Susceptibilities of human ACE2 genetic variants in coronavirus infection

The COVID-19 pandemic, caused by SARS-CoV-2, has resulted in more than 1603 million cases worldwide and 3.4 million deaths (as of May 2021), with varying incidences and death rates among regions/ethnicities. Human genetic variation can affect disease progression and outcome, but little is known about genetic risk factors for SARS-CoV-2 infection. The coronaviruses SARS-CoV, SARS-CoV-2 and HCoV-NL63 all utilize the human protein angiotensin-converting enzyme 2 (ACE2) as the receptor to enter cells. We hypothesized that the genetic variability in ACE2 may contribute to the variable clinical outcomes of COVID-19. To test this hypothesis, we first conducted an in silico investigation of single-nucleotide polymorphisms (SNPs) in the coding region of ACE2 gene. We then applied an integrated approach of genetics, biochemistry and virology to explore the capacity of select ACE2 variants to bind coronavirus spike protein and mediate viral entry. We identified the ACE2 D355N variant that restricts the spike protein-ACE2 interaction and consequently limits infection both in vitro and in vivo. In conclusion, ACE2 polymorphisms could modulate susceptibility to SARS-CoV-2, which may lead to variable disease severity.

Differential interferon-α subtype immune signatures suppress SARS-CoV-2 infection

Type I interferons (IFN-I) exert pleiotropic biological effects during viral infections, balancing virus control versus immune-mediated pathologies and have been successfully employed for the treatment of viral diseases. Humans express twelve IFN-alpha () subtypes, which activate downstream signalling cascades and result in distinct patterns of immune responses and differential antiviral responses. Inborn errors in type I IFN immunity and the presence of anti-IFN autoantibodies account for very severe courses of COVID-19, therefore, early administration of type I IFNs may be protective against life-threatening disease. Here we comprehensively analysed the antiviral activity of all IFN subtypes against SARS-CoV-2 to identify the underlying immune signatures and explore their therapeutic potential. Prophylaxis of primary human airway epithelial cells (hAEC) with different IFN subtypes during SARS-CoV-2 infection uncovered distinct functional classes with high, intermediate and low antiviral IFNs. In particular IFN5 showed superior antiviral activity against SARS-CoV-2 infection. Dose-dependency studies further displayed additive effects upon co-administered with the broad antiviral drug remdesivir in cell culture. Transcriptomics of IFN-treated hAEC revealed different transcriptional signatures, uncovering distinct, intersecting and prototypical genes of individual IFN subtypes. Global proteomic analyses systematically assessed the abundance of specific antiviral key effector molecules which are involved in type I IFN signalling pathways, negative regulation of viral processes and immune effector processes for the potent antiviral IFN5. Taken together, our data provide a systemic, multi-modular definition of antiviral host responses mediated by defined type I IFNs. This knowledge shall support the development of novel therapeutic approaches against SARS-CoV-2.

Ultrapotent SARS-CoV-2 neutralizing antibodies with protective efficacy against newly emerged mutational variants

Accumulating mutations in the SARS-CoV-2 Spike (S) protein can increase the possibility of immune escape, challenging the present COVID-19 prophylaxis and clinical interventions. Here, 3 receptor binding domain (RBD) specific monoclonal antibodies (mAbs), 58G6, 510A5 and 13G9, with high neutralizing potency blocking authentic SARS-CoV-2 virus displayed remarkable efficacy against authentic B.1.351 virus. Each of these 3 mAbs in combination with one neutralizing Ab recognizing non-competing epitope exhibited synergistic effect against authentic SARS-CoV-2 virus. Surprisingly, structural analysis revealed that 58G6 and 13G9, encoded by the IGHV1-58 and the IGKV3-20 germline genes, both recognized the steric region S470-495 on the RBD, overlapping the E484K mutation presented in B.1.351. Also, 58G6 directly bound to another region S450-458 in the RBD. Significantly, 58G6 and 510A5 both demonstrated prophylactic efficacy against authentic SARS-CoV-2 and B.1.351 viruses in the transgenic mice expressing human ACE2 (hACE2), protecting weight loss and reducing virus loads. These 2 ultrapotent neutralizing Abs can be promising candidates to fulfill the urgent needs for the prolonged COVID-19 pandemic.

