Peritumoral SPARC expression induced by exosomes from nasopharyngeal carcinoma infected Epstein-Barr virus: A poor prognostic marker.

Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) cells have high metastatic potential. Recent research has revealed that the interaction of between tumor cells and the surrounding stroma plays an important role in tumor invasion and metastasis. In this study, we showed the prognostic value of expression of SPARC, an extracellular matrix protein with multiple cellular functions, in normal adjacent tissues (NAT) surrounding NPC. In the immunohistochemical analysis of 51 NPC biopsy specimens, SPARC expression levels were significantly elevated in the NAT of EBER (EBV-encoded small RNA)-positive NPC compared to that in the NAT of EBER-negative NPC. Moreover, increased SPARC expression in NAT was associated with a worsening of overall survival. The enrichment analysis of RNA-seq of publicly available NPC and NAT surrounding NPC data showed that high SPARC expression in NPC was associated with epithelial mesenchymal transition promotion, and there was a dynamic change in the gene expression profile associated with interference of cellular proliferation in NAT, including SPARC expression. Furthermore, EBV-positive NPC cells induce SPARC expression in normal nasopharyngeal cells via exosomes. Induction of SPARC in cancer-surrounding NAT cells reduced intercellular adhesion in normal nasopharyngeal structures and promoted cell competition between cancer cells and normal epithelial cells. These results suggest that epithelial cells loosen their own binding with the extracellular matrix as well as stromal cells, facilitating the invasion of tumor cells into the adjacent stroma by activating cell competition. Our findings reveal a new mechanism by which EBV creates a pro-metastatic microenvironment by upregulating SPARC expression in NPC.

Estrogen induces the expression of EBV lytic protein ZEBRA, a marker of poor prognosis in nasopharyngeal carcinoma.

Several epidemiological studies have suggested that Epstein-Barr virus (EBV) lytic infection is essential for the development of nasopharyngeal carcinoma (NPC), as the elevation of antibody titers against EBV lytic proteins is a common feature of NPC. Although ZEBRA protein is a key trigger for the initiation of lytic infection, whether its expression affects the prognosis and pathogenesis of NPC remains unclear. In this study, 64 NPC biopsy specimens were analyzed using immunohistochemistry. We found that ZEBRA was significantly associated with a worsening of progression-free survival in NPC (adjusted hazard ratio, 3.58; 95% confidence interval, 1.08-11.87; p = 0.037). Moreover, ZEBRA expression positively correlated with key endocrinological proteins, estrogen receptor α, and aromatase. The transcriptional level of ZEBRA is activated by estrogen in an estrogen receptor α-dependent manner, resulting in an increase in structural gene expression levels and extracellular virus DNA copy number in NPC cell lines, reminiscent of lytic infection. Interestingly, it did not suppress cellular proliferation or increase apoptosis, in contrast with cells treated with 12-O-tetradecanoylphorbol-13-acetate and sodium butyrate, indicating that viral production induced by estrogen is not a cell lytic phenomenon. Our results suggest that intratumoral estrogen overproduced by aromatase could induce ZEBRA expression and EBV reactivation, contributing to the progression of NPC.

Purification of Emu IgY for Therapeutic and Diagnostic Use Based on Monoclonal Secondary Antibodies Specific to Emu IgY.

The emu is the second largest ratite; thus, their sera and egg yolks, obtained after immunization, could provide therapeutic and diagnostically important immunoglobulins with improved production efficiency. Reliable purification tools are required to establish a pipeline for supplying practical emu-derived antibodies, the majority of which belongs to the immunoglobulin Y (IgY) class. Therefore, we generated a monoclonal secondary antibody specific to emu IgY. Initially, we immunized an emu with bovine serum albumin multiply haptenized with 2,4-dinitrophenyl (DNP) groups. Polyclonal emu anti-DNP antibodies were partially purified using conventional precipitation method and used as antigen for immunizing a BALB/c mouse. Splenocytes were fused with myeloma cells and a hybridoma clone secreting a desirable secondary antibody (mAb#2-16) was established. The secondary antibody bound specifically to emu-derived IgY, distinguishing IgYs from chicken, duck, ostrich, quail, and turkey, as well as human IgGs. Affinity columns immobilizing the mAb#2-16 antibodies enabled purification of emu IgY fractions from sera and egg yolks via simple protocols, with which we succeeded in producing IgYs specific to the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) spike protein with a practical binding ability. We expect that the presented purification method, and the secondary antibody produced in this study, will facilitate the utilization of emus as a novel source of therapeutic and diagnostic antibodies.

