Molecular Architecture of the SARS-CoV-2 Virus.

SARS-CoV-2 is an enveloped virus responsible for the COVID-19 pandemic. Despite recent advances in the structural elucidation of SARS-CoV-2 proteins, the detailed architecture of the intact virus remains to be unveiled. Here we report the molecular assembly of the authentic SARS-CoV-2 virus using cryoelectron tomography (cryo-ET) and subtomogram averaging (STA). Native structures of the S proteins in pre- and postfusion conformations were determined to average resolutions of 8.7-11 Å. Compositions of the N-linked glycans from the native spikes were analyzed by mass spectrometry, which revealed overall processing states of the native glycans highly similar to that of the recombinant glycoprotein glycans. The native conformation of the ribonucleoproteins (RNPs) and their higher-order assemblies were revealed. Overall, these characterizations revealed the architecture of the SARS-CoV-2 virus in exceptional detail and shed light on how the virus packs its ∼30-kb-long single-segmented RNA in the ∼80-nm-diameter lumen.

In situ architecture and membrane fusion of SARS-CoV-2 Delta variant.

Among the current five Variants of Concern, infections caused by SARS-CoV-2 B.1.617.2 (Delta) variant are often associated with the greatest severity. Despite recent advances on the molecular basis of elevated pathogenicity using recombinant proteins, the architecture of intact Delta virions remains veiled. Moreover, pieces of molecular evidence for the detailed mechanism of S-mediated membrane fusion are missing. Here, we showed the pleomorphic nature of Delta virions from electron beam inactivated samples and reported the in situ structure and distribution of S on the authentic Delta variant. We also captured the virus-virus fusion events, which provided pieces of structural evidence for Delta's attenuated dependency on cellular factors for fusion activation, and proposed a model of S-mediated membrane fusion. Besides, site-specific glycan analysis revealed increased oligomannose-type glycosylation of native Delta S than that of the WT S. Together, these results disclose distinctive factors of Delta being the most virulent SARS-CoV-2 variant.

Intracellular MHC class II molecules promote TLR-triggered innate immune responses by maintaining activation of the kinase Btk.

The molecular mechanisms involved in the full activation of innate immunity achieved through Toll-like receptors (TLRs) remain to be fully elucidated. In addition to their classical antigen-presenting function, major histocompatibility complex (MHC) class II molecules might mediate reverse signaling. Here we report that deficiency in MHC class II attenuated the TLR-triggered production of proinflammatory cytokines and type I interferon in macrophages and dendritic cells, which protected mice from endotoxin shock. Intracellular MHC class II molecules interacted with the tyrosine kinase Btk via the costimulatory molecule CD40 and maintained Btk activation, but cell surface MHC class II molecules did not. Then, Btk interacted with the adaptor molecules MyD88 and TRIF and thereby promoted TLR signaling. Therefore, intracellular MHC class II molecules can act as adaptors, promoting full activation of TLR-triggered innate immune responses.

The microRNA miR-29 controls innate and adaptive immune responses to intracellular bacterial infection by targeting interferon-γ.

Interferon-γ (IFN-γ) has a critical role in immune responses to intracellular bacterial infection. MicroRNAs (miRNAs) are important in the regulation of innate and adaptive immunity. However, whether miRNAs can directly target IFN-γ and regulate IFN-γ production post-transcriptionally remains unknown. Here we show that infection of mice with Listeria monocytogenes or Mycobacterium bovis bacillus Calmette-Guérin (BCG) downregulated miR-29 expression in IFN-γ-producing natural killer cells, CD4(+) T cells and CD8(+) T cells. Moreover, miR-29 suppressed IFN-γ production by directly targeting IFN-γ mRNA. We developed mice with transgenic expression of a 'sponge' target to compete with endogenous miR-29 targets (GS29 mice). We found higher serum concentrations of IFN-γ and lower L. monocytogenes burdens in L. monocytogenes-infected GS29 mice than in their littermates. GS29 mice had enhanced T helper type 1 (T(H)1) responses and greater resistance to infection with BCG or Mycobacterium tuberculosis. Therefore, miR-29 suppresses immune responses to intracellular pathogens by targeting IFN-γ.

Development of a multi-recombinase polymerase amplification assay for rapid identification of COVID-19, influenza A and B.