Comparative analysis reveals the species-specific genetic determinants of ACE2 required for SARS-CoV-2 entry

Coronavirus interaction with its viral receptor is a primary genetic determinant of host range and tissue tropism. SARS-CoV-2 utilizes ACE2 as the receptor to enter host cell in a species-specific manner. We and others have previously shown that ACE2 orthologs from New World monkey, koala and mouse cannot interact with SARS-CoV-2 to mediate viral entry, and this defect can be restored by humanization of the restrictive residues in New World monkey ACE2. To better understand the genetic determinants behind the ability of ACE2 orthologs to support viral entry, we compared koala and mouse ACE2 sequences with that of human and identified the key residues in koala and mouse ACE2 that restrict viral receptor activity. Humanization of these critical residues rendered both koala and mouse ACE2 capable of binding the spike protein and facilitating viral entry. The single mutation that allowed for mouse ACE2 to serve as a viral receptor provides a potential avenue for the development of SARS-CoV-2 mouse model.

A bacterial artificial chromosome (BAC)-vectored noninfectious replicon of SARS-CoV-2

Vaccines and antiviral agents are in urgent need to stop the COVID-19 pandemic. To facilitate antiviral screening against SARS-CoV-2 without requirement for high biosafety level facility, we developed a bacterial artificial chromosome (BAC)-vectored replicon of SARS-CoV-2, nCoV-SH01 strain, in which secreted Gaussia luciferase (sGluc) was encoded in viral subgenomic mRNA as a reporter gene. The replicon was devoid of structural genes spike (S), membrane (M), and envelope (E). Upon transfection, the replicon RNA replicated in various cell lines, and was sensitive to interferon alpha (IFN-), remdesivir, but was resistant to hepatitis C virus inhibitors daclatasvir and sofosbuvir. Replication of the replicon was also sensitive overexpression of zinc-finger antiviral protein (ZAP). We also constructed a four-plasmid in-vitro ligation system that is compatible with the BAC system, which makes it easy to introduce desired mutations into the assembly plasmids for in-vitro ligation. This replicon system would be helpful for performing antiviral screening and dissecting virus-host interactions.

A key linear epitope for a potent neutralizing antibody to SARS-CoV-2 S-RBD

The spread of SARS-CoV-2 confers a serious threat to the public health without effective intervention strategies1-3. Its variant carrying mutated Spike (S) protein D614G (SD614G) has become the most prevalent form in the current global pandemic4,5. We have identified a large panel of potential neutralizing antibodies (NAbs) targeting the receptor-binding domain (RBD) of SARS-CoV-2 S6. Here, we focused on the top 20 potential NAbs for the mechanism study. Of them, the top 4 NAbs could individually neutralize both authentic SARS-CoV-2 and SD614G pseudovirus efficiently. Our epitope mapping revealed that 16/20 potent NAbs overlapped the same steric epitope. Excitingly, we found that one of these potent NAbs (58G6) exclusively bound to a linear epitope on S-RBD (termed as 58G6e), and the interaction of 58G6e and the recombinant ACE2 could be blocked by 58G6. We confirmed that 58G6e represented a key site of vulnerability on S-RBD and it could positively react with COVID-19 convalescent patients plasma. We are the first, as far as we know, to provide direct evidences of a linear epitope that can be recognized by a potent NAb against SARS-CoV-2 S-RBD. This study paves the way for the applications of these NAbs and the potential safe and effective vaccine design.

