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Background: Corona Virus Disease 2019(COVID-19)is a global pandemic novel coronavirus infection disease caused by Severe acute respiratory syndrome Coronavirus 2 (SARS-CoV-2). Although rapid, large-scale testing plays an important role in patient management and slowing the spread of the disease. However, there has been no good and widely used drug treatment for infection and transmission of SARS-CoV-2. Key findings: Therefore, this review updates the body of knowledge on viral structure, infection routes, detection methods, and clinical treatment, with the aim of responding to the large-section caused by SARS-CoV-2. This paper focuses on the structure of SARS-CoV-2 viral protease, RNA polymerase, serine protease and main proteinase-like protease as well as targeted antiviral drugs. Conclusion: In vitro or clinical trials have been carried out to provide deeper thinking for the pathogenesis, clinical diagnosis, vaccine development and treatment of SARS-CoV-2.

Host-virus interactions can significantly impact the viral life cycle and pathogenesis; however, our understanding of the specific host factors involved in highly pathogenic avian influenza A virus H7N9 (HPAI H7N9) infection is currently restricted. Herein, we designed and synthesized 65 small interfering RNAs targeting host genes potentially associated with various aspects of RNA virus life cycles. Afterward, HPAI H7N9 viruses were isolated and RNA interference was used to screen for host factors likely to be involved in the life cycle of HPAI H7N9. Moreover, the research entailed assessing the associations between host proteins and HPAI H7N9 proteins. Twelve key host proteins were identified: Annexin A (ANXA)2, ANXA5, adaptor related protein complex 2 subunit sigma 1 (AP2S1), adaptor related protein complex 3 subunit sigma 1 (AP3S1), ATP synthase F1 subunit alpha (ATP5A1), COPI coat complex subunit alpha (COP)A, COPG1, heat shock protein family A (Hsp70) member 1A (HSPA)1A, HSPA8, heat shock protein 90 alpha family class A member 1 (HSP90AA1), RAB11B, and RAB18. Co-immunoprecipitation revealed intricate interactions between viral proteins (hemagglutinin, matrix 1 protein, neuraminidase, nucleoprotein, polymerase basic 1, and polymerase basic 2) and these host proteins, presumably playing a crucial role in modulating the life cycle of HPAI H7N9. Notably, ANXA5, AP2S1, AP3S1, ATP5A1, HSP90A1, and RAB18, were identified as novel interactors with HPAI H7N9 proteins rather than other influenza A viruses (IAVs). These findings underscore the significance of host-viral protein interactions in shaping the dynamics of HPAI H7N9 infection, while highlighting subtle variations compared with other IAVs. Deeper understanding of these interactions holds promise to advance disease treatment and prevention strategies.

Abstract Background The highly pathogenic Influenza H7N9 virus is believed to cause multiple organ infections. However, there have been few systematic animal experiments demonstrating the virus distribution after H7N9 virus infection. The present study was carried out to investigate the viral distribution and pathological changes in the main organs of mice after experimental infection with highly pathogenic H7N9 virus. Methods Infection of mice with A/Guangdong/GZ8H002/2017(H7N9) virus was achieved via nasal inoculation. Mice were killed at 2, 3, and 7 days post infection. The other mice were used to observe their illness status and weight changes. Reverse transcription polymerase chain reaction and viral isolation were used to analyse the characteristics of viral invasion. The pathological changes of the main organs were observed using haematoxylin and eosin staining and immunohistochemistry. Results The weight of H7N9 virus-infected mice increased slightly in the first two days. However, the weight of the mice decreased sharply in the following days, by up to 20%. All the mice had died by the 8th day post infection and showed multiple organ injury. The emergence of viremia in mice was synchronous with lung infection. On the third day post infection, except in the brain, the virus could be isolated from all organs (lung, heart, kidney, liver, and spleen). On the seventh day post infection, the virus could be detected in all six organs. Brain infection was detected in all mice, and the viral titre in the heart, kidney, and spleen infection was high. Conclusion Acute diffuse lung injury was the initial pathogenesis in highly pathogenic H7N9 virus infection. In addition to lung infection and viremia, the highly pathogenic H7N9 virus could cause multiple organ infection and injury.

