Frequency of Viral Replication Markers in Chronic HBV Carriers in N’Djamena, Chad

Introduction: According to the WHO, Chad is located in an area of high endemicity for hepatitis B virus (HBV). The aim of this study was to analyze the virological profiles of chronic HBV carriers. Materials and Method: This was a retrospective and analytical study including outpatients with chronic HBV of any age received in Department of Internal Medicine and Gastroenterology of National General Reference Hospital of N’Djamena from 2013 to 2017. Results: During the study period, 401 chronic hepatitis B virus carriers (mean age was 35 years ± 11 years with sex ratio of 3.26) were seen in outpatient clinic with a frequency of 3.34% (401/11,988). HBeAg (+) patients were 12.8% and HBeAg negative 87.2%. HBeAg-negative patients were significantly older (p = 0.000) than those with HBeAg (+). The mean HBV-DNA level was higher in HBeAg (+) patients. The HBV-DNA level in HBeAg-positive patients was higher than in HBeAg-negative patients with a significant difference (p = 0.043). The mean ALT level was 45 IU/L in HBeAg (−) patients. It was 49 in HBeAg (+) patients. Conclusion: HBeAg (−) chronic hepatitis B is the most predominant form in our study. This observation is important because it can help to adapt the management strategies.

Naturally occurring PA^(E206K)point mutation in 2009 H1N1 pandemic influenza viruses impairs viral replication at high temperatures

The emergence of influenza virus A pandemic H1N1 in April 2009 marked the first pandemic of the 21st century.In this study,we observed significant differences in the polymerase activities of two clinical 2009 H1N1 influenza A virus isolates from Chinese and Japanese patients.Sequence comparison of the three main protein subunits(PB2,PB1,and PA)of the viral RNA-dependent RNA polymerase complex and subsequent mutational analysis revealed that a single amino acid substitution(E206K)was responsible for the observed impaired replication phenotype.Further in vitro experiments showed that presence of PAE206K decreased the replication of influenza A/WSN/33 virus in mammalian cells and a reduction in the virus’s pathogenicity in vivo.Mechanistic studies revealed that PAE206K is a temperature-sensitive mutant associated with the inability to transport PB1–PA complex to the nucleus at high temperature(39.5℃).Hence,this naturally occurring variant in the PA protein represents an ideal candidate mutation for the development of live attenuated influenza vaccines.

Ebola virus VP35 perturbs type I interferon signaling to facilitate viral replication

As one of the deadliest viruses,Ebola virus(EBOV)causes lethal hemorrhagic fevers in humans and nonhuman primates.The suppression of innate immunity leads to robust systemic virus replication of EBOV,leading to enhanced transmission.However,the mechanism of EBOV-host interaction is not fully understood.Here,we identified multiple dysregulated genes in early stage of EBOV infection through transcriptomic analysis,which are highly clustered to Jak-STAT signaling.EBOV VP35 and VP30 were found to inhibit type I interferon(IFN)signaling.Moreover,exogenous expression of VP35 blocks the phosphorylation of endogenous STAT1,and suppresses nuclear translocation of STAT1.Using serial truncated mutations of VP35,N-terminal 1–220amino acid residues of VP35 were identified to be essential for blocking on type I IFN signaling.Remarkably,VP35 of EBOV suppresses type I IFN signaling more efficiently than those of Bundibugyo virus(BDBV)and Marburg virus(MARV),resulting in stable replication to facilitate the pathogenesis.Altogether,this study enriches understanding on EBOV evasion of innate immune response,and provides insights into the interplay between filoviruses and host.

Melanization induced by Heliothis virescens ascovirus 3h promotes viral replication

Melanization is an important innate immune defense mechanism of insects,which can kill invading pathogens.Most pathogens,for their survival and reproduction,inhibit the melanization of the host.Interestingly,our results suggested that after infection with Heliothis virescens ascovirus 3h(HvAV-3h),the speed of melanization in infected Spodoptera exigiia larval hemolymph was accelerated and that the phenoloxidase(PO)activity of hemolymph in larvae infected with HvAV-3h increased significantly(1.20-fold at 96 hpi,1.52-fold at 120 hpi,1.23-fold at 144 hpi,1.12-fold at 168 hpi).The transcription level of the gene encoding S.exigua prophenoloxidase-1(SePPO-1 gene)was upregulated dramatically in the fat body during the middle stage of infection.In addition,when melanization was inhibited or promoted,the replication of HvAV-3h was inhibited or promoted,respectively.In conclusion,infection with HvAV-3h can markedly induce melanization in the middle stage of infection,and melanization is helpful for HvAV-3h viral replication.