The S1/S2 boundary of SARS-CoV-2 spike protein modulates cell entry pathways and transmission

The global spread of SARS-CoV-2 is posing major public health challenges. One unique feature of SARS-CoV-2 spike protein is the insertion of multi-basic residues at the S1/S2 subunit cleavage site, the function of which remains uncertain. We found that the virus with intact spike (Sfull) preferentially enters cells via fusion at the plasma membrane, whereas a clone (Sdel) with deletion disrupting the multi-basic S1/S2 site instead utilizes a less efficient endosomal entry pathway. This idea was supported by the identification of a suite of endosomal entry factors specific to Sdel virus by a genome-wide CRISPR-Cas9 screen. A panel of host factors regulating the surface expression of ACE2 was identified for both viruses. Using a hamster model, animal-to-animal transmission with the Sdel virus was almost completely abrogated, unlike with Sfull. These findings highlight the critical role of the S1/S2 boundary of the SARS-CoV-2 spike protein in modulating virus entry and transmission.

Functional mapping of B-cell linear epitopes 1 of SARS-CoV-2 in COVID-19 convalescent population

Pandemic SARS-CoV-2 has infected over 10 million people and caused over 500,000 mortalities. Vaccine development is in urgent need to stop the pandemic. Despite great progresses on SARS-CoV-2 vaccine development, the efficacy of the vaccines remains to be determined. Deciphering the interactions of the viral epitopes with their elicited neutralizing antibodies in the convalescent COVID-19 population inspires the vaccine development. In this study, we devised a peptide array composed of 20-mer overlapped peptides of spike (S), membrane (M) and envelope (E) proteins, and performed a screening with 120 COVID-19 convalescent serums and 24 non-COVID-19 serums. We identified five SARS-CoV-2-specific dominant epitopes that reacted with above 40% COVID-19 convalescent serums. Epitopes in the receptor-binding domain (RBD) of S ill reacted with the convalescent serums. Of note, two peptides non-specifically interacted with most of the non-COVID-19 serums. Neutralization assay indicated that only five serums completely blocked viral infection at the dilution of 1:200. By using a peptide-compete neutralizing assay, we found that three dominant epitopes partially competed the neutralization activity of several convalescent serums, suggesting antibodies elicited by these epitopes played an important role in neutralizing viral infection. The epitopes we identified in this study may serve as vaccine candidates to elicit neutralizing antibodies in most vaccinated people or specific antigens for SARS-CoV-2 diagnosis.

Immunization with the receptor-binding domain of SARS-CoV-2 elicits antibodies cross-neutralizing SARS-CoV-2 and SARS-CoV without antibody-dependent enhancement

Recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen responsible for the ongoing coronavirus disease 2019 (COVID-19) pandemic. Currently, there is no vaccine available for preventing SARS-CoV-2 infection. Like closely related severe acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2 also uses its receptor-binding domain (RBD) on the spike (S) protein to engage the host receptor, human angiotensin-converting enzyme 2 (ACE2), facilitating subsequent viral entry. Here we report the immunogenicity and vaccine potential of SARS-CoV-2 RBD (SARS2-RBD)-based recombinant proteins. Immunization with SARS2-RBD recombinant proteins potently induced a multi-functional antibody response in mice. The resulting antisera could efficiently block the interaction between SARS2-RBD and ACE2, inhibit S-mediated cell-cell fusion, and neutralize both SARS-CoV-2 pseudovirus entry and authentic SARS-CoV-2 infection. In addition, the anti-RBD sera also exhibited cross binding, ACE2-blockade, and neutralization effects towards SARS-CoV. More importantly, we found that the anti-RBD sera did not promote antibody-dependent enhancement of either SARS-CoV-2 pseudovirus entry or authentic virus infection of Fc receptor-bearing cells. These findings provide a solid foundation for developing RBD-based subunit vaccines for SARS-CoV2.

Potent antiviral effect of protoporphyrin IX and verteporfin on SARS-CoV-2 infection

The SARS-CoV-2 infection is spreading rapidly worldwide. Efficacious antiviral therapeutics against SARS-CoV-2 is urgently needed. Here, we discovered that protoporphyrin IX (PpIX) and verteporfin, two FDA-approved drugs, completely inhibited the cytopathic effect produced by SARS-CoV-2 infection at 1.25 M and 0.31 M respectively, and their EC50 values of reduction of viral RNA were at nanomolar concentrations. The selectivity indices of PpIX and verteporfin were 952.74 and 368.93, respectively, suggesting broad margin of safety. Importantly, PpIX and verteporfin prevented SARS-CoV-2 infection in mice adenovirally transduced with human ACE2. The compounds, sharing a porphyrin ring structure, were shown to bind viral receptor ACE2 and interfere with the interaction between ACE2 and the receptor-binding domain of viral S protein. Our study suggests that PpIX and verteporfin are potent antiviral agents against SARS-CoV-2 infection and sheds new light on developing novel chemoprophylaxis and chemotherapy against SARS-CoV-2.