[Introduction of high containment laboratories in abroad].

Recently, outbreaks of highly pathogenic viruses, such as those of Ebola and Lassa viruses, have become a global public health issue. Such viruses must be handled in biosafety level 4 (BSL-4) laboratories. Currently, 62 BSL-4 laboratories are in operation, under construction, or planned in 24 counties. In this review, I provide an overview of the current status and characteristics of BSL-4 facilities in abroad and introduce my research on the wild-type Ebola virus at the BSL-4 facility in the USA.

[Developing a Biosafety Level 4 Laboratory user training program].

In recent years, numerous emerging and reemerging infectious diseases have occurred worldwide and have seriously threatened our society. As a countermeasure against the pathogens responsible for serious diseases (classified as class 4 pathogens), we are preparing for full operation of the first suit-type biosafety level 4 (BSL-4) facility available for basic and applied research at Nagasaki University. For the safe operation of these facilities, experienced and qualified personnel with appropriate skills and knowledge of biorisk management must be certified. Developing an appropriate training system is a prerequisite for ensuring the safety of users involved in research in a BSL-4 laboratory. Here, we introduce an overview of the content of the training program that we are currently establishing for the BSL-4 facility at Nagasaki University. We are designing this program to follow national and international guidelines and regulations in part by referring to experiences and materials derived from multiple BSL-4 facilities in other countries. The established training program system, including the formulation processes, will serve as a reference and will provide practical materials for other research organizations to develop their own high-containment laboratory training programs.

Ubiquitin Modification of the Epstein-Barr Virus Immediate Early Transactivator Zta.

The Epstein-Barr virus (EBV) immediate early transactivator Zta plays a key role in regulating the transition from latency to the lytic replication stages of EBV infection. Regulation of Zta is known to be controlled through a number of transcriptional and posttranscriptional events. Here, we show that Zta is targeted for ubiquitin modification and that this can occur in EBV-negative and in EBV-infected cells. Genetic studies show critical roles for both an amino-terminal region of Zta and the basic DNA binding domain of Zta in regulating Zta ubiquitination. Pulse-chase experiments demonstrate that the bulk population of Zta is relatively stable but that at least a subset of ubiquitinated Zta molecules are targeted for degradation in the cell. Mutation of four out of a total of nine lysine residues in Zta largely abrogates its ubiquitination, indicating that these are primary ubiquitination target sites. A Zta mutant carrying mutations at these four lysine residues (lysine 12, lysine 188, lysine 207, and lysine 219) cannot induce latently infected cells to produce and/or release infectious virions. Nevertheless, this mutant can induce early gene expression, suggesting a possible defect at the level of viral replication or later in the lytic cascade. As far as we know, this is the first study that has investigated the targeting of Zta by ubiquitination or its role in Zta function.IMPORTANCE Epstein-Barr virus (EBV) is a ubiquitous human pathogen and associated with various human diseases. EBV undergoes latency and lytic replication stages in its life cycle. The transition into the lytic replication stage, at which virus is produced, is mainly regulated by the viral gene product, Zta. Therefore, the regulation of Zta function becomes a central issue regarding viral biology and pathogenesis. Known modifications of Zta include phosphorylation and sumoylation. Here, we report the role of ubiquitination in regulating Zta function. We found that Zta is subjected to ubiquitination in both EBV-infected and EBV-negative cells. The ubiquitin modification targets 4 lysine residues on Zta, leading to both mono- and polyubiquitination of Zta. Ubiquitination of Zta affects the protein's stability and likely contributes to the progression of viral lytic replication. The function and fate of Zta may be determined by the specific lysine residue being modified.