Sumoylation of Human Parainfluenza Virus Type 3 Phosphoprotein Correlates with A Reduction in Viral Replication

Human parainfluenza virus type 3(HPIV3), a member of the Paramyxoviridae family, can cause lower respiratory disease in infants and young children. The phosphoprotein(P) of HPIV3 is an essential cofactor of the viral RNA-dependent RNA polymerase large protein(L). P connects nucleocapsid protein(N) with L to initiate genome transcription and replication.Sumoylation influences many important pathways of the target proteins, and many viral proteins are also themselves sumoylated. In this study, we found that the P of HPIV3 could be sumoylated, and mutation of K492 and K532 to arginine(PK492 R/K532 R) failed to be sumoylated within P, which enhances HPIV3 minigenome activity. Biochemical studies showed that PK492 R/K532 Rhad no effect on its interactions with N, formation of homo-tetramers and formation of inclusion bodies.Finally, we found that incorporation of K492 R/K532 R into a recombinant HPIV3(rHPIV3-PK492 R/K532 R) increased viral production in culture cells, suggesting that sumoylation attenuates functions of P and down-regulates viral replication.

Selective recruitment of host factors by HSV-1 replication centers

Herpes simplex virus type 1（HSV-1） enters productive infection after infecting epithelial cells, where it controls the host nucleus to make viral proteins, starts viral DNA synthesis and assembles infectious virions. In this process, replicating viral genomes are organized into replication centers to facilitate viral growth. HSV-1 is known to use host factors, including host chromatin and host transcription regulators, to transcribe its genes; however, the invading virus also encounters host defense and stress responses to inhibit viral growth. Recently, we found that HSV-1 replication centers recruit host factor CTCF but exclude γH2A.X. Thus, HSV-1 replication centers may selectively recruit cellular factors needed for viral growth, while excluding host factors that are deleterious for viral transcription or replication. Here we report that the viral replication centers selectively excluded modified histone H3, including heterochromatin mark H3K9me3, H3S10 P and active chromatin mark H3K4me3, but not unmodified H3. We found a dynamic association between the viral replication centers and host RNA polymerase II. The centers also recruited components of the DNA damage response pathway, including 53BP1, BRCA1 and host antiviral protein SP100. Importantly, we found that ATM kinase was needed for the recruitment of CTCF to the viral centers. These results suggest that the HSV-1 replication centers took advantage of host signaling pathways to actively recruit or exclude host factors to benefit viral growth.

LncRNA NKILA inhibits HBV replication by repressing NF-κB signalling activation

Hepatitis B virus(HBV)infection results in liver cirrhosis and hepatocellular carcinoma(HCC).HBx/nuclear factor(NF)-κB pathway plays a role in HBV replication.However,whether NF-κB-interacting long noncoding RNA(NKILA),a suppressor of NF-κB activation,regulates HBV replication remains largely unknown.In this study,gain-and-loss experiments showed that NKILA inhibited HBV replication by inhibiting NF-κB activity.In turn,HBV infection down-regulated NKILA expression.In addition,expression levels of NKILA were lower in the peripheral blood-derived monocytes(PBMCs)of HBV-positive patients than in healthy individuals,which were correlated with HBV viral loads.And a negative correlation between NKILA expression level and HBV viral loads was observed in blood serum from HBV-positive patients.Lower levels of endogenous NKILA were also observed in HepG2 cells expressing a 1.3-fold HBV genome,HBV-infected HepG2-NTCP cells,stable HBV-producing HepG2.2.15 and HepAD38 cells,compared to those HBV-negative cells.Furthermore,HBx was required for NKILA-mediated inhibition on HBV replication.NKILA decreased HBx-induced NF-κB activation by interrupting the interaction between HBx and p65,whereas NKILA mutants lack of essential domains for NF-κB inhibition,lost the ability to inhibit HBV replication.Together,our data demonstrate that NKILA may serve as a suppressor of HBV replication via NF-κB signalling.