The coronavirus disease 2019 (COVID-19) pandemic caused extensive loss of life worldwide. Further, the COVID-19 and influenza mix-infection had caused great distress to the diagnosis of the disease. To control illness progression and limit viral spread within the population, a real-time reverse-transcription PCR (RT-PCR) assay for early diagnosis of COVID-19 was developed, but detection was time-consuming (4-6 h). To improve the diagnosis of COVID-19 and influenza, we herein developed a recombinase polymerase amplification (RPA) method for simple and rapid amplification of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19 and Influenza A (H1N1, H3N2) and B (influenza B). Genes encoding the matrix protein (M) for H1N1, and the hemagglutinin (HA) for H3N2, and the polymerase A (PA) for Influenza B, and the nucleocapsid protein (N), the RNA-dependent-RNA polymerase (RdRP) in the open reading frame 1ab (ORF1ab) region, and the envelope protein (E) for SARS-CoV-2 were selected, and specific primers were designed. We validated our method using SARS-CoV-2, H1N1, H3N2 and influenza B plasmid standards and RNA samples extracted from COVID-19 and Influenza A/B (RT-PCR-verified) positive patients. The method could detect SARS-CoV-2 plasmid standard DNA quantitatively between 102 and 105 copies/ml with a log linearity of 0.99 in 22 min. And this method also be very effective in simultaneous detection of H1N1, H3N2 and influenza B. Clinical validation of 100 cases revealed a sensitivity of 100% for differentiating COVID-19 patients from healthy controls when the specificity was set at 90%. These results demonstrate that this nucleic acid testing method is advantageous compared with traditional PCR and other isothermal nucleic acid amplification methods in terms of time and portability. This method could potentially be used for detection of SARS-CoV-2, H1N1, H3N2 and influenza B, and adapted for point-of-care (POC) detection of a broad range of infectious pathogens in resource-limited settings.

A multiplex real-time RT-PCR assay for the detection of H1, H2 and H3 subtype avian influenza viruses.

Avian influenza viruses (AIVs) are influenza A viruses, of which subtypes H1, H2 and H3 are highly transmissible in poultry and have the risk of transmission to human as well. It is important to establish an accurate, sensitive and convenient means of virus detection. In this study, we developed a multiplex real-time RT-PCR assay based on conserved sequences of the virus hemagglutinin and matrix, and designed primers and probes for the simultaneous and rapid detection of AIV subtypes H1, H2 and H3. We used different subtypes of AIVs and other avian respiratory viruses for evaluation of the specificity of this method. The results showed good sensitivity, specificity and reproducibility. The detection limit was 10-100 copies per reaction. The method also achieved good concordance with the virus isolation method when compared to 81 poultry samples evaluated. It provides a new method for detecting mixed infections of AIVs.

Generation, characterization, and protective ability of mouse monoclonal antibodies against the HA of A (H1N1) influenza virus.

Influenza virus infections pose a continuous threat to human health. Although vaccines function as a preventive and protective tool, they may not be effective due to antigen drift or an inaccurate prediction of epidemic strains. Monoclonal antibodies (mAbs) have attracted wide attention as a promising therapeutic method for influenza virus infections. In this study, three hemagglutinin (HA)-specific mAbs, named 2A1, 2H4, and 2G2, respectively, were derived from mice immunized with the HA protein from A/Michigan/45/2015(H1N1). The isolated mAbs all displayed hemagglutination inhibition activity and the 2G2 mAb exhibited the strongest neutralization effect. Two amino acid mutations (A198E and G173E), recognized in the process of selection of mAb-resistant mutants, were located in antigenic site Sb and Ca1, respectively. In prophylactic experiments, all three mAbs could achieve 100% protection in mice infected with a lethal dose of A/Michigan/45/2015(H1N1). A dose of 1 mg/kg for 2H4 and 2G2 was sufficient to achieve a full protective effect. Therapeutic experiments showed that all three mAbs could protect mice from death if they received the mAb administration at 6 h postinfection, and 2G2 was still protective after 24 h. Our findings indicate that these three mAbs may have potential prevention and treatment value in an H1N1 epidemic, as well as in the study of antigen epitope recognition.

Rapid emergence of a PB2 D701N substitution during adaptation of an H9N2 avian influenza virus in mice.

H9N2 avian influenza viruses (AIVs) have been isolated frequently from multiple avian species and, occasionally, from humans. To explore the potential molecular basis of cross-species transmission of H9N2 AIVs, an H9N2 AIV (A/chicken/Zhejiang/221/2016) was serially passaged in mouse lung. The results showed that the mouse-adapted H9N2 virus exhibited higher virulence and replicated more efficiently in mouse lung and liver. Whole-genome sequencing showed an amino acid substitution, D701N, in the PB2 protein, which is likely associated with the increased replicative ability of H9N2 virus in mice. The rapid emergence of adaptive substitutions indicates the necessity of continuous monitoring of H9N2 virus in poultry.

