biology.Vaccinia virus, the well-studied prototype of the Orthopoxvirus genus in the family Poxviridae, has a wide range of infectivity in many cell lines and animals (20). It produces several forms of infectious particles, of which the vaccinia intracellular mature virus (IMV) is the most abundant in cells (see reference 14 and references therein). An IMV is enclosed by a single envelope and contains more than 70 viral proteins (11,45,65).The molecular mechanism of vaccinia IMV entry remains largely unknown. IMV binds to ubiquitous cellular attachment factors, such as glycosaminoglycans (12, 27) and the extracellular matrix protein laminin (10). It is not known whether IMV recognizes additional cellular coreceptors to trigger the postbinding fusion step, although virus entry through fusion with the plasma membrane (3, 9, 19, 37) or intracellular compartments (16, 58) has been reported. Interestingly, IMV has been shown to trigger cellular signaling during virus entry (2, 37, 44), but the molecular pathway of virus uptake has not been characterized.In this study, we characterized the mechanism by whi...

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor. Patients diagnosed with GBM have a poor prognosis, and it has been reported that tumor malignancy and GBM recurrence are promoted by STAT3 signaling. As resveratrol (RV), a polyphenol in grapes, is reported to be a potent and non-toxic cancer-preventive compound, the aim of this study was to investigate the therapeutic effect and molecular mechanisms of RV on GBM-derived radioresistant tumor initiating cells (TIC). Firstly, our results showed that primary GBM-CD133(+) TIC presented high tumorigenic and radiochemoresistant properties as well as increased protein levels of phosphorylated STAT3. We consistently observed that treatment with shRNA-STAT3 (sh-STAT3) or AG490, a STAT3 inhibitor, significantly inhibited the cancer stem-like cell properties and radioresistance of GBM-CD133(+) in vitro and in vivo. Furthermore, treatment of GBM-CD133(+) with 100 µM RV induced apoptosis and enhanced radiosensitivity by suppressing STAT3 signaling. Microarray results suggested that RV or AG490 inhibited the stemness gene signatures of GBM-CD133(+) and facilitated the differentiation of GBM-CD133(+) into GBM-CD133(-) or astrocytoma cells. Finally, xenotransplant experiments indicated that RV or sh-STAT3 therapy could significantly improve the survival rate and synergistically enhance the radiosensitivity of radiation-treated GBM-TIC. In summary, RV can reduce in vivo tumorigenicity and enhance the sensitivity of GBM-TIC to radiotherapies through the STAT3 pathway.

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Purpose: Allelic loss at chromosome 4q21-23 occurs frequently in human hepatocellular carcinoma, and the putative tumor suppressor gene (TSG) has not yet been identified.We studied the Fas-associated phosphatase-1 (FAP-1) gene as a potential candidateTSG in this region. Experimental Design: The expression level of FAP-1 RNA in hepatocellular carcinomas was evaluated by RNase protection and quantitative PCR. Sodium bisulfite modification and subsequent single-strand conformational polymorphism and sequence analyses were used to assay the methylation of CpGs at FAP-1promoter. Direct sequencing of the FAP-1coding region was conducted for detecting the genetic mutations. Two common single nucleotide polymorphisms of FAP-1were selected for evaluating their association with the hepatocellular carcinoma trait in sporadic and familial hepatocellular carcinomas. Moreover, the functional effect of FAP-1on cellular proliferation has been evaluated by small interfering RNA approach. Results: Around 50% of hepatocellular carcinomas showed significantly decreased expression of FAP-1 compared with the corresponding nontumorous liver tissues. In most cases, the RNA level was well correlated with the methylation status of promoter CpGs, suggesting that the promoter methylation may contribute to the down-regulation. Several genetic mutations of FAP1have been identified in hepatocellular carcinomas. The G/G genotype of FAP-1cSNP6304 was significantly associated with the increased risk of multiplex familial hepatocellular carcinomas (odds ratio, 2.44; 95% confidence interval, 1.19-5.01). Finally, knockdown expression of FAP-1 was shown to enhance the cellular proliferation in PLC5 cells. Conclusions: FAP-1 could be inactivated during hepatocarcinogenesis, mainly attributed by allelic loss and promoter methylation. The genetic mutations and polymorphisms may also confront with the higher hepatocellular carcinoma risk. These results first suggested FAP-1 as a putativeTSG in hepatocarcinogenesis.