Ebola virus requires a host scramblase for externalization of phosphatidylserine on the surface of viral particles.

Cell surface receptors for phosphatidylserine contribute to the entry of Ebola virus (EBOV) particles, indicating that the presence of phosphatidylserine in the envelope of EBOV is important for the internalization of EBOV particles. Phosphatidylserine is typically distributed in the inner layer of the plasma membrane in normal cells. Progeny virions bud from the plasma membrane of infected cells, suggesting that phosphatidylserine is likely flipped to the outer leaflet of the plasma membrane in infected cells for EBOV virions to acquire it. Currently, the intracellular dynamics of phosphatidylserine during EBOV infection are poorly understood. Here, we explored the role of XK-related protein (Xkr) 8, which is a scramblase responsible for exposure of phosphatidylserine in the plasma membrane of apoptotic cells, to understand its significance in phosphatidylserine-dependent entry of EBOV. We found that Xkr8 and transiently expressed EBOV glycoprotein GP often co-localized in intracellular vesicles and the plasma membrane. We also found that co-expression of GP and viral major matrix protein VP40 promoted incorporation of Xkr8 into ebolavirus-like particles (VLPs) and exposure of phosphatidylserine on their surface, although only a limited amount of phosphatidylserine was exposed on the surface of the cells expressing GP and/or VP40. Downregulating Xkr8 or blocking caspase-mediated Xkr8 activation did not affect VLP production, but they reduced the amount of phosphatidylserine on the VLPs and their uptake in recipient cells. Taken together, our findings indicate that Xkr8 is trafficked to budding sites via GP-containing vesicles, is incorporated into VLPs, and then promote the entry of the released EBOV to cells in a phosphatidylserine-dependent manner.

A Peptide Derived from Phosphoinositide 3-kinase Inhibits Endocytosis and Influenza Virus Infection.

Endocytosis mediates the internalization and ingestion of a variety of endogenous or exogenous substances, including virus particles, under the control of intracellular signaling pathways. We have previously reported that the complex formed between the small GTPase Ras and phosphoinositide 3-kinase (PI3K) translocates from the plasma membrane to endosomes, signaling from which thereby regulates clathrin-independent endocytosis, endosome maturation, influenza virus internalization, and infection. However, the molecular mechanism by which the Ras-PI3K complex is recruited to endosomes remains unclear. Here, we have identified the amino acid sequence responsible for endosomal localization of the Ras-PI3K complex. PI3K lacking this sequence failed to translocate to endosomes, and expression of the peptide comprising this PI3K-derived sequence inhibited clathrin-independent endocytosis, influenza virus internalization, and infection. Moreover, treatment of cells with this peptide in an arginine-rich, cell-penetrating form successfully suppressed influenza virus infection in vitro and ex vivo, making this peptide a potential therapeutic agent against influenza virus infection.Key words: signal transduction, endocytosis, endosome, imaging, influenza virus.

Budding of Ebola Virus Particles Requires the Rab11-Dependent Endocytic Recycling Pathway.

The Ebola virus-encoded major matrix protein VP40 traffics to the plasma membrane, which leads to the formation of filamentous viral particles and subsequent viral egress. However, the cellular machineries underlying this process are not fully understood. In the present study, we have assessed the role of host endocytic recycling in Ebola virus particle formation. We found that a small GTPase Rab11, which regulates recycling of molecules among the trans-Golgi network, recycling endosomes, and the plasma membrane, was incorporated in Ebola virus-like particles. Although Rab11 predominantly localized in the perinuclear region, it distributed diffusely in the cytoplasm and partly localized in the periphery of the cells transiently expressing VP40. In contrast, Rab11 exhibited a perinuclear distribution when 2 VP40 derivatives that lack ability to traffic to the plasma membrane were expressed. Finally, expression of a dominant-negative form of Rab11 or knockdown of Rab11 inhibited both VP40-induced clusters at the plasma membrane and release of viral-like particles. Taken together, our findings demonstrate that Ebola virus exploits host endocytic recycling machinery to facilitate the trafficking of VP40 to the cell surface and the subsequent release of viral-like particles for its establishment of efficient viral egress.

