Preventive and therapeutic benefits of nelfinavir in rhesus macaques and human beings infected with SARS-CoV-2.

Effective drugs with broad spectrum safety profile to all people are highly expected to combat COVID-19 caused by SARS-CoV-2. Here we report that nelfinavir, an FDA approved drug for the treatment of HIV infection, is effective against SARS-CoV-2 and COVID-19. Preincubation of nelfinavir could inhibit the activity of the main protease of the SARS-CoV-2 (IC50 = 8.26 µM), while its antiviral activity in Vero E6 cells against a clinical isolate of SARS-CoV-2 was determined to be 2.93 µM (EC50). In comparison with vehicle-treated animals, rhesus macaque prophylactically treated with nelfinavir had significantly lower temperature and significantly reduced virus loads in the nasal and anal swabs of the animals. At necropsy, nelfinavir-treated animals had a significant reduction of the viral replication in the lungs by nearly three orders of magnitude. A prospective clinic study with 37 enrolled treatment-naive patients at Shanghai Public Health Clinical Center, which were randomized (1:1) to nelfinavir and control groups, showed that the nelfinavir treatment could shorten the duration of viral shedding by 5.5 days (9.0 vs. 14.5 days, P = 0.055) and the duration of fever time by 3.8 days (2.8 vs. 6.6 days, P = 0.014) in mild/moderate COVID-19 patients. The antiviral efficiency and clinical benefits in rhesus macaque model and in COVID-19 patients, together with its well-established good safety profile in almost all ages and during pregnancy, indicated that nelfinavir is a highly promising medication with the potential of preventative effect for the treatment of COVID-19.

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.

Adenovirus Vaccine Containing Truncated SARS-CoV-2 Spike Protein S1 Subunit Leads to a Specific Immune Response in Mice.

The development of an efficient and safe coronavirus disease 2019 (COVID-19) vaccine is a crucial approach for managing the severe acute respiratory disease coronavirus 2 (SARS-CoV-2) pandemic in light of current conditions. In this study, we produced a shortened segment of the optimized SARS-CoV-2 spike gene (2043 bp, termed S1) that was able to encode a truncated S1 protein. The protein was tested to determine if it could elicit efficient immunization in mice against SARS-CoV-2. The presence of the S1 protein was confirmed with immunofluorescence and Western blotting. An adenovirus vaccine bearing the S1 gene fragment (Ad-S1) was administered intramuscularly to mice four times over 4 weeks. SARS-CoV-2 S1 protein humoral immunity was demonstrated in all immunized mice. The serum from immunized mice demonstrated excellent anti-infection activity in vitro. A robust humoral immune response against SARS-CoV-2 was observed in the mice after vaccination with Ad-S1, suggesting that the adenovirus vaccine may aid the development of vaccines against SARS-CoV-2 and other genetically distinct viruses.

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.

Unusual global outbreak of monkeypox: what should we do?

Recently, monkeypox has become a global concern amid the ongoing COVID-19 pandemic. Monkeypox is an acute rash zoonosis caused by the monkeypox virus, which was previously concentrated in Africa. The re-emergence of this pathogen seems unusual on account of outbreaks in multiple nonendemic countries and the incline to spread from person to person. We need to revisit this virus to prevent the epidemic from getting worse. In this review, we comprehensively summarize studies on monkeypox, including its epidemiology, biological characteristics, pathogenesis, and clinical characteristics, as well as therapeutics and vaccines, highlighting its unusual outbreak attributed to the transformation of transmission. We also analyze the present situation and put forward countermeasures from both clinical and scientific research to address it.

Potent Antiviral and Antimicrobial Polymers as Safe and Effective Disinfectants for the Prevention of Infections.

Disinfection using effective antimicrobials is essential in preventing the spread of infectious diseases. This COVID-19 pandemic has brought the need for effective disinfectants to greater attention due to the fast transmission of SARS-CoV-2. Current active ingredients in disinfectants are small molecules that microorganisms can develop resistance against after repeated long-term use and may penetrate the skin, causing harmful side-effects. To this end, a series of membrane-disrupting polyionenes that contain quaternary ammoniums and varying hydrophobic components is synthesized. They are effective against bacteria and fungi. They are also fast acting against clinically isolated drug resistant strains of bacteria. Formulating them with thickeners and nonionic surfactants do not affect their killing efficiency. These polyionenes are also effective in preventing infections caused by nonenveloped and enveloped viruses. Their effectiveness against mouse coronavirus (i.e., mouse hepatitis virus-MHV) depends on their hydrophobicity. The polyionenes with optimal compositions inactivates MHV completely in 30 s. More importantly, the polyionenes are effective in inhibiting SARS-CoV-2 by >99.999% within 30 s. While they are effective against the microorganisms, they do not cause damage to the skin and have a high oral lethal dose. Overall, these polyionenes are promising active ingredients for disinfection and prevention of viral and microbial infections.

