The genomic landscape of swine influenza A viruses in Southeast Asia.

Swine are a primary source for the emergence of pandemic influenza A viruses. The intensification of swine production, along with global trade, has amplified the transmission and zoonotic risk of swine influenza A virus (swIAV). Effective surveillance is essential to uncover emerging virus strains; however gaps remain in our understanding of the swIAV genomic landscape in Southeast Asia. More than 4,000 nasal swabs were collected from pigs in Cambodia, yielding 72 IAV-positive samples by RT-qPCR and 45 genomic sequences. We unmasked the cocirculation of multiple lineages of genetically diverse swIAV of pandemic concern. Genomic analyses revealed a novel European avian-like H1N2 swIAV reassortant variant with North American triple reassortant internal genes, that emerged approximately seven years before its first detection in pigs in 2021. Using phylogeographic reconstruction, we identified south central China as the dominant source of swine viruses disseminated to other regions in China and Southeast Asia. We also identified nine distinct swIAV lineages in Cambodia, which diverged from their closest ancestors between two and 15 B.P., indicating significant undetected diversity in the region, including reverse zoonoses of human H1N1/2009 pandemic and H3N2 viruses. A similar period of cryptic circulation of swIAVs occurred in the decades before the H1N1/2009 pandemic. The hidden diversity of swIAV observed here further emphasizes the complex underlying evolutionary processes present in this region, reinforcing the importance of genomic surveillance at the human-swine interface for early warning of disease emergence to avoid future pandemics.

Spike-Independent Infection of Human Coronavirus 229E in Bat Cells.

Bats are the reservoir for numerous human pathogens, including coronaviruses. Despite many coronaviruses having descended from bat ancestors, little is known about virus-host interactions and broader evolutionary history involving bats. Studies have largely focused on the zoonotic potential of coronaviruses with few infection experiments conducted in bat cells. To determine genetic changes derived from replication in bat cells and possibly identify potential novel evolutionary pathways for zoonotic virus emergence, we serially passaged six human 229E isolates in a newly established Rhinolophus lepidus (horseshoe bat) kidney cell line. Here, we observed extensive deletions within the spike and open reading frame 4 (ORF4) genes of five 229E viruses after passaging in bat cells. As a result, spike protein expression and infectivity of human cells was lost in 5 of 6 viruses, but the capability to infect bat cells was maintained. Only viruses that expressed the spike protein could be neutralized by 229E spike-specific antibodies in human cells, whereas there was no neutralizing effect on viruses that did not express the spike protein inoculated on bat cells. However, one isolate acquired an early stop codon, abrogating spike expression but maintaining infection in bat cells. After passaging this isolate in human cells, spike expression was restored due to acquisition of nucleotide insertions among virus subpopulations. Spike-independent infection of human coronavirus 229E may provide an alternative mechanism for viral maintenance in bats that does not rely on the compatibility of viral surface proteins and known cellular entry receptors. IMPORTANCE Many viruses, including coronaviruses, originated from bats. Yet, we know little about how these viruses switch between hosts and enter human populations. Coronaviruses have succeeded in establishing in humans at least five times, including endemic coronaviruses and the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In an approach to identify requirements for host switches, we established a bat cell line and adapted human coronavirus 229E viruses by serial passage. The resulting viruses lost their spike protein but maintained the ability to infect bat cells, but not human cells. Maintenance of 229E viruses in bat cells appears to be independent of a canonical spike receptor match, which in turn might facilitate cross-species transmission in bats.

Genetic Characterization of Highly Pathogenic Avian Influenza A(H5N8) Virus in Pakistani Live Bird Markets Reveals Rapid Diversification of Clade 2.3.4.4b Viruses.

The highly pathogenic (HPAI) avian influenza A(H5N1) viruses have undergone reassortment with multiple non-N1-subtype neuraminidase genes since 2008, leading to the emergence of H5Nx viruses. H5Nx viruses established themselves quickly in birds and disseminated from China to Africa, the Middle East, Europe and North America. Multiple genetic clades have successively evolved through frequent mutations and reassortment, posing a continuous threat to domestic poultry and causing substantial economic losses. Live bird markets are recognized as major sources of avian-to-human infection and for the emergence of zoonotic influenza. In Pakistan, the A(H5N1) virus was first reported in domestic birds in 2007; however, avian influenza surveillance is limited and there is a lack of knowledge on the evolution and transmission of the A(H5) virus in the country. We collected oropharyngeal swabs from domestic poultry and environmental samples from six different live bird markets during 2018-2019. We detected and sequenced HPAI A(H5N8) viruses from two chickens, one quail and one environmental sample in two markets. Temporal phylogenetics indicated that all novel HPAI A(H5N8) viruses belonged to clade 2.3.4.4b, with all eight genes of Pakistan A(H5N8) viruses most closely related to 2017 Saudi Arabia A(H5N8) viruses, which were likely introduced via cross-border transmission from neighboring regions approximately three months prior to virus detection into domestic poultry. Our data further revealed that clade 2.3.4.4b viruses underwent rapid lineage expansion in 2017 and acquired significant amino acid mutations, including mutations associated with increased haemagglutinin affinity to human α-2,6 receptors, prior to the first human A(H5N8) infection in Russian poultry workers in 2020. These results highlight the need for systematic avian influenza surveillance in live bird markets in Pakistan to monitor for potential A(H5Nx) variants that may arise from poultry populations.