Abstract Advanced colorectal adenocarcinoma (CRAC), featured by distinctive histopathological appearance, distant organ metastasis, acquired chemoresistance, and tumorigenic stemness is a group of heterogeneous cancers with unique genetic signatures and malignant phenotypes. Treatment of CRAC is a daunting task for oncologists. Currently, various strategies including molecular targeting using therapeutic monoclonal antibodies, small molecule kinase inhibitors and immunoregulatory checkpoint therapy have been applied to combat this deadly disease. However, these therapeutic modalities and approaches achieve only limited success. Thus, there is a pharmaceutical need to discover new targets and develop novel therapeutics for CRAC therapy. MET and RON receptor tyrosine kinases have been implicated in CRAC pathogenesis. Clinical studies have revealed that aberrant MET and/or RON expression and signaling are critical in regulating CRAC progression and malignant phenotypes. Increased MET and/or RON expression also has prognostic value for CRAC progression and patient survival. These features provide the rationale to target MET and RON for clinical CRAC intervention. At present, the use of small molecule kinase inhibitors targeting MET for CRAC treatment has achieved significant progress with several approvals for clinical application. Nevertheless, antibody-based biotherapeutics, although under clinical trials for more than 8 years, have made very little progress. In this review, we discuss the importance of MET and/or RON in CRAC tumorigenesis and development of anti-MET, anti-RON, and MET and RON-dual targeting antibody-drug conjugates for clinical application. The findings from both preclinical studies and clinical trials highlight the potential of this novel type of biotherapeutics for CRAC therapy in the future.

ABSTRACTThe H7N9 virus mutated in 2017, resulting in new cases of highly pathogenic avian influenza (HPAI) H7N9 virus infection. H7N9 was found in a viraemic patient in Guangdong province, China. The present study aimed to clarify the pathogenic characteristics of HPAI H7N9. Virus was isolated from the plasma and sputum of the patient with HPAI H7N9. Liquid phase chip technology was used to detect the plasma cytokines from the infected patient and healthy controls. Mice were infected with strains A/Guangdong/GZ8H002/2017(H7N9) and A/Zhejiang/DTID-ZJU01/2013(H7N9) to observe the virus’s pathogenic characteristics. Serum and brain tissue were collected at 2, 4, and 6 days after infection. The viruses in serum and brain tissue were detected and isolated. The two strains were infected into A549 cells, exosomes were extracted, and virus genes in the exosomes were assessed. Live virus was isolated from the patient’s plasma. An acute cytokine storm was detected during the whole course of the disease. In animal experiments, A/Guangdong/GZ8H002/2017(H7N9) was more pathogenic than A/Zhejiang /DTID-ZJU01/2013(H7N9) and resulted in the death of mice. Live virus was isolated from infected mouse serum. Virus infection was also detected in the brain of mice. Under viral stress, A549 cells secreted exosomes containing the entire viral genome. The viraemic patient was confirmed to have an HPAI H7N9 infection. A/Guangdong/GZ8H002/2017(H7N9) showed significantly enhanced toxicity. Patient deaths might result from cytokine storms and brain infections. Extrapulmonary tissue infection might occur via the exosome pathway. The determined pathogenic characteristics of HPAI H7N9 will contribute to its future treatment.

Middle East respiratory syndrome (MERS) is a viral respiratory disease caused by a de novo coronavirus—MERS-CoV—that is associated with high mortality. However, the mechanism by which MERS-CoV infects humans remains unclear. To date, there is no effective vaccine or antibody for human immunity and treatment, other than the safety and tolerability of the fully human polyclonal Immunoglobulin G (IgG) antibody (SAB-301) as a putative therapeutic agent specific for MERS. Although rapid diagnostic and public health measures are currently being implemented, new cases of MERS-CoV infection are still being reported. Therefore, various effective measures should be taken to prevent the serious impact of similar epidemics in the future. Further investigation of the epidemiology and pathogenesis of the virus, as well as the development of effective therapeutic and prophylactic anti-MERS-CoV infections, is necessary. For this purpose, detailed information on MERS-CoV proteins is needed. In this review, we describe the major structural and nonstructural proteins of MERS-CoV and summarize different potential strategies for limiting the outbreak of MERS-CoV. The combination of computational biology and virology can accelerate the advanced design and development of effective peptide therapeutics against MERS-CoV. In summary, this review provides important information about the progress of the elimination of MERS, from prevention to treatment. Keywords: MERS-CoV, Spike protein, Structural protein, Accessory protein, Nonstructural protein