Fcγ-receptor IIa-mediated Src Signaling Pathway Is Essential for the Antibody-Dependent Enhancement of Ebola Virus Infection.

Antibody-dependent enhancement (ADE) of Ebola virus (EBOV) infection has been demonstrated in vitro, raising concerns about the detrimental potential of some anti-EBOV antibodies. ADE has been described for many viruses and mostly depends on the cross-linking of virus-antibody complexes to cell surface Fc receptors, leading to enhanced infection. However, little is known about the molecular mechanisms underlying this phenomenon. Here we show that Fcγ-receptor IIa (FcγRIIa)-mediated intracellular signaling through Src family protein tyrosine kinases (PTKs) is required for ADE of EBOV infection. We found that deletion of the FcγRIIa cytoplasmic tail abolished EBOV ADE due to decreased virus uptake into cellular endosomes. Furthermore, EBOV ADE, but not non-ADE infection, was significantly reduced by inhibition of the Src family protein PTK pathway, which was also found to be important to promote phagocytosis/macropinocytosis for viral uptake into endosomes. We further confirmed a significant increase of the Src phosphorylation mediated by ADE. These data suggest that antibody-EBOV complexes bound to the cell surface FcγRIIa activate the Src signaling pathway that leads to enhanced viral entry into cells, providing a novel perspective for the general understanding of ADE of virus infection.

Inside the Cell: Assembly of Filoviruses.

This chapter reviews our current knowledge about the spatiotemporal assembly of filoviral particles. We will follow particles from nucleocapsid entry into the cytoplasm until the nucleocapsids are enveloped at the plasma membrane. We will also highlight the currently open scientific questions surrounding filovirus assembly.

Improved FRET Biosensor for the Measurement of BCR-ABL Activity in Chronic Myeloid Leukemia Cells.

Although the co-development of companion diagnostics with molecular targeted drugs is desirable, truly efficient diagnostics are limited to diseases in which chromosomal translocations or overt mutations are clearly correlated with drug efficacy. Moreover, even for such diseases, few methods are available to predict whether drug administration is effective for each individual patient whose disease is expected to respond to the drug(s). We have previously developed a biosensor based on the principle of Förster resonance energy transfer to measure the activity of the tyrosine kinase BCR-ABL and its response to drug treatment in patient-derived chronic myeloid leukemia cells. The biosensor harbors CrkL, one of the major substrates of BCR-ABL, and is therefore named Pickles after phosphorylation indicator of CrkL en substrate. The efficacy of this technique as a clinical test has been demonstrated, but the number of cells available for analysis is limited in a case-dependent manner, owing to the cleavage of the biosensor in patient-derived leukemia cells. Here, we describe an improved biosensor with an amino acid substitution and a nuclear export signal being introduced. Of the two predicted cleavage positions in CrkL, the mutations inhibited one cleavage completely and the other cleavage partially, thus collectively increasing the number of cells available for drug evaluation. This improved version of the biosensor holds promise in the future development of companion diagnostics to predict responses to tyrosine kinase inhibitors in patients with chronic myeloid leukemia.

Epstein-Barr virus exploits host endocytic machinery for cell-to-cell viral transmission rather than a virological synapse.