Functional and Genetic Analysis of Viral Receptor ACE2 Orthologs Reveals Broad Potential Host Range of SARS-CoV-2

The pandemic of Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a major global health threat. Epidemiological studies suggest that bats are the natural zoonotic reservoir for SARS-CoV-2. However, the host range of SARS-CoV-2 and intermediate hosts that facilitate its transmission to humans remain unknown. The interaction of coronavirus with its host receptor is a key genetic determinant of host range and cross-species transmission. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as the receptor to enter host cells in a species-dependent manner. It has been shown that human, palm civet, pig and bat ACE2 can support virus entry, while the murine ortholog cannot. In this study, we characterized the ability of ACE2 from diverse species to support viral entry. We found that ACE2 is expressed in a wide range of species, with especially high conservation in mammals. By analyzing amino acid residues of ACE2 critical for virus entry, based on structure of SARS-CoV spike protein interaction with human, bat, palm civet, pig and ferret ACE2, we identified approximately eighty ACE2 proteins from mammals that could potentially mediate SARS-CoV-2 entry. We chose 48 representative ACE2 orthologs among eighty orthologs for functional analysis and it showed that 44 of these mammalian ACE2 orthologs, including those of domestic animals, pets, livestock, and animals commonly found in zoos and aquaria, could bind SARS-CoV-2 spike protein and support viral entry. In contrast, New World monkey ACE2 orthologs could not bind SARS-CoV-2 spike protein and support viral entry. We further identified the genetic determinant of New World monkey ACE2 that restricts viral entry using genetic and functional analyses. In summary, our study demonstrates that ACE2 from a remarkably broad range of species can facilitate SARS-CoV-2 entry. These findings highlight a potentially broad host tropism of SARS-CoV-2 and suggest that SARS-CoV-2 might be distributed much more widely than previously recognized, underscoring the necessity to monitor susceptible hosts to prevent future outbreaks.

Recapitulation of SARS-CoV-2 Infection and Cholangiocyte Damage with Human Liver Organoids

The newly emerged pandemic coronavirus, SARS-CoV-2, has posed a significant public health threat worldwide. However, the mode of virus transmission and tissue tropism is not well established yet. Recent findings of substantial liver damage in patients and ACE2+ cholangiocytes in healthy liver tissues prompted us to hypothesize that human liver ductal organoids could serve as a model to determine the susceptibility and mechanisms underlining the liver damage upon SARS-CoV-2 infection. By single-cell RNA sequencing, we found that long-term liver ductal organoid culture preserved the human specific ACE2+ population of cholangiocytes. Moreover, human liver ductal organoids were permissive to SARS-CoV-2 infection and support robust replication. Notably, virus infection impaired the barrier and bile acid transporting functions of cholangiocytes through dysregulation of genes involved in tight junction formation and bile acid transportation, which could explain the bile acid accumulation and consequent liver damage in patients. These results indicate that control of liver damage caused directly by viral infection should be valued in treating COVID-19 patients. Our findings also provide an application of human organoids in investigating the tropism and pathogenesis of SARS-CoV-2, which would facilitate novel drug discovery.

Regulatory mechanisms of the transcription and metabolism of hepatitis B virus covalently closed circular DNA and strategies for silencing and elimination / 临床肝胆病杂志

It is known that hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) persists in the nucleus of infected hepatocytes in the form of minichromosome and is difficult to target and eliminate. Studies on the mechanisms and strategies for persistent silencing or elimination of HBV cccDNA are the focus achieving for “functional cure” of chronic hepatitis B. This article introduces the current knowledge on the basic biological features of cccDNA, regulatory mechanisms of transcription and metabolism, and related host factors, with a focus on the potential pathways and strategies for cccDNA silencing or elimination.