Abstract Background Antibody-drug conjugates (ADCs) targeting the RON receptor, a tumorigenic factor contributing to cancer malignancy, has been considered as a novel strategy for cancer therapy. Here we describe a humanized antibody recognizing the RON plexin-semaphorin-integrin (PSI) domain with increased drug delivery capability for potential clinical application. Method Monoclonal antibody PCM5B14 specific to the human and monkey RON PSI domain was generated and characterized by various immunological methods. Humanized antibody H5B14 was created by grafting PCM5B14 complementarity-determining regions into human IgG1/κ acceptor frameworks and conjugated with monomethyl auristatin E and duocarmycin to form two H5B14-based ADCs. Stability of H5B14-based ADCs in human plasma was measured using hydrophobic interaction chromatography. Various biochemical and biological assays were used to determine ADC- regulated RON internalization, cell viability, spheroid formation, and death of cancer stem-like cells. Efficacies of H5B14-based ADCs in vivo were validated using tumor xenograft models. Maximal tolerated doses of H5B14-based ADCs were established in mice. Results H5B14 was highly specific to the human RON PSI domain and superior over other anti-RON ADCs in induction of RON internalization in various cancer cell lines tested. H5B14-based ADCS had a drug to antibody ratio of ~ 3.70:1 and were stable in human plasma with a minimal dissociation within a 10-day period. Functionally, H5B14-mediated drug delivery decreased cell viability at early stages with an average IC50 at ~ 20 nM in multiple cancer cell lines examined. H5B14-based ADCs also inhibited spheroid formation and caused death of cancer stem-like cells with RON+/CD44+/ESA+ phenotypes. In vivo, H5B14-based ADCs in a single injection inhibited tumor xenograft growth mediated by multiple cancer cell lines. Tumoristatic concentrations calculated from xenograft tumor models were in the range of 0.63 to 2.0 mg/kg bodyweight. Significantly, H5B14-based ADCs were capable of eradicating tumors at variable levels across multiple xenograft models regardless their malignant statuses. Toxicologically, H5B14-based ADCs were well tolerated in mice up to 60 mg/kg. Conclusion H5B14-based ADCs targeting the RON PSI domain are superior in inducing RON internalization, leading to robust drug delivery and overall inhibition and eradication of tumors in multiple xenograft models. These findings warrant H5B14-based ADCs for clinical trials in the future.

Abstract Background Aberrant expression of the RON receptor tyrosine kinase is a pathogenic feature and a validated drug target in various types of cancers. Currently, therapeutic antibodies targeting RON for cancer therapy are under intensive evaluation. Here we report the development and validation of a novel humanized anti-RON antibody-drug conjugate for cancer therapy. Methods Antibody humanization was achieved by grafting sequences of complementarity-determining regions from mouse monoclonal antibody Zt/g4 into human IgG1/κ acceptor frameworks. The selected humanized Zt/g4 subclone H1L3 was conjugated with monomethyl auristatin E using a dipeptide linker to form H-Zt/g4-MMAE. Pharmacokinetic analysis of H-Zt/g4-MMAE was determined using hydrophobic interaction chromatography and a MMAE ADC ELISA kit. Biochemical and biological assays were used for measuring RON expression, internalization, cell viability and death. Therapeutic efficacies of H-Zt/g4-MMAE were validated in vivo using three pancreatic cancer xenograft models. Toxicological activities of H-Zt/g4-MMAE were determined in mouse and cynomolgus monkey. Results H-Zt/g4-MMAE had a drug to antibody ratio of 3.77:1 and was highly stable in human plasma with a dissociation rate less than 5% within a 20 day period. H-Zt/g4-MMAE displayed a favorable pharmacokinetic profile in both mouse and cynomolgus monkey. In vitro, H-Zt/g4-MMAE induced RON internalization, which results in killing of pancreatic cancer cells with IC50 values at 10–20 nM. In vivo, H-Zt/g4-MMAE inhibited pancreatic cancer xenograft growth with tumoristatic concentrations at 1~3 mg/kg bodyweight. Significantly, H-Zt/g4-MMAE eradicated tumors across multiple xenograft models regardless their chemoresistant and metastatic statuses. Moreover, H-Zt/g4-MMAE inhibited and eradicated xenografts mediated by pancreatic cancer stem-like cells and by primary cells from patient-derived tumors. Toxicologically, H-Zt/g4-MMAE is well tolerated in mice up to 60 mg/kg. In cynomolgus monkey, H-Zt/g4-MMAE up to 30 mg/kg had a manageable and reversible toxicity profile. Conclusions H-Zt/g4-MMAE is superior in eradication of pancreatic cancer xenografts with favorable pharmacokinetic profiles and manageable toxicological activities. These findings warrant the transition of H-Zt/g4-MMAE into clinical trials in the future.