Interactome Analysis of the Nucleocapsid Protein of SARS-CoV-2 Virus.

SARS-CoV-2 infection has caused a global pandemic that has severely damaged both public health and the economy. The nucleocapsid protein of SARS-CoV-2 is multifunctional and plays an important role in ribonucleocapsid formation and viral genome replication. In order to elucidate its functions, interaction partners of the SARS-CoV-2 N protein in human cells were identified via affinity purification and mass spectrometry. We identified 160 cellular proteins as interaction partners of the SARS-CoV-2 N protein in HEK293T and/or Calu-3 cells. Functional analysis revealed strong enrichment for ribosome biogenesis and RNA-associated processes, including ribonucleoprotein complex biogenesis, ribosomal large and small subunits biogenesis, RNA binding, catalysis, translation and transcription. Proteins related to virus defence responses, including MOV10, EIF2AK2, TRIM25, G3BP1, ZC3HAV1 and ZCCHC3 were also identified in the N protein interactome. This study comprehensively profiled the viral-host interactome of the SARS-CoV-2 N protein in human cells, and the findings provide the basis for further studies on the pathogenesis and antiviral strategies for this emerging infection.

Rational development of a human antibody cocktail that deploys multiple functions to confer Pan-SARS-CoVs protection.

Structural principles underlying the composition and synergistic mechanisms of protective monoclonal antibody cocktails are poorly defined. Here, we exploited antibody cooperativity to develop a therapeutic antibody cocktail against SARS-CoV-2. On the basis of our previously identified humanized cross-neutralizing antibody H014, we systematically analyzed a fully human naive antibody library and rationally identified a potent neutralizing antibody partner, P17, which confers effective protection in animal model. Cryo-EM studies dissected the nature of the P17 epitope, which is SARS-CoV-2 specific and distinctly different from that of H014. High-resolution structure of the SARS-CoV-2 spike in complex with H014 and P17, together with functional investigations revealed that in a two-antibody cocktail, synergistic neutralization was achieved by S1 shielding and conformational locking, thereby blocking receptor attachment and viral membrane fusion, conferring high potency as well as robustness against viral mutation escape. Furthermore, cluster analysis identified a hypothetical 3rd antibody partner for further reinforcing the cocktail as pan-SARS-CoVs therapeutics.

Dynamic Characteristic Analysis of Antibodies in Patients With COVID-19: A 13-Month Study.

There is a worldwide pandemic of Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection; yet our understanding remains limited on the characteristic of antibodies, especially for dynamic long-term tracking. Sequential serum samples were collected up to 416 days post onset of symptoms (POS) from 102 patients who were hospitalized with coronavirus disease 2019 (COVID-19). Immunoglobulin (Ig)G, IgM, and IgA levels targeting SARS-CoV-2 spike 1 receptor-binding domain (S1-RBD), spike 2 extracellular domain (S2-ECD), and nucleocapsid protein (N) were quantified as well as neutralizing activity. We were pleasantly surprised to find that the antibody remained detective and effective for more than a year POS. We also found the varied reactions of different antibodies as time passed: N-IgA rose most rapidly in the early stage of infection, while S2-IgG was present at a high level in the long time of observation. This study described the long traceable antibody response of the COVID-19 and offered hints about targets to screen for postinfectious immunity and for vaccination development of SARS-CoV-2.

Patient-derived SARS-CoV-2 mutations impact viral replication dynamics and infectivity in vitro and with clinical implications in vivo.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread globally with more than 33 million patients diagnosed, taking more than a million lives. Abundant mutations were observed but the functional consequences of these mutations are largely unknown. We report the mutation spectrum, replication dynamics, and infectivity of 11 patient-derived viral isolates in diverse cell lines, including the human lung cancer cell line Calu-3. We observed 46 mutations, including 9 different mutations in the spike gene. Importantly, these viral isolates show significant and consistent variations in replication dynamics and infectivity in tested cell lines, up to a 1500-fold difference in viral titers at 24 h after infecting Calu-3 cells. Moreover, we show that the variations in viral titers among viral isolates are positively correlated with blood clotting function but inversely correlated with the amount of red blood cell and hemoglobin in patients. Therefore, we provide direct evidence that naturally occurring mutations in SARS-CoV-2 can substantially change its replication dynamics and infectivity in diverse human cell lines, with clinical implications in vivo.

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.