Application of in situ hybridization in the detection of hepatitis B virus nucleic acids and cccDNA / 临床肝胆病杂志

In situ hybridization (ISH) is a new technique which combines molecular biology, histochemistry, and cytology. It can quantify and locate specific nucleic acids at the cellular and chromosomal levels and is widely used in virological research. ISH is of great significance for the detection of hepatitis B virus (HBV) nucleic acids (RNA and replicative intermediate DNA) and covalently closed circular DNA. This article reviews the development of ISH and its application in HBV research.

Influence of interferon and nucleos(t)ide analogues on HBV cccDNA and functional cure of chronic hepatitis B / 临床肝胆病杂志

At present, interferon (IFN) and nucleos(t)ide analogues (NAs) remain the most important methods for the treatment of chronic hepatitis B in clinical practice, but neither of them can effectively eliminate the virus and cure hepatitis B. As the template for HBV transcription and replication, HBV covalently closed circular DNA (cccDNA) persistently exists in the nucleus in the form of minichromosome and is considered the most important reason for chronic and refractory HBV infection. Since it is hard to completely eliminate cccDNA, functional cure of chronic hepatitis B through sustained silencing of cccDNA has become a major goal of clinical and basic research in recent years. This article reviews the influence of current treatment methods on cccDNA, the factors regulating the amount and activity of cccDNA, and the key obstacles to eradication of cccDNA pool, with perspectives of cccDNA research towards a functional cure of chronic hepatitis B.

Association of exosomes with viral infection and hepatitis B virus-related liver diseases / 临床肝胆病杂志

Hepatitis B virus (HBV) infection causes pathological changes of the liver,including liver inflammation,hepatocyte necrosis,and even liver fibrosis,and promotes the progression from chronic hepatitis to liver cirrhosis and liver cancer,but related mechanisms remain unclear.The mechanism for the interaction between hepatocytes infected by HBV and uninfected hepatocytes/host immune system might be exosomes-mediated cell-cell communication in liver microenvironment.Many studies have demonstrated that viral infection can regulate the production of exosomes and affect their composition,and viral microRNAs,proteins,and even the entire virion can be incorporated into the exosomes,which can affect the immune recognition of viruses or regulate the function of adjacent cells.This article elaborates on the production and composition of exosomes and their roles in viral infection,as well as the research advances in the association between exosomes and HBV infection.

Analysis of epidemiology characteristics of norovirus among diarrheal outpatients in 27 provinces in China, 2009-2013 / 中华流行病学杂志

<p><b>OBJECTIVE</b>To investigate the epidemiology characteristics of norovirus among diarrheal outpatients in China.</p><p><b>METHODS</b>Diarrhea cases were monitored at emergency/outpatient departments at 173 hospitals in 27 provinces of China, with clinical and epidemiological data, and fecal specimens collected and sent to 58 network-laboratories to detect norovirus by RT-PCR method, and to analyze the positive rate of norovirus in various regions, population and time during 2009-2013.</p><p><b>RESULTS</b>11.6% of the 34 031 diarrheal cases under surveillance were found with norovirus. Age group of 6-23 month-old children and that of people over 45 years old were found with the highest positive percentage, 13.7% and 12.4% respectively. Positive percentage of norovirus peaks in autumn and winter in a year; it peaks in mid-temperate zones (10.7%) and warm-temperate zones (11.6%) in winter. It peaks in sub-tropical zones in autumn (14.3%). The most prevalent genogroups detected were norovirus G II, accounting for 89.9% of identified strains.</p><p><b>CONCLUSION</b>Norovirus affects all ages and was most prevalent in children and the elderly among diarrhea outpatients. Norovirus' positive percentage showed strong seasonal pattern, and peaks at different times of a year in different climate zones of China. Since no effective preventive measures existed, further study on norovirus epidemiology and intervention strategies should be conducted in future.</p>

Inhibition of hepatitis B virus replication by MyD88 is mediated by nuclear factor-kappaB activation.