Aberrant expression and/or activation of the MET receptor tyrosine kinase is characterized by genomic recombination, gene amplification, activating mutation, alternative exon-splicing, increased transcription, and their different combinations. These dysregulations serve as oncogenic determinants contributing to cancerous initiation, progression, malignancy, and stemness. Moreover, integration of the MET pathway into the cellular signaling network as an addiction mechanism for survival has made this receptor an attractive pharmaceutical target for oncological intervention. For the last 20 years, MET-targeting small-molecule kinase inhibitors (SMKIs), conventional therapeutic monoclonal antibodies (TMABs), and antibody-based biotherapeutics such as bispecific antibodies, antibody–drug conjugates (ADC), and dual-targeting ADCs have been under intensive investigation. Outcomes from preclinical studies and clinical trials are mixed with certain successes but also various setbacks. Due to the complex nature of MET dysregulation with multiple facets and underlying mechanisms, mechanism-based validation of MET-targeting therapeutics is crucial for the selection and validation of lead candidates for clinical trials. In this review, we discuss the importance of various types of mechanism-based pharmaceutical models in evaluation of different types of MET-targeting therapeutics. The advantages and disadvantages of these mechanism-based strategies for SMKIs, conventional TMABs, and antibody-based biotherapeutics are analyzed. The demand for establishing new strategies suitable for validating novel biotherapeutics is also discussed. The information summarized should provide a pharmaceutical guideline for selection and validation of MET-targeting therapeutics for clinical application in the future.

The recepteur d’origine nantais (RON) receptor tyrosine kinase, belonging to the mesenchymal-to-epithelial transition proto-oncogene family, has been implicated in the pathogenesis of cancers derived from the colon, lung, breast, and pancreas. These findings lay the foundation for targeting RON for cancer treatment. However, development of RON-targeted therapeutics has not gained sufficient attention for the last decade. Although therapeutic monoclonal antibodies (TMABs) targeting RON have been validated in preclinical studies, results from clinical trials have met with limited success. This outcome diminishes pharmaceutical enthusiasm for further development of RON-targeted therapeutics. Recently, antibody–drug conjugates (ADCs) targeting RON have drawn special attention owing to their increased therapeutic activity. The rationale for developing anti-RON ADCs is based on the observation that cancer cells are not sufficiently addicted to RON signaling for survival. Thus, TMAB-mediated inhibition of RON signaling is ineffective for clinical application. In contrast, anti-RON ADCs combine a target-specific antibody with potent cytotoxins for cancer cell killing. This approach not only overcomes the shortcomings in TMAB-targeted therapies but also holds the promise for advancing anti-RON ADCs into clinical trials. In this review, we discuss the latest advancements in the development of anti-RON ADCs for targeted cancer therapy including drug conjugation profile, pharmacokinetic properties, cytotoxic effect in vitro , efficacy in tumor models, and toxicological activities in primates.

RON (recepteur d'origine nantais) and MET (hepatocyte growth factor receptor) are tyrosine kinase receptors. Various cancers have aberrant RON and MET expression and activation, which contribute to cancer cell proliferation, invasiveness, and metastasis. Here, we explored RON and MET expression in pancreatic cancer and their relationship with overall survival (OS) time, and evaluated their significance as therapeutic targets of tyrosine kinase inhibitors in pancreatic cancer. We enrolled 227 patients with pancreatic cancer in the study. RON and MET expression was analyzed by immunohistochemical staining. Four human pancreatic cancer cell lines expressing variable levels of RON or MET and four MET superfamily inhibitors (BMS777607, PHA665752, INCB28060, Tivantinib) were used. The effect of the four tyrosine kinase inhibitors on cell viability, migration, and apoptosis were determined using cell viability, scratch wound healing, and Caspase-Glo 3/7 assays. Cellular signaling was analyzed by immunoprecipitation and western blotting. The therapeutic efficacy of the tyrosine kinase inhibitors was determined with mouse xenograft pancreatic cancer models in vivo. There was wide aberrant RON and MET expression in the cancer tissues. In 227 pancreatic cancer samples, 33% had RON overexpression, 41% had MET overexpression, and 15.4% had RON and MET co-overexpression. RON and MET expression were highly correlated. RON and MET expression levels were significantly related to OS. Patients with RON and MET co-overexpression had poorer OS. BMS777607 and PHA665752 inhibited pancreatic cancer cell viability and migration, and promoted apoptosis by inhibiting RON and MET phosphorylation and further inhibiting the downstream signaling pathways in vitro. They also inhibited tumor growth and further inhibited phosphorylated (phosphor)-RON and phospho-MET expression in the mouse xenograft models in vivo effectively. INCB28060, which inhibits the MET signaling pathway alone, was not effective. RON and MET can be important indicators of prognosis in pancreatic cancer. Tyrosine kinase inhibitors targeting RON and MET in pancreatic cancer are a novel and potential approach for pancreatic cancer therapy.