Corilagin inhibits SARS-CoV-2 replication by targeting viral RNA-dependent RNA polymerase

Severe acute respiratory syndrome coronavirus-2(SARS-CoV-2) has become one major threat to human population health.The RNA-dependent RNA polymerase(RdRp) presents an ideal target of antivirals,whereas nucleoside analogs inhibitor is hindered by the proofreading activity of coronavirus.Herein,we report that corilagin(RAI-S-37) as a non-nucleoside inhibitor of SARS-CoV-2 RdRp,binds directly to RdRp,effectively inhibits the polymerase activity in both cell-free and cell-based assays,fully resists the proofreading activity and potently inhibits SARS-CoV-2 infection with a low 50% effective concentration(EC50) value of 0.13 μmol/L.Computation modeling predicts that RAI-S-37 lands at the palm domain of RdRp and prevents conformational changes required for nucleotide incorporation by RdRp.In addition,combination of RAI-S-37 with remdesivir exhibits additive activity against antiSARS-CoV-2 RdRp.Together with the current data available on the safety and pharmacokinetics of corilagin as a medicinal herbal agent,these results demonstrate the potential of being developed into one of the much-needed SARS-CoV-2 therapeutics.

Identification of the strain-specifically truncated nonstructural protein 10 of porcine reproductive and respiratory syndrome virus in infected cells

The nonstructural protein 10（nsp10） of porcine reproductive and respiratory syndrome virus（PRRSV） encodes for helicase which plays a vital role in viral replication. In the present study, a truncated form of nsp10, termed nsp10 a, was found in PRRSV-infected cells and the production of nsp10 a was strain-specific. Mass spectrometric analysis and deletion mutagenesis indicated that nsp10 a may be short of about 70 amino acids in the N terminus of nsp10. Further studies by rescuing recombinant viruses showed that the Glu-69 in nsp10 was the key amino acid for nsp10 a production. Finally, we demonstrated that nsp10 a exerted little influence on the growth kinetics of PRRSV in vitro.

In vitro inhibition of HIV-1 replication in autologous CD4*T cells indicates viral containment by multifactorial mechanisms

HIV-1-specific cytotoxic T lymphocytes(CTLs) and neutralizing antibodies(NAbs) are present during chronic infection, but the relative contributions of these effector mechanisms to viral containment remain unclear. Here, using an in vitro model involving autologous CD4+ T cells,primary HIV-1 isolates, HIV-1-specific CTLs, and neutralizing monoclonal antibodies, we show that b12, a potent and broadly neutralizing monoclonal antibody to HIV-1, was able to block viral infection when preincubated with virus prior to infection, but was much less effective than CTLs at limiting virus replication when added to infected cell cultures. However, the same neutralizing antibody was able to contain viruses by antibody-dependent cell-mediated virus inhibition in vitro,which was mediated by natural killer cells(NKs) and dependent on an Fc-Fc receptor interaction.Meanwhile, bulk CTLs from HIV-1 controllers were more effective in suppression of virus replication than those from progressors. These findings indicate that control of HIV-1 replication in activated CD4^+ T cells is ineffectively mediated by neutralizing antibodies alone, but that both CTLs and antibody-dependent NK-mediated immune mechanisms contribute to viral containment. Our study systemically compared three major players in controlling HIV-1 infection, CTLs, NAbs, and NKs, in an autologous system and highlighted the multifactorial mechanisms for viral containment and vaccine success.

The Role of Autophagy in Hepatitis C Virus Infection

Hepatitis C virus(HCV) infects ≈2%of the world's population.HCV infection not only causes acute and chronic hepatitis,but also leads to liver cirrhosis and hepatocellular carcinoma(HCC).The molecular pathogenesis of HCV infection has been explored and many evidence indicated that autophagy is an important process for its life cycle,although autophagy was thought as a mechanism to eliminate invaded HCV from hepatocyte.Structural and non-structural proteins of HCV are important regulators of autophagy,and HCV uses autophagy as a necessary step in its replication.Down-regulation of innate immune response by HCV through unfolded protein response(UPR) and autophagy induction was used as a pathway to establish chronic HCV infection in the liver.Meanwhile,the infected hepatocyte is also using autophagy mechanism to eradicate HCV virus from liver.The study on relationship between HCV and autophagy will pave the new way to understand HCV life cycle and to find new strategy for prevention and treatment of liver diseases caused by HCV infection.