In our previous paper, we reported that myeloid differential primary response protein (MyD88), a key adaptor in the signaling cascade of the innate immune response, inhibits hepatitis B virus (HBV) replication. The MyD88 activated nuclear factor-kappaB (NF-kappaB) signaling pathway and the intracellular upregulation of NF-kappaB signaling can induce an antiviral effect. Therefore, the association between the inhibition of HBV replication by MyD88 and NF-kappaB activation was investigated further. The results show that NF-kappaB activation was moderately increased after MyD88 expression. The strong activation of NF-kappaB by the IkappaB kinase complex IKKalpha/IKKbeta dramatically suppressed HBV replication; the MyD88 dominant negative mutant that abrogated NF-kappaB activity did not inhibit HBV replication. Furthermore, the IkappaBalpha dominant negative mutant restored the inhibition of HBV replication by MyD88. These results support a role for NF-kappaB activation in the inhibition of HBV replication and suggest a novel mechanism for the inhibition of HBV replication by MyD88 protein.

Subproteomic analysis of the cellular proteins associated with the 3' untranslated region of the hepatitis C virus genome in human liver cells.

The 3' untranslated region (UTR) of the hepatitis C virus (HCV) is believed to function in the initiation and regulation of viral RNA replication and protein translation by interacting with the viral and host components. To examine host proteins interacting with the HCV 3'UTR, biotinylated 3'(+)UTR, and its reverse complementary 5'(-)UTR were used in RNA pull-down assay. Cellular proteins from Huh7 cells pulled down by biotinylated RNAs were identified by 2DE/MALDI-TOF MS and 1DE/LC/MS methods. Totally, 10 proteins could be identified from both methods, among which six bound specifically to the 3'(+)UTR, three proteins to the 5'(-)UTR only, and one protein bound to both. Three identified proteins (PCBP2, G3BP1, and DDX1) were selected for further investigation into their possible roles on the HCV replication. Differently regulating effects on HCV replication by siRNA-mediated silencing of these proteins were observed, indicating a complex role of 3'UTR binding proteins on HCV replication.

Association between NS5A gene sequence and response to interferon therapy in chronic hepatitis C patients in Shanghai / 中华实验和临床病毒学杂志

<p><b>BACKGROUND</b>To elucidate relationship between amino acid sequence of non-structural protein 5A (NS5A) and outcome of HCV (1 b) patients after interferon (IFNa) therapy.</p><p><b>METHODS</b>Sera of 24 patients were collected before, during and after IFNa therapy. Pretreatment RNA levels and the sequences of HCV NS5A interferon sensitivity determining region (ISDR) were determined. NS5A full-length sequences of 5 HCV isolates from 3 patients with different response types were also analyzed. Phylogenetic tree analysis and protein secondary structure prediction were undertaken.</p><p><b>RESULTS</b>Pretreatment RNA levels of sustained response group were significantly lower than that of non-response group and relapse group (4.50X104 copies/ml versus 1.82X107 copies/ml, P < 0.01).ISDR sequences of NS5A from pretreatment sera were compared with HCV-J strain (prototype). Thirteen of 24 isolates were wild type,11 of 24 were intermediate type and none of them was mutant type. 3 of 6 sustained responders were infected with wild-type isolates, the rest with intermediate type isolates. Phylogenetic tree based on NS5A full-length sequences classified 5 isolates with 3 different response types into 3 groups. Non-response isolates belonged to the same group as HCV-J. Secondary structure prediction of 5 isolates revealed significant differences existing in 2 255- 2 289. This region was partly overlapped with PKR-binding domain.</p><p><b>CONCLUSIONS</b>Low HCV RNA levels in serum are associated with favorable outcome of IFNa therapy. ISDR sequence alone could not predict outcome of IFN treatment. Combination of determination of HCV RNA levels in serum with sequence analysis of PKR-binding domain may be helpful in predicting the efficacy of IFN therapy.</p>