Ebolavirus (EBOV) life cycle involves interactions with numerous host factors, but it remains poorly understood, as does pathogenesis. Herein, we synthesized 65 siRNAs targeting host genes mostly connected with aspects of the negative-sense RNA virus life cycle (including viral entry, uncoating, fusion, replication, assembly, and budding). We produced EBOV transcription- and replication-competent virus-like particles (trVLPs) to mimic the EBOV life cycle. After screening host factors associated with the trVLP life cycle, we assessed interactions of host proteins with trVLP glycoprotein (GP), VP40, and RNA by co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP). The results demonstrate that RNAi silencing with 11 siRNAs (ANXA5, ARFGAP1, FLT4, GRP78, HSPA1A, HSP90AB1, HSPA8, MAPK11, MEK2, NTRK1, and YWHAZ) decreased the replication efficiency of trVLPs. Co-IP revealed nine candidate host proteins (FLT4, GRP78, HSPA1A, HSP90AB1, HSPA8, MAPK11, MEK2, NTRK1, and YWHAZ) potentially interacting with trVLP GP, and four (ANXA5, GRP78, HSPA1A, and HSP90AB1) potentially interacting with trVLP VP40. Ch-IP identified nine candidate host proteins (ANXA5, ARFGAP1, FLT4, GRP78, HSPA1A, HSP90AB1, MAPK11, MEK2, and NTRK1) interacting with trVLP RNA. This study was based on trVLP and could not replace live ebolavirus entirely; in particular, the interaction between trVLP RNA and host proteins cannot be assumed to be identical in live virus. However, the results provide valuable information for further studies and deepen our understanding of essential host factors involved in the EBOV life cycle.

Ebolaviruses are highly infectious pathogens that cause lethal Ebola virus disease (EVD) in humans and non-human primates (NHPs). Due to their high pathogenicity and transmissibility, as well as the potential to be misused as a bioterrorism agent, ebolaviruses would threaten the health of global populations if not controlled. In this review, we describe the origin and structure of ebolaviruses and the development of vaccines from the beginning of the 1980s, including conventional ebolavirus vaccines, DNA vaccines, Ebola virus-like particles (VLPs), vaccinia virus-based vaccines, Venezuelan equine encephalitis virus (VEEV)-like replicon particles, Kunjin virus-based vaccine, recombinant Zaire Ebolavirus∆VP30, recombinant cytomegalovirus (CMV)-based vaccines, recombinant rabies virus (RABV)-based vaccines, recombinant paramyxovirus-based vaccines, adenovirus-based vaccines and vesicular stomatitis virus (VSV)-based vaccines. No licensed vaccine or specific treatment is currently available to counteract ebolavirus infection, although DNA plasmids and several viral vector approaches have been evaluated as promising vaccine platforms. These vaccine candidates have been confirmed to be successful in protecting NHPs against lethal infection. Moreover, these vaccine candidates were successfully advanced to clinical trials. The present review provides an update of the current research on Ebola vaccines, with the aim of providing an overview on current prospects in the fight against EVD.

AIM: To investigate the mechanism underlying the anti-inflammatory effects of epigallocatechin-3-gallate (EGCG) in lipopolysaccharide (LPS)-stimulated human retinal endothelial cells (HRECs).METHODS: HRECs pre-treated with EGCG (0-100 μmol/L) were stimulated with LPS (250 ng/mL). Levels of tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1) and nitric oxide (NO) in the supernatants were determined by enzyme-linked immunosorbent assay (ELISA) and Griess assay. The protein expression of phosphorylated extracellular signal-regulated kinase (ERK) 1/2 and p38 mitogen-activated protein kinases (p38) were determined by Western blot analysis.RESULTS: EGCG pre-treatment significantly inhibited the secretion of TNF-α, VEGF, MCP-1 and NO in LPS-stimulated HRECs. Moreover, EGCG effectively attenuated LPS-induced activation and phosphorylation of ERK1/2 and p38 in HRECs in a dose-dependent manner.CONCLUSION: EGCG exhibited inhibitory effects on LPS-induced pro-inflammatory cytokines production by modulating ERK1/2 and p38 pathways in HRECs, suggesting EGCG as a potential candidate for anti-inflammatory intervention.

Following publication of the original article [1], the author reported two errors in the authors affiliations: