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Variants of SARS-CoV-2 continue to emerge, posing great challenges in outbreak prevention and control. It is important to understand in advance the impact of possible variants of concern (VOCs) on infectivity and antigenicity. Here, we constructed one or more of the 15 high-frequency naturally occurring amino acid changes in the receptor-binding domain (RBD) of Alpha, Beta, and Gamma variants. A single mutant of A520S, V367F, and S494P in the above three VOCs enhanced infectivity in ACE2-overexpressing 293T cells of different species, LLC-MK2 and Vero cells. Aggregation of multiple RBD mutations significantly reduces the infectivity of the possible three VOCs. Regarding neutralization, it is noteworthy that E484K, N501Y, K417N, and N439K predispose to monoclonal antibodies (mAbs) protection failure in the 15 high-frequency mutations. Most importantly, almost all possible VOCs (single RBD mutation or aggregation of multiple mutations) showed no more than a fourfold decrease in neutralizing activity with convalescent sera, vaccine sera, and immune sera of guinea pigs with different immunogens, and no significant antigenic drift was formed. In conclusion, our pseudovirus results could reduce the concern that the aggregation of multiple high-frequency mutations in the RBD of the spike protein of the three VOCs would lead to severe antigenic drift, and this would provide value for vaccine development strategies.

The emergence of Omicron/BA.1 has brought new challenges to fight against SARS-CoV-2. A large number of mutations in the Spike protein suggest that its susceptibility to immune protection elicited by the existing COVID-19 infection and vaccines may be altered. In this study, we constructed the pseudotyped SARS-CoV-2 variant Omicron. The sensitivity of 28 serum samples from COVID-19 convalescent patients infected with SARS-CoV-2 original strain was tested against pseudotyped Omicron as well as the other variants of concern (VOCs, Alpha, Beta, Gamma, Delta) and variants of interest (VOIs, Lambda, Mu). Our results indicated that the mean neutralization ED50 of these sera against Omicron decreased to 66, which is about 8.4-folds compared to the D614G reference strain (ED50 = 556), whereas the neutralization activity of other VOC and VOI pseudotyped viruses decreased only about 1.2–4.5-folds. The finding from our in vitro assay suggest that Omicron variant may lead to more significant escape from immune protection elicited by previous SARS-CoV-2 infection and perhaps even by existing COVID-19 vaccines.

Severe acute respiratory syndrome coronavirus 2 variants have continued to emerge in diverse geographic locations with a temporal distribution. The Lambda variant containing multiple mutations in the spike protein, has thus far appeared mainly in South America. The variant harbours two mutations in the receptor binding domain, L452Q and F490S, which may change its infectivity and antigenicity to neutralizing antibodies. In this study, we constructed 10 pseudoviruses to study the Lambda variant and each individual amino acid mutation's effect on viral function, and used eight cell lines to study variant infectivity. In total, 12 monoclonal antibodies, 14 convalescent sera, and 23 immunized sera induced by mRNA vaccines, inactivated vaccine, and adenovirus type 5 vector vaccine were used to study the antigenicity of the Lambda variant. We found that compared with the D614G reference strain, Lambda demonstrated enhanced infectivity of Calu-3 and LLC-MK2 cells by 3.3-fold and 1.6-fold, respectively. Notably, the sensitivity of the Lambda variant to 5 of 12 neutralizing monoclonal antibodies, 9G11, AM180, R126, X593, and AbG3, was substantially diminished. Furthermore, convalescent- and vaccine-immunized sera showed on average 1.3–2.5-fold lower neutralizing titres against the Lambda variant. Single mutation analysis revealed that this reduction in neutralization was caused by L452Q and F490S mutations. Collectively, the reduced neutralization ability of the Lambda variant suggests that the efficacy of monoclonal antibodies and vaccines may be compromised during the current pandemic.