Background Nasopharyngeal carcinoma (NPC) is a common head and neck cancer in Taiwan. Early detection is the best way to improve survival for this disease. A prospective study was designed to assess the feasibility of a new screening method for NPC by use of polymerase chain reaction (PCR) based on the close association of NPC and Epstein‐Barr virus (EBV). Methods One hundred thirty‐three different tissues from nasopharynx, nose, and sinus were investigated by use of PCR to check for the presence of EBV genome. Subsequently, from April 1996 to March 1997, 55 patients were enrolled in a prospective screening study. All patients underwent nasopharyngeal swabs before biopsy. Polymerase chain reaction detection of EBV genome was conducted on swab samples. Anti‐EBV viral capsid antigen (VCA) in IgA and IgG class were checked at the same visit. Results The EBV genome was present in 91.4% (85/93) of NPC tissues and in 25.0% (10/40) of non‐NPC tissues (p< .001, χ2 test). Of the 55 swabs, 49 (89.1%) specimens obtained enough cells for PCR examination. Thirty of these 49 patients were pathologically proved NPC. The presence of EBV were 86.7% (26/30) in NPC group and 42.1% (8/19) in non‐NPC group. The sensitivity and specificity were 86.7% and 57.9%, respectively, which were similar to those of serologic method (87.5% and 43.5%) when the cut‐off point was set at anti‐VCA IgG ≥ 160 and IgA ≥ 10. Combining both methods elevates the specificity to 84.2%. Conclusions The sensitivity of this PCR screening method is similar to that of the serologic method. Combining both methods can greatly increase the specificity, indicating this new method is a good supplement to the serologic screening of this endemic disease. © 1998 John Wiley & Sons, Inc. Head Neck 20: 732–738, 1998.

The real-world efficacy of epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) in patients with advanced non-small cell lung cancer (NSCLC) harboring EGFR-activating mutations remains unclear. We conducted a retrospective cohort study using data from the claims database of Taipei Veterans General Hospital to perform direct comparisons of these three EGFR-TKIs (gefitinib, erlotinib, and afatinib) combined with co-medications (metformin, statins, antacids, and steroids). Stage IIIB and IV NSCLC patients with EGFR mutations receiving EGFR-TKIs as first-line treatment for > 3 months between 2011 and 2016 were enrolled. The primary endpoint was time to treatment failure (TTF). Patients who had received co-medications (≥ 28 defined daily doses) in the first 3 months of EGFR-TKI therapy were assigned to co-medications groups. A total of 853 patients treated with gefitinib (n = 534), erlotinib (n = 220), and afatinib (n = 99) were enrolled. The median duration of TTF was 11.5 months in the gefitinib arm, 11.7 months in the erlotinib arm, and 16.1 months in the afatinib arm (log-rank test, P < 0.001). After adjustments, afatinib showed lower risk of treatment failure compared with gefitinib (hazard ratio [HR] 0.54, 95% confidence interval [CI] 0.41–0.71) and erlotinib (HR 0.62, 95% CI 0.46–0.83). The risk of treatment failure in patients treated with EGFR-TKIs who received concomitant systemic glucocorticoid therapy was higher than in those treated with EGFR-TKI monotherapy (HR 1.47, 95% CI 1.08–2.01). Afatinib or erlotinib use was associated with a lower risk of treatment failure in patients with advanced NSCLC harboring EGFR mutations compared to gefitinib use. Concurrent use of systemic glucocorticoids was linked to higher risk of treatment failure.