FBXW5 Promotes Tumorigenesis and Metastasis in Gastric Cancer via Activation of the FAK-Src Signaling Pathway.

: F-box/WD repeat-containing protein 5 (FBXW5) is a member of the FBXW subclass of F-box proteins. Despite its known function as a component of the Skp1-Cullin-F-box (SCF) ubiquitin ligase complex, the role of FBXW5 in gastric cancer tumorigenesis and metastasis has not been investigated. The present study investigates the role of FBXW5 in tumorigenesis and metastasis, as well as the regulation of key signaling pathways in gastric cancer; using in-vitro FBXW5 knockdown/overexpression cell line and in-vivo models. In-vitro knockdown of FBXW5 results in a decrease in cell proliferation and cell cycle progression, with a concomitant increase in cell apoptosis and caspase-3 activity. Furthermore, knockdown of FBXW5 also leads to a down regulation in cell migration and adhesion, characterized by a reduction in actin polymerization, focal adhesion turnover and traction forces. This study also delineates the mechanistic role of FBXW5 in oncogenic signaling as its inhibition down regulates RhoA-ROCK 1 (Rho-associated protein kinase 1) and focal adhesion kinase (FAK) signaling cascades. Overexpression of FBXW5 promotes in-vivo tumor growth, whereas its inhibition down regulates in-vivo tumor metastasis. When considered together, our study identifies the novel oncogenic role of FBXW5 in gastric cancer and draws further interest regarding its clinical utility as a potential therapeutic target.

Anti-tumor efficacy of Selinexor (KPT-330) in gastric cancer is dependent on nuclear accumulation of p53 tumor suppressor.

Exportin-1 (XPO1) controls the nucleo-cytoplasmic trafficking of several key growth regulatory and tumor suppressor proteins. Nuclear export blockade through XPO1 inhibition is a target for therapeutic inhibition in many cancers. Studies have suggested XPO1 upregulation as an indicator of poor prognosis in gastric cancer. In the current study, we investigated the anti-tumor efficacy of selective inhibitors of nuclear export (SINE) compounds KPT-185, KTP-276 and clinical stage selinexor (KPT-330) in gastric cancer. XPO1 was found to be overexpressed in gastric cancer as compared to adjacent normal tissues and was correlated with poor survival outcomes. Among the 3 SINE compounds, in vitro targeting of XPO1 with selinexor resulted in greatest potency with significant anti-proliferative effects at nano molar concentrations. XPO1 inhibition by selinexor resulted in nuclear accumulation of p53, causing cell cycle arrest and apoptosis. Also, inhibition of XPO1 lead to the cytoplasmic retention of p21 and suppression of survivin. Orally administered selienxor caused significant inhibition of tumor growth in xenograft models of gastric cancer. Furthermore, combination of selinexor with irinotecan exhibited greater anti-tumor effect compared to individual treatment. Taken together, our study underscores the therapeutic utility of XPO1 targeting in gastric cancer and suggests the potential benefits of XPO1 inhibition in-combination with chemotherapy.

Melanoma associated antigen (MAGE)-A3 promotes cell proliferation and chemotherapeutic drug resistance in gastric cancer.

BACKGROUND: Melanoma-associated antigen (MAGE)-A3 is a member of the family of cancer-testis antigens and has been found to be epigenetically regulated and aberrantly expressed in various cancer types. It has also been found that MAGE-A3 expression may correlate with an aggressive clinical course and with chemo-resistance. The objectives of this study were to assess the relationship between MAGE-A3 promoter methylation and expression and (1) gastric cancer patient survival and (2) its functional consequences in gastric cancer-derived cells. METHODS: Samples from two independent gastric cancer cohorts (including matched non-malignant gastric samples) were included in this study. MAGE-A3 methylation and mRNA expression levels were determined by methylation-specific PCR (MSP) and quantitative real-time PCR (qPCR), respectively. MAGE-A3 expression was knocked down in MKN1 gastric cancer-derived cells using miRNAs. In addition, in vitro cell proliferation, colony formation, apoptosis, cell cycle, drug treatment, immunohistochemistry and Western blot assays were performed. RESULTS: Clinical analysis of 223 primary patient-derived samples (ntumor = 161, nnormal = 62) showed a significant inverse correlation between MAGE-A3 promoter methylation and expression in the cancer samples (R = -0.63, p = 5.99e-19). A lower MAGE-A3 methylation level was found to be associated with a worse patient survival (HR: 1.5, 95 % CI: 1.02-2.37, p = 0.04). In addition, we found that miRNA-mediated knockdown of MAGE-A3 expression in MKN1 cells caused a reduction in its proliferation and colony forming capacities, respectively. Under stress conditions MAGE-A3 was found to regulate the expression of Bax and p21. MAGE-A3 knock down also led to an increase in Puma and Noxa expression, thus contributing to an enhanced docetaxel sensitivity in the gastric cancer-derived cells. CONCLUSIONS: From our results we conclude that MAGE-A3 expression is regulated epigenetically by promoter methylation, and that its expression contributes to gastric cell proliferation and drug sensitivity. This study underscores the potential implications of MAGE-A3 as a therapeutic target and prognostic marker in gastric cancer patients.