Emerging SARS-CoV-2 variants are the most serious problem for COVID-19 prophylaxis and treatment. To determine whether the SARS-CoV-2 vaccine strain should be updated following variant emergence like seasonal flu vaccine, the changed degree on antigenicity of SARS-CoV-2 variants and H3N2 flu vaccine strains was compared. The neutralization activities of Alpha, Beta and Gamma variants’ spike protein-immunized sera were analysed against the eight current epidemic variants and 20 possible variants combining the top 10 prevalent RBD mutations based on the Delta variant, which were constructed using pseudotyped viruses. Meanwhile, the neutralization activities of convalescent sera and current inactivated and recombinant protein vaccine-elicited sera were also examined against all possible Delta variants. Eight HA protein-expressing DNAs elicited-animal sera were also tested against eight pseudotyped viruses of H3N2 flu vaccine strains from 2011–2019. Our results indicate that the antigenicity changes of possible Delta variants were mostly within four folds, whereas the antigenicity changes among different H3N2 vaccine strains were approximately 10–100-fold. Structural analysis of the antigenic characterization of the SARS-CoV-2 and H3N2 mutations supports the neutralization results. This study indicates that the antigenicity changes of the current SARS-CoV-2 may not be sufficient to require replacement of the current vaccine strain.

We evaluated the association between human pegivirus-2 (HPgV-2) infection and hepatitis C virus (HCV)/hepatitis B virus (HBV) co-infection in 745 plasma samples collected from HCV-positive but human immunodeficiency virus type one (HIV-1)-negative people who inject drugs in Hunan, China. The prevalence of anti-HPgV-2 was 4.43 % (33/745) and, within this, the HCV 6a genotype showed significantly higher prevalence as compared with the HCV non-6a genotypes, 6.29 % (18/286) vs. 1.69 % (4/236), respectively (P=0.009). HPgV-2 RNA was detected in 2.15 % (16/745), and was not significantly different between the HCV 6a and non-6a genotypes, 2.45 % (7/286) vs. 2.54 % (6/236), respectively (P =0.945). HBV single infection did not increase the risk of HPgV-2 infection. Compared with HCV single infection, HCV/HBV co-infection increased the risk of HPgV-2 infection by about three-fold: odds ratio (OR)=3.24 [95 % confidence interval (CI) 1.34-7.82, P=0.014] according to anti-HPgV-2 positivity or OR=3.51 (95 % CI 1.15-10.74, P=0.051) according to HPgV-2 viraemia. HPgV-2 infection did not increase the levels of liver-specific enzymes. Our study provides new findings regarding the association between HPgV-2 and HCV genotypes as well as HCV/HBV co-infection.

Coronavirus disease 2019 (COVID-19) is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)1-3 and individuals with COVID-19 have symptoms that can be asymptomatic, mild, moderate or severe4,5. In the early phase of infection, T- and B-cell counts are substantially decreased6,7; however, IgM8-11 and IgG12-14 are detectable within 14 d after symptom onset. In COVID-19-convalescent individuals, spike-specific neutralizing antibodies are variable3,15,16. No specific drug or vaccine is available for COVID-19 at the time of writing; however, patients benefit from treatment with serum from COVID-19-convalescent individuals17,18. Nevertheless, antibody responses and cross-reactivity with other coronaviruses in COVID-19-convalescent individuals are largely unknown. Here, we show that the majority of COVID-19-convalescent individuals maintained SARS-CoV-2 spike S1- and S2-specific antibodies with neutralizing activity against the SARS-CoV-2 pseudotyped virus, and that some of the antibodies cross-neutralized SARS-CoV, Middle East respiratory syndrome coronavirus or both pseudotyped viruses. Convalescent individuals who experienced severe COVID-19 showed higher neutralizing antibody titres, a faster increase in lymphocyte counts and a higher frequency of CXCR3+ T follicular help (TFH) cells compared with COVID-19-convalescent individuals who experienced non-severe disease. Circulating TFH cells were spike specific and functional, and the frequencies of CXCR3+ TFH cells were positively associated with neutralizing antibody titres in COVID-19-convalescent individuals. No individuals had detectable autoantibodies. These findings provide insights into neutralizing antibody responses in COVID-19-convalescent individuals and facilitate the treatment and vaccine development for SARS-CoV-2 infection.