Curcumin inhibits the replication of enterovirus 71 in vitro

Human enterovirus 71(EV71)is the main causative pathogen of hand,foot,and mouth disease(HFMD)in children.The epidemic of HFMD has been a public health problem in Asia-Pacific region for decades,and no vaccine and effective antiviral medicine are available.Curcumin has been used as a traditional medicine for centuries to treat a diversity of disorders including viral infections.In this study,we demonstrated that curcumin showed potent antiviral effect again EV71.In Vero cells infected with EV71,the addition of curcumin significantly suppressed the synthesis of viral RNA,the expression of viral protein,and the overall production of viral progeny.Similar with the previous reports,curcumin reduced the production of ROS induced by viral infection.However,the antioxidant property of curcumin did not contribute to its antiviral activity,since N-acetyl-L-cysteine,the potent antioxidant failed to suppress viral replication.This study also showed that extracellular signal-regulated kinase(ERK)was activated by either viral infection or curcumin treatment,but the activated ERK did not interfere with the antiviral effect of curcumin,indicating ERK is not involved in the antiviral mechanism of curcumin.Unlike the previous reports that curcumin inhibited protein degradation through ubiquitin–proteasome system(UPS),we found that curcumin had no impact on UPS in control cells.However,curcumin did reduce the activity of proteasomes which was increased by viral infection.In addition,the accumulation of the short-lived proteins,p53 and p21,was increased by the treatment of curcumin in EV71-infected cells.We further probed the antiviral mechanism of curcumin by examining the expression of GBF1 and PI4KB,both of which are required for the formation of viral replication complex.We found that curcumin significantly reduced the level of both proteins.Moreover,the decreased expression of either GBF1 or PI4KB by the application of siRNAs was sufficient to suppress viral replication.We also demonstrated that curcumin showed anti-apoptotic activity at the early stage of viral infection.The results of this study provide solid evidence that curcumin has potent anti-EV71 activity.Whether or not the down-regulated GBF1 and PI4KB by curcumin contribute to its antiviral effect needs further studies.

Three-dimensional reconstruction and comparison of vacuolar membranes in response to viral infection

The vacuole is a unique plant organelle that plays an important role in maintaining cellular homeostasis under various environmental stress conditions. However, the effects of biotic stress on vacuole structure has not been examined using three-dimensional(3D) visualization. Here, we performed 3D electron tomography to compare the ultrastructural changes in the vacuole during infection with different viruses. The 3D models revealed that vacuoles are remodeled in cells infected with cucumber mosaic virus(CMV) or tobacco necrosis virus A Chinese isolate(TNV-AC), resulting in the formation of spherules at the periphery of the vacuole. These spherules contain neck-like channels that connect their interior with the cytosol. Confocal microscopy of CMV replication proteins 1 a and 2 a and TNV-AC auxiliary replication protein p23 showed that all of these proteins localize to the tonoplast.Electron microscopy revealed that the expression of these replication proteins alone is sufficient to induce spherule formation on the tonoplast, suggesting that these proteins play prominent roles in inducing vacuolar membrane remodeling. This is the first report of the 3D structures of viral replication factories built on the tonoplasts. These findings contribute to our understanding of vacuole biogenesis under normal conditions and during assembly of plant(+) RNA virus replication complexes.

A novel IFNbeta-induced long non-coding RNA ZAP-IT1 interrupts Zika virus replication in A549 cells

Zika virus(ZIKV) infection can cause severe neurological diseases including neonatal microcephaly and GuillainBarre syndrome. Long noncoding RNAs(lncRNAs) are the by-products of the transcription process, which are considered to affect viral infection. However, it remains largely unexplored whether host lncRNAs play a role in ZIKV infection. Here, we identified a group of human lncRNAs that were up-regulated upon ZIKV infection and were dependent on the type I interferon(IFN) signaling. Overexpression of lncRNA ZAP-IT1 leads to an impairment of ZIKV infection. Correspondently, deficiency of ZAP-IT1 led to an enhancement of ZIKV infection.We further confirmed that ZAP-IT1, an intronic lncRNA with total 551 nt in length, is mainly located in the nuclear upon ZIKV infection. Knockout of ZAP-IT1 also led to the increase of dengue virus(DENV), Japanese encephalitis virus(JEV), or vesicular stomatitis virus(VSV) infection. Mechanically, we found that the antiviral effect of ZAP-IT1 was independent of the type I IFN signaling pathway. Therefore, our data unveiled that host lncRNA ZAP-IT1 induced by the type I IFN signaling, showed robust restriction on ZIKV infection, and even on DENV, JEV, and VSV infection, which may benefit the development of antiviral therapeutics.