GTSE1 expression represses apoptotic signaling and confers cisplatin resistance in gastric cancer cells.

BACKGROUND: Platinum based therapy is commonly used in the treatment of advanced gastric cancer. However, resistance to chemotherapy is a major challenge that causes marked variation in individual response rate and survival rate. In this study, we aimed to identify the expression of GTSE1 and its correlation with cisplatin resistance in gastric cancer cells. METHODS: Methylation profiling was carried out in tissue samples from gastric cancer patients before undergoing neoadjuvent therapy using docetaxel, cisplatin and 5FU (DCX) and in gastric cancer cell lines. The correlation between GTSE1 expression and methylation in gastric cancer cells was determined by RT-PCR and MSP respectively. GTSE1 expression was knocked-down using shRNA's and its effects on cisplatin cytotoxicity and cell survival were detected by MTS, proliferation and clonogenic survival assays. Additionally, the effect of GTSE1 knock down in drug induced apoptosis was determined by western blotting and apoptosis assays. RESULTS: GTSE1 exhibited a differential methylation index in gastric cancer patients and in cell lines that correlated with DCX treatment response and cisplatin sensitivity, respectively. In-vitro, GTSE1 expression showed a direct correlation with hypomethylation. Interestingly, Cisplatin treatment induced a dose dependent up regulation as well as nuclear translocation of GTSE1 expression in gastric cancer cells. Knock down of GTSE1 enhanced cisplatin cytotoxity and led to a significant reduction in cell proliferation and clonogenic survival. Also, loss of GTSE1 expression caused a significant increase in P53 mediated apoptosis in cisplatin treated cells. CONCLUSION: Our study identifies GTSE1 as a biomarker for cisplatin resistance in gastric cancer cells. This study also suggests the repressive role of GTSE1 in cisplatin induced apoptosis and signifies its potential utility as a therapeutic target for better clinical management of gastric cancer patients.

An integrative approach identified genes associated with drug response in gastric cancer.

Gastric cancer (GC) is the second leading cause of global cancer mortality worldwide. However, the molecular mechanism underlying its carcinogenesis and drug resistance is not well understood. To identify novel functionally important genes that were differentially expressed due to combinations of genetic and epigenetic changes, we analyzed datasets containing genome-wide mRNA expression, DNA copy number alterations and DNA methylation status from 154 primary GC samples and 47 matched non-neoplastic mucosa tissues from Asian patients. We used concepts of 'within' and 'between' statistical analysis to compare the difference between tumors and controls within each platform, and assessed the correlations between platforms. This 'multi-regulated gene (MRG)' analysis identified 126 differentially expressed genes that underwent a combination of copy number and DNA methylation changes. Most genes were located at genomic loci associated with GC. Statistical enrichment analysis showed that MRGs were enriched for cancer, GC and drug response. We analysed several MRGs that previously had not been associated with GC. Knockdown of DDX27, TH1L or IDH3G sensitized cells to epirubicin or cisplatin, and knockdown of RAI14 reduced cell proliferation. Further studies showed that overexpression of DDX27 reduced epirubicin-induced DNA damage and apoptosis. Levels of DDX27 mRNA and protein were increased in early-stage gastric tumors, and may be a potential diagnostic and prognostic marker for GC. In summary, we used an integrative bioinformatics strategy to identify novel genes that are altered in GC and regulate resistance of GC cells to drugs in vitro.

Combination therapy with gossypol reveals synergism against gemcitabine resistance in cancer cells with high BCL-2 expression.

Although gemcitabine is highly active in several cancer types, intrinsic and acquired drug resistance remains a major challenge. Overexpression of Bcl-2 has been associated with gemcitabine resistance. The aim of this study is to determine whether gossypol can overcome gemcitabine resistance in cell lines with high level of Bcl-2 expression in combination drug therapy. Our study demonstrated that in 10 cell lines derived from different cancers, high Bcl-2 baseline expression was observed in cell lines that were resistant to gemcitabine (GEM-R). Furthermore, synergistic effect of combination therapy was observed in gemcitabine-resistant (GEM-R) cell lines with high Bcl-2 expression, but not in a gemcitabine-sensitive (GEM-S) cell lines regardless of Bcl-2 expression. Gossypol treatment resulted in the decrease of anti-apoptotic genes such as Bcl-2 and Bcl-xl and an upregulation of the pro-apoptotic gene, Noxa. Furthermore, the addition of gossypol to gemcitabine resulted in lower expressions of anti-apoptotic genes compared to gemcitabine alone. Gene expression profiling in GEM-R and GEM-S cell lines suggest that anti-apoptotic genes such as pAkt and PI3KR2 may play important role in gemcitabine resistance, while pro-apoptotic Bcl-2 related genes (Bad, Caspase-6 and Calpain-1) may regulate synergistic interaction in combination therapy.