Hepatitis C virus (HCV) and hepatitis B virus (HBV) coinfection reciprocally influences viral replication and host defence responses. This study aimed to investigate the impact of HBV coinfection on circulating T follicular helper cell (cTfh) distribution and the HCV neutralizing antibody (nAb) response. HCV neutralizing antibody responses were measured in individuals with HCV monoinfection (n = 83) and HBV/HCV coinfection (n = 78) using the HCV pseudoparticle neutralization assay. The frequencies of cTfh cells and their subsets in HCV monoinfection (n = 34) and HBV/HCV coinfection (n = 30) were analysed by flow cytometry. The correlations of clinical parameters, cTfh cells and neutralizing antibody responses were analysed. Compared with HCV monoinfection, the HBV coinfection group showed significantly lower HCV neutralizing antibody responses (P < 0.001) and a decreased frequency of circulating Th1-like Tfh cells (Tfh1) (P = 0.004). In HCV monoinfection, the frequency of the Tfh1 subset was positively correlated with HCV neutralizing antibody responses (R = 0.378, P = 0.03), but this correlation was lost under HBV/HCV coinfection (R = 0.115, P = 0.551). In contrast, the frequency of circulating Th2-like Tfh cells (Tfh2) was negatively correlated with the HCV neutralizing antibody responses (R = 0.404, P = 0.003). Further analysis showed that HBV coinfection enhanced the Tfh2 subset composition within cTfh cells (P < 0.001), which was associated with serum HBsAg in HBV/HCV coinfection (R = 0.521, P = 0.003). As expected, HBsAg also exhibited an inverse association with HCV neutralizing antibody responses in HBV/HCV coinfection (R = 0.59, P < 0.001). In contrast to HCV monoinfection, HBV/HCV coinfection leads to altered cTfh cell distribution and impaired HCV neutralizing antibody responses, which are associated with HBsAg. These findings will be helpful for better understanding the immunopathogenesis of HBV/HCV coinfection.

The 501Y.V2 variants of SARS-CoV-2 containing multiple mutations in Spike are now dominant in South Africa and are rapidly spreading to other countries. Here, experiments with 18 pseudotyped viruses showed that the 501Y.V2 variants do not confer increased infectivity in multiple cell types except for murine ACE2-overexpressing cells, where a substantial increase in infectivity was observed. Notably, the susceptibility of the 501Y.V2 variants to 12 of 17 neutralizing monoclonal antibodies was substantially diminished, and the neutralization ability of the sera from convalescent patients and immunized mice was also reduced for these variants. The neutralization resistance was mainly caused by E484K and N501Y mutations in the receptor-binding domain of Spike. The enhanced infectivity in murine ACE2-overexpressing cells suggests the possibility of spillover of the 501Y.V2 variants to mice. Moreover, the neutralization resistance we detected for the 501Y.V2 variants suggests the potential for compromised efficacy of monoclonal antibodies and vaccines., Experiments with pseudotyped viruses show that the 501Y.V2 variant of SARS-CoV-2 exhibits resistance to neutralization from monoclonal antibodies and sera from convalescent as well as immunized individuals, predominantly due to the E484K and N501Y mutations in the receptor-binding domain of the viral spike protein.

Lymphocyte activation gene-3 (LAG-3), an inhibitory molecule, which has been shown co-expressed with multiple inhibitory receptors on CD8

Objectives Regulatory cytokines are associated with viral infection. The objective of this study was to evaluate the relation between serum regulatory cytokines concentrations and respiratory syncytial virus (RSV) disease. Methods We enrolled 325 children aged

The antiviral effects of hepatitis C virus (HCV)-specific CD8 T cells have been shown in an HCV replicon system but not in an authentic infectious HCV cell culture (HCVcc) system. Here, we developed tools to examine the antigenicity of HCV-infected HLA-A2-positive Huh7.5 hepatoma cells (Huh7.5A2 cells) in activating HCV-specific CD8 T cells and the downstream antiviral effects. Infectious HCV epitope mutants encoding the well-defined genotype 1a-derived HLA-A2-restricted HCV NS3-1073 or NS5-2594 epitope were generated from a genotype 2a-derived HCV clone (Jc1Gluc2A) by site-directed mutagenesis. CD8 T-cell lines specific for NS3-1073 and NS5-2594 were expanded from HCV-seropositive persons by peptide stimulation in vitro or engineered from HCV-seronegative donor T cells by transduction of a lentiviral vector expressing HCV-specific T-cell receptors. HCV-specific CD8 T cells were cocultured with Huh7