Epstein-Barr virus (EBV) establishes a lifelong latent infection in B lymphocytes and often is found in epithelial cells. Several lines of evidence indicate that viral transmission mediated by cell-to-cell contact is the dominant mode of infection by EBV for epithelial cells. However, its detailed molecular mechanism has not been fully elucidated. We investigated the role of host membrane trafficking machinery in this process. We have found that adhesion molecules critical for this process are expressed in EBV-positive and -negative Burkitt's lymphoma (BL) cells and multiple epithelial cell lines. Treatment with blocking antibodies against ß1 and ß2 integrin families and their ligands suppressed EBV transmission in a dose-dependent manner. We also confirmed that adhesion molecules are upregulated in co-cultured BL cells. Immunofluorescence staining revealed that the intracellular adhesion molecule 1 (ICAM-1) distributed to the cell surface and partially co-localized with recycling endosomes in co-cultured BL cells. Moreover, cell-to-cell EBV transmission was inhibited upon blocking endocytic recycling by expression of a dominant-negative form of a small GTPase Rab11 or by knockdown of Rab11, supporting the notion that the endocytic pathway-dependent trafficking of ICAM-1 to the cell surface of BL cells contributes to viral transmission by stabilizing cell-to-cell contact between the donor cells and recipient cells. Finally, we demonstrated that co-cultivation upregulated clathrin-mediated endocytosis in the recipient cells, allowing EBV to be internalized. Taken together, our findings demonstrate that EBV exploits host endocytic machinery in both donor and recipient cells, a process which is facilitated by cell-to-cell contact, thereby promoting successful viral transmission.

Interaction between TIM-1 and NPC1 Is Important for Cellular Entry of Ebola Virus.

UNLABELLED: Multiple host molecules are known to be involved in the cellular entry of filoviruses, including Ebola virus (EBOV); T-cell immunoglobulin and mucin domain 1 (TIM-1) and Niemann-Pick C1 (NPC1) have been identified as attachment and fusion receptors, respectively. However, the molecular mechanisms underlying the entry process have not been fully understood. We found that TIM-1 and NPC1 colocalized and interacted in the intracellular vesicles where EBOV glycoprotein (GP)-mediated membrane fusion occurred. Interestingly, a TIM-1-specific monoclonal antibody (MAb), M224/1, prevented GP-mediated membrane fusion and also interfered with the binding of TIM-1 to NPC1, suggesting that the interaction between TIM-1 and NPC1 is important for filovirus membrane fusion. Moreover, MAb M224/1 efficiently inhibited the cellular entry of viruses from all known filovirus species. These data suggest a novel mechanism underlying filovirus membrane fusion and provide a potential cellular target for antiviral compounds that can be universally used against filovirus infections. IMPORTANCE: Filoviruses, including Ebola and Marburg viruses, cause rapidly fatal diseases in humans and nonhuman primates. There are currently no approved vaccines or therapeutics for filovirus diseases. In general, the cellular entry step of viruses is one of the key mechanisms to develop antiviral strategies. However, the molecular mechanisms underlying the entry process of filoviruses have not been fully understood. In this study, we demonstrate that TIM-1 and NPC1, which serve as attachment and fusion receptors for filovirus entry, interact in the intracellular vesicles where Ebola virus GP-mediated membrane fusion occurs and that this interaction is important for filovirus infection. We found that filovirus infection and GP-mediated membrane fusion in cultured cells were remarkably suppressed by treatment with a TIM-1-specific monoclonal antibody that interfered with the interaction between TIM-1 and NPC1. Our data provide new insights for the development of antiviral compounds that can be universally used against filovirus infections.

Comprehensive high-throughput RNA sequencing analysis reveals contamination of multiple nasopharyngeal carcinoma cell lines with HeLa cell genomes.