The RNA Modification N^6-methyladenosine and Its Implications in Human Disease

Impaired gene regulation lies at the heart of many disorders, including developmental diseases and cancer. Furthermore, the molecular pathways that control gene expression are often the target of cellular parasites, such as viruses. Gene expression is controlled through multiple mechanisms that are coordinated to ensure the proper and timely expression of each gene. Many of these mechanisms target the life cycle of the RNA molecule, from transcription to translation. Recently, another layer of regulation at the RNA level involving RNA modifications has gained renewed interest of the scientific community. The discovery that N6-methyladenosine （m6A）, a mod- ification present in mRNAs and long noncoding RNAs, can be removed by the activity of RNA demethylases, launched the field of epitranscriptomics; the study of how RNA function is regulated through the addition or removal of post-transcriptional modifications, similar to strategies used to regulate gene expression at the DNA and protein level. The abundance of RNA post-transcriptional modifications is determined by the activity of writer complexes （methylase） and eraser （RNA demethylase） proteins. Subsequently, the effects of RNA modifications materialize as changes in RNA structure and/or modulation of interactions between the modified RNA and RNA binding proteins or regulatory RNAs. Disruption of these pathways impairs gene expression and cellular function. This review focuses on the links between the RNA modification m6A and its implications in human diseases.

Compositional Variability and Mutation Spectra of Monophyletic SARS-CoV-2 Clades

COVID-19 and its causative pathogen SARS-CoV-2 have rushed the world into a staggering pandemic in a few months,and a global fight against both has been intensifying.Here,we describe an analysis procedure where genome composition and its variables are related,through the genetic code to molecular mechanisms,based on understanding of RNA replication and its feedback loop from mutation to viral proteome sequence fraternity including effective sites on the replicase-transcriptase complex.Our analysis starts with primary sequence information,identity-based phylogeny based on 22,051 SARS-CoV-2 sequences,and evaluation of sequence variation patterns as mutation spectra and its 12 permutations among organized clades.All are tailored to two key mechanisms:strand-biased and function-associated mutations.Our findings are listed as follows:1)The most dominant mutation is C-to-U permutation,whose abundant second-codon-position counts alter amino acid composition toward higher molecular weight and lower hydrophobicity,albeit assumed most slightly deleterious.2)The second abundance group includes three negative-strand mutations(U-to-C,A-to-G,and G-to-A)and a positive-strand mutation(G-to-U)due to DNA repair mechanisms after cellular abasic events.3)A clade-associated biased mutation trend is found attributable to elevated level of negative-sense strand synthesis.4)Within-clade permutation variation is very informative for associating non-synonymous mutations and viral proteome changes.These findings demand a platform where emerging mutations are mapped onto mostly subtle but fast-adjusting viral proteomes and transcriptomes,to provide biological and clinical information after logical convergence for effective pharmaceutical and diagnostic applications.Such actions are in desperate need,especially in the middle of the War against COVID-19.

Insights into biological therapeutic strategies for COVID-19

The worldwide pandemic of novel coronavirus disease 2019(COVID-19)caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)that emerged in late December 2019 requires the urgent development of therapeutic options.So far,numerous studies have investigated and uncovered the underlying epidemiology and clinical characteristics of COVID-19 infections in order to develop effective drugs.Compared with antiviral small-molecule inhibitors,biotherapeutics have unique advantages such as fewer side effects by virtue of their high specificity,and thus can be rapidly developed for promising treatments of COVID-19.Here,we summarize potential biotherapeutics and their mechanisms of action,including convalescent plasma,therapeutic antibodies,peptides,engineered ACE2,interferons,cytokine inhibitors,and RNAi-based therapeutics,and discuss in depth the advancements and precautions for each type of biotherapeutics in the treatment of COVID-19.

Phosphatidylserine-Specific Phospholipase A1 is the Critical Bridge for Hepatitis C Virus Assembly

The phosphatidylserine-specific phospholipase A1(PLA1A)is an essential host factor in hepatitis C virus(HCV)assembly.In this study,we mapped the E2,NS2 and NS5A involved in PLA1A interaction to their lumenal domains and membranous parts,through which they form oligomeric protein complexes to participate in HCV assembly.Multiple regions of PLA1A were involved in their interaction and complex formation.Furthermore,the results represented structures with PLA1A and E2 in closer proximity than NS2 and NS5A,and strongly suggest PLA1 A-E2,s physical interaction in cells.Meanwhile,we mapped the NS5A sequence which participated in PLA1A interaction with the C-terminus of domain 1.Interestingly,these amino acids in the sequence are also essential for viral RNA replication.Further experiments revealed that these four proteins interact with each other.Moreover,PLA1A expression levels were elevated in livers from HCV-infected patients.In conclusion,we exposed the structural determinants of PLA1A,E2,NS2 and NS5A proteins which were important for HCV assembly and provided a detailed characterization of PLA1A in HCV assembly.