Increased virulence of a novel reassortant H1N3 avian influenza virus in mice as a result of adaptive amino acid substitutions.

In this study, a novel multiple-gene reassortant H1N3 subtype avian influenza virus (AIV) (A/chicken/Zhejiang/81213/2017, CK81213) was isolated in Eastern China, whose genes were derived from H1 (H1N3), H7 (H7N3 and H7N9), and H10 (H10N3 and H10N8) AIVs. This AIV belongs to the avian Eurasian-lineage and exhibits low pathogenicity. Serial lung-to-lung passages of CK81213 in mice was performed to study the amino acid substitutions potentially related to the adaptation of H1 AIVs in mammals. And the mouse-adapted H1N3 virus showed greater virulence than wild-type H1N3 AIV in mice and the genomic analysis revealed a total of two amino acid substitutions in the PB2 (E627K) and HA (L67V) proteins. Additionally, the results of the animal study indicate that CK81213 could infect mice without prior adaption and become highly pathogenic to mice after continuous passage. Our findings show that routine surveillance of H1 AIVs is important for the prediction of influenza epidemics.

Proteomic Analysis Identifies Prolonged Disturbances in Pathways Related to Cholesterol Metabolism and Myocardium Function in the COVID-19 Recovery Stage.

The COVID-19 pandemic has become a worldwide health crisis. So far, most studies have focused on the epidemiology and pathogenesis of this infectious disease. Little attention has been given to the disease sequelae in patients recovering from COVID-19, and nothing is known about the mechanisms underlying these sequelae. Herein, we profiled the serum proteome of a cohort of COVID-19 patients in the disease onset and recovery stages. Based on the close integration of our proteomic analysis with clinical data, we propose that COVID-19 is associated with prolonged disorders in cholesterol metabolism and myocardium, even in the recovery stage. We identify potential biomarkers for these disorders. Moreover, severely affected patients presented more serious disturbances in these pathways. Our findings potentially support clinical decision-making to improve the prognosis and treatment of patients.

Development of a monoclonal antibody-based antigen capture enzyme-linked immunosorbent assay for detection of H7N9 subtype avian influenza virus.

To establish a rapid detection method for H7N9 avian influenza virus (AIV), monoclonal antibodies (mAbs) against hemagglutinin (HA) of H7N9 were developed to establish an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA). AC-ELISA achieved high specificity and sensitivity, with a detection limit of 3.9 ng/mL for H7N9 HA protein (A/Zhejiang/DTID-ZJU01/2013), and 2-2 HA unit/100 µL for live H7N9 AIV. The inter- and intra-assay coefficient of variation was less than 10%. Compared with conventional virus isolation detection, the sensitivity and specificity were 94.96% and 88.24%, respectively. AC-ELISA proved to be a rapid and practical technique for the detection of H7N9 AIV.

Antigen-capture ELISA and immunochromatographic test strip to detect the H9N2 subtype avian influenza virus rapidly based on monoclonal antibodies.

BACKGROUND: The H9N2 subtype of avian influenza virus (AIV) has become the most widespread subtype of AIV among birds in Asia, which threatens the poultry industry and human health. Therefore, it is important to establish methods for the rapid diagnosis and continuous surveillance of H9N2 subtype AIV. METHODS: In this study, an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) and a colloidal gold immunochromatographic test (ICT) strip using monoclonal antibodies (MAbs) 3G4 and 2G7 were established to detect H9N2 subtype AIV. RESULTS: The AC-ELISA method and ICT strip can detect H9N2 subtype AIV quickly, and do not cross-react with other subtype AIVs or other viruses. The detection limit of AC-ELISA was a hemagglutinin (HA) titer of 4 for H9N2 subtype AIV per 100 µl sample, and the limit of detection of the HA protein of AIV H9N2 was 31.5 ng/ml. The ICT strip detection limit was an HA titer of 4 for H9N2 subtype AIV per 100 µl sample. Moreover, both detection methods exhibited good reproducibility and repeatability, with coefficients of variation < 5%. For detection in 200 actual poultry samples, the sensitivities and specificities of AC-ELISA were determined as 93.2% and 98.1%, respectively. The sensitivities and specificities of the ICT strips were determined as 90.9% and 97.4%, respectively. CONCLUSIONS: The developed AC-ELISA and ICT strips displayed high specificity, sensitivity, and stability, making them suitable for rapid diagnosis and field investigation of H9N2 subtype AIV.