UNLABELLED: In an attempt to explore infectious agents associated with nasopharyngeal carcinomas (NPCs), we employed our high-throughput RNA sequencing (RNA-seq) analysis pipeline, RNA CoMPASS, to investigate the presence of ectopic organisms within a number of NPC cell lines commonly used by NPC and Epstein-Barr virus (EBV) researchers. Sequencing data sets from both CNE1 and HONE1 were found to contain reads for human papillomavirus 18 (HPV-18). Subsequent real-time reverse transcription-PCR (RT-PCR) analysis on a panel of NPC cell lines identified HPV-18 in CNE1 and HONE1 as well as three additional NPC cell lines (CNE2, AdAH, and NPC-KT). Further analysis of the chromosomal integration arrangement of HPV-18 in NPCs revealed patterns identical to those observed in HeLa cells. Clustering based on human single nucleotide variation (SNV) analysis of two separate HeLa cell lines and several NPC cell lines demonstrated two distinct clusters with CNE1, as well as HONE1 clustering with the two HeLa cell lines. In addition, duplex-PCR-based genotyping showed that CNE1, CNE2, and HONE1 do not have a HeLa cell-specific L1 retrotransposon insertion, suggesting that these three HPV-18(+) NPC lines are likely products of a somatic hybridization with HeLa cells, which is also consistent with our RNA-seq-based gene level SNV analysis. Taking all of these findings together, we conclude that a widespread HeLa contamination may exist in many NPC cell lines, and authentication of these cell lines is recommended. Finally, we provide a proof of concept for the utility of an RNA-seq-based approach for cell authentication. IMPORTANCE: Nasopharyngeal carcinoma (NPC) cell lines are important model systems for analyzing the complex life cycle and pathogenesis of Epstein-Barr virus (EBV). Using an RNA-seq-based approach, we found HeLa cell contamination in several NPC cell lines that are commonly used in the EBV and related fields. Our data support the notion that contamination resulted from somatic hybridization with HeLa cells, likely occurring at the point of cell line establishment. Given the rarity of NPCs, the long history of NPC cell lines, and the lack of rigorous cell line authentication, it is likely that the actual prevalence and impact of HeLa cell contamination on the EBV field might be greater. We therefore recommend cell line authentication prior to performing experiments using NPC cell lines to avoid inaccurate conclusions. The novel RNA-seq-based cell authentication approach reported here can serve as a comprehensive method for validating cell lines.

Exosomes derived from Epstein-Barr virus-infected cells are internalized via caveola-dependent endocytosis and promote phenotypic modulation in target cells.

Epstein-Barr virus (EBV), a human gammaherpesvirus, establishes a lifelong latent infection in B lymphocytes and epithelial cells following primary infection. Several lines of evidence suggest that exosomes derived from EBV-infected cells are internalized and transfer viral factors, including EBV-encoded latent membrane protein and microRNAs, to the recipient cells. However, the detailed mechanism by which exosomes are internalized and their physiological impact on the recipient cells are still poorly understood. In this study, we visualized the internalization of fluorescently labeled exosomes derived from EBV-uninfected and EBV-infected B cells of type I and type III latency into EBV-negative epithelial cells. In this way, we demonstrated that exosomes derived from all three cell types were internalized into the target cells in a similar fashion. Internalization of exosomes was significantly suppressed by treatment with an inhibitor of dynamin and also by the knockdown of caveolin-1. Labeled exosomes were colocalized with caveolae and subsequently trafficked through endocytic pathways. Moreover, we observed that exosomes derived from type III latency cells upregulated proliferation and expression of intercellular adhesion molecule 1 (ICAM-1) in the recipient cells more significantly than did those derived from EBV-negative and type I latency cells. We also identified the EBV latent membrane protein 1 (LMP1) gene as responsible for induction of ICAM-1 expression. Taken together, our data indicate that exosomes released from EBV-infected B cells are internalized via caveola-dependent endocytosis, which, in turn, contributes to phenotypic changes in the recipient cells through transferring one or more viral factors.

Current Insights into the Maturation of Epstein-Barr Virus Particles.

The three subfamilies of herpesviruses (alphaherpesviruses, betaherpesviruses, and gammaherpesviruses) appear to share a unique mechanism for the maturation and egress of virions, mediated by several budding and fusion processes of various organelle membranes during replication, which prevents cellular membrane disruption. Newly synthesized viral DNA is packaged into capsids within the nucleus, which are subsequently released into the cytoplasm via sequential fusion (primary envelopment) and budding through the inner and outer nuclear membranes. Maturation concludes with tegumentation and the secondary envelopment of nucleocapsids, which are mediated by budding into various cell organelles. Intracellular compartments containing mature virions are transported to the plasma membrane via host vesicular trafficking machinery, where they fuse with the plasma membrane to extracellularly release mature virions. The entire process of viral maturation is orchestrated by sequential interactions between viral proteins and intracellular membranes. Compared with other herpesvirus subfamilies, the mechanisms of gammaherpesvirus maturation and egress remain poorly understood. This review summarizes the major findings, including recently updated information of the molecular mechanism underlying the maturation and egress process of the Epstein-Barr virus, a ubiquitous human gammaherpesvirus subfamily member that infects most of the population worldwide and is associated with a number of human malignancies.

Micro-second time-resolved X-ray single-molecule internal motions of SARS-CoV-2 spike variants.

Single-molecule intramolecular dynamics were successfully measured for three variants of SARS-CoV-2 spike protein, alpha: B.1.1.7, delta: B.1.617, and omicron: B.1.1.529, with a time resolution of 100 µs using X-rays. The results were then compared with respect to the magnitude and directions of motions for the three variants. The largest 3-D intramolecular movement was observed for the omicron variant irrespective of ACE2 receptor binding. A more detailed analysis of the intramolecular motions revealed that the distribution state of intramolecular motion for the three variants was completely different with and without ACE2 receptor binding. The molecular dynamics for the trimeric spike protein of the omicron variant increased when ACE2 binding occurred. At that time, the diffusion constant increased from 71.0 [mrad2/ms] to 91.1 [mrad2/ms].

Exploring indoor and outdoor dust as a potential tool for detection and monitoring of COVID-19 transmission.

This study investigated the potential of using SARS-CoV-2 viral concentrations in dust as an additional surveillance tool for early detection and monitoring of COVID-19 transmission. Dust samples were collected from 8 public locations in 16 districts of Bangkok, Thailand, from June to August 2021. SARS-CoV-2 RNA concentrations in dust were quantified, and their correlation with community case incidence was assessed. Our findings revealed a positive correlation between viral concentrations detected in dust and the relative risk of COVID-19. The highest risk was observed with no delay (0-day lag), and this risk gradually decreased as the lag time increased. We observed an overall decline in viral concentrations in public places during lockdown, closely associated with reduced human mobility. The effective reproduction number for COVID-19 transmission remained above one throughout the study period, suggesting that transmission may persist in locations beyond public areas even after the lockdown measures were in place.

Roles of cell signaling pathways in cell-to-cell contact-mediated Epstein-Barr virus transmission.

Epstein-Barr virus (EBV), a human gamma herpesvirus, establishes a life-long latent infection in B lymphocytes and epithelial cells following primary infection. Several lines of evidence indicate that the efficiency of EBV infection in epithelial cells is accelerated up to 10(4)-fold by coculturing with EBV-infected Burkitt's lymphoma (BL) cells compared to infection with cell-free virions, indicating that EBV infection into epithelial cells is mainly mediated via cell-to-cell contact. However, the molecular mechanisms involved in this pathway are poorly understood. Here, we establish a novel assay to assess cell-to-cell contact-mediated EBV transmission by coculturing an EBV-infected BL cell line with an EBV-negative epithelial cell line under stimulation for lytic cycle induction. By using this assay, we confirmed that EBV was transmitted from BL cells to epithelial cells via cell-to-cell contact but not via cell-to-cell fusion. The inhibitor treatments of extracellular signal-regulated kinase (ERK) and nuclear factor (NF)-κB pathways blocked EBV transmission in addition to lytic induction. The blockage of the phosphoinositide 3-kinase (PI3K) pathway impaired EBV transmission coupled with the inhibition of lytic induction. Knockdown of the RelA/p65 subunit of NF-κB reduced viral transmission. Moreover, these signaling pathways were activated in cocultured BL cells and in epithelial cells. Finally, we observed that viral replication was induced in cocultured BL cells. Taken together, our data suggest that cell-to-cell contact induces multiple cell signaling pathways in BL cells and epithelial cells, contributing to the induction of the viral lytic cycle in BL cells and the enhancement of viral transmission to epithelial cells.