The viral distribution and pathological characteristics of BALB/c mice infected with highly pathogenic Influenza H7N9 virus.

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.

The MSP-RON pathway regulates liver fibrosis through transforming growth factor beta-dependent epithelial-mesenchymal transition.

BACKGROUND: Liver fibrosis is pathologically important in the liver cirrhosis progression. The epithelial-mesenchymal transition (EMT) is crucial for organ fibrosis. Macrophage-stimulating protein (MSP) and its receptor tyrosine kinase, RON, promote cellular EMT. However, their role in liver fibrosis is unclear. Here, we clarify the biological profile, potential mechanisms and therapeutic targets of the MSP-RON pathway in liver fibrosis. MATERIALS AND METHODS: Macrophage-stimulating protein expression and its correlation with clinicopathological characteristics of cirrhosis were evaluated in 57 clinical cases and a control group. The effect of MSP-RON pathway in liver fibrosis was determined in vitro and in vivo. The therapeutic effects of MSP or RON inhibition on liver fibrosis were evaluated in a mouse liver fibrosis model. RESULTS: Macrophage-stimulating protein is upregulated in liver cirrhosis, which was associated with poor patient prognosis. The MSP-RON pathway promoted hepatocytes EMT. MSP-RON-induced EMT depends on the transforming growth factor beta (TGF-ß) pathway and is regulated by TGF-ß inhibitors. In animal models, an MSP blocking antibody and a small molecule inhibitor of RON, BMS-777607, both inhibited liver fibrosis progression. CONCLUSION: Our study revealed that MSP is an important biomarker in liver cirrhosis progression and can be used to prognose patients. The MSP-RON pathway promotes the EMT of hepatocytes and the progress of fibrosis via a TGF-ß related pathway. Consequently, we identified a new treatment strategy for liver cirrhosis through targeted inhibition of MSP/RON. This research increases the understanding of EMT-modulated liver fibrosis and provides new insights into biomarkers and therapeutic targets of liver fibrosis.

Isolation and characterization of two novel reassortant H5N6 avian influenza viruses from waterfowl in eastern China.

Waterfowl are considered to be the natural hosts of avian influenza virus. In 2017, two reassortant highly pathogenic H5N6 avian influenza viruses of clade 2.3.4.4, subclade II, were identified in wild birds in eastern China. Genome sequencing and phylogenetic and antigenicity analysis showed that the viruses originated from multiple reassortments. To evaluate their pathogenicity in mammals, 15 BALB/c mice were infected with these viruses, and survival and weight loss were monitored for 14 days. Infection was associated with moderate pathogenicity in the mice, and the viruses could replicate in the lungs without prior adaptation. Thus, the existence of these viruses poses a continuous threat to both birds and humans.

Methylene blue photochemical treatment as a reliable SARS-CoV-2 plasma virus inactivation method for blood safety and convalescent plasma therapy for COVID-19.

BACKGROUND: In 2020, a new coronavirus, SARS-CoV-2, quickly spread worldwide within a few months. Although coronaviruses typically infect the upper or lower respiratory tract, the virus RNA can be detected in plasma. The risk of transmitting coronavirus via transfusion of blood products remains. As more asymptomatic infections are identified in COVID-19 cases, blood safety has become particularly important. Methylene blue (MB) photochemical technology has been proven to inactivate lipid-enveloped viruses with high efficiency and safety. The present study aimed to investigate the SARS-CoV-2 inactivation effects of MB in plasma. METHODS: The SARS-CoV-2 virus strain was isolated from Zhejiang University. The live virus was harvested from cultured VERO-E6 cells, and mixed with MB in plasma. The MB final concentrations were 0, 1, 2, and 4 µM. The "BX-1 AIDS treatment instrument" was used at room temperature, the illumination adjusted to 55,000 ± 0.5 million Lux, and the plasma was irradiated for 0, 2, 5, 10, 20, and 40 mins using light at a single wavelength of 630 nm. Virus load changes were measured using quantitative reverse transcription- PCR. RESULTS: BX-1 could effectively eliminate SARS-CoV-2 within 2 mins in plasma, and the virus titer declined to 4.5 log10 TCID50 (median tissue culture infectious dose)/mL. CONCLUSION: BX-1 is based on MB photochemical technology, which was designed to inactivate HIV-1 virus in plasma. It was proven to be safe and reliable in clinical trials of HIV treatment. In this study, we showed that BX-1 could also be applied to inactivate SARS-CoV-2. During the current outbreak, this technique it has great potential for ensuring the safety of blood transfusions, for plasma transfusion therapy in recovering patients, and for preparing inactivated vaccines.

Involvement of the circular RNA/microRNA/glucose-6-phosphate dehydrogenase axis in the pathological mechanism of hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death worldwide with high mortality. The incidence of HCC is increasing in China. Abnormal activation of glucose-6-phosphate dehydrogenase (G6PD) exists in all malignant tumors, including HCC, and is closely related to the development of HCC. In addition, the differential expression of non-coding RNAs is closely related to the development of HCC. This systematic review focuses on the relationship between G6PD, HCC, and non-coding RNA, which form the basis for the circRNA/miRNA/G6PD axis in HCC. The circular RNA (circRNA)/microRNA (miRNA)/G6PD axis is involved in development of HCC. We proposed that non-coding RNA molecules of the circRNA/miRNA/G6PD axis may be novel biomarkers for the pathological diagnosis, prognosis, and targeted therapy of HCC.

Development of an antigen-capture enzyme-linked immunosorbent assay and immunochromatographic strip based on monoclonal antibodies for detection of H6 avian influenza viruses.

Continuous surveillance has shown that H6 subtype avian influenza viruses (AIVs) are prevalent in poultry and occasionally break the species barrier to infect humans. It is therefore necessary to establish a specific, rapid and sensitive method to screen H6 AIVs. In this study, a panel of monoclonal antibodies (mAbs) against the hemagglutinin (HA) of an H6 AIV isolate was produced. The purified mAbs have high affinity and specificity for H6 AIVs. An antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) and immunochromatographic strip were developed based on two mAbs (1D7 and 1F12). The AC-ELISA results showed high sensitivity with a limit of detection (LOD) of 3.9 ng/ml for H6 HA protein and 0.5 HAU (HA units)/100 µl for live H6 subtype AIVs. The average recovery of the AC-ELISA with allantoic fluid, respiratory specimens, and cloacal swabs was 91.907 ± 1.559%, 82.977 ± 1.497% and 73.791 ± 2.588%, respectively. The intra- and inter-assay coefficient of variation was less than 10%. The LOD of immunochromatographic strip was 1 HAU when evaluated by the naked eye, and the detection time was less than 10 min without any equipment. Storage at room temperature or 4 °C for 30 days or 60 days had no effect on sensitivity and specificity of the strip. Thus, the AC-ELISA and immunochromatographic strips described here could be a secondary method to diagnose H6 AIV infections with high specificity, sensitivity, and stability.

Development of a colloidal gold-based immunochromatographic strip test using two monoclonal antibodies to detect H7N9 avian influenza virus.

H7N9 low pathogenic avian influenza viruses (AIVs) emerged in China in 2013 and mutated into highly pathogenic strains in 2017, causing disease in infected birds and humans. Thus, the development of rapid, specific, and sensitive detection methods is urgently required. Herein, two specific monoclonal antibodies against H7N9 AIV were produced to develop a colloidal gold-based immunochromatographic test strip to detect H7N9 AIV. High specificity, repeatability, and sensitivity were achieved, with a detection limit of two hemagglutinin units or 102.55 50% tissue culture infective dose. This assay may represent a powerful tool to rapidly detect H7N9 influenza viruses in the future.

Development and evaluation of a TaqMan MGB RT-PCR assay for detection of H5 and N8 subtype influenza virus.

BACKGROUND: Highly pathogenic influenza A (H5N8) viruses have caused several worldwide outbreaks in birds and are of potential risk to humans. Thus, a specific, rapid and sensitive method for detection is urgently needed. METHODS: In the present study, TaqMan minor groove binder probes and multiplex real-time RT-PCR primers were designed to target the H5 hemagglutinin and N8 neuraminidase genes. A total of 38 strains of avian influenza viruses and other viruses were selected to test the performance of the assay. RESULTS: The results showed that only H5 and N8 avian influenza viruses yielded a positive signal, while all other subtypes avian influenza viruses and other viruses were negative. High specificity, repeatability, and sensitivity were achieved, with a detection limit of 10 copies per reaction. CONCLUSIONS: The developed assay could be a powerful tool for rapid detection of H5N8 influenza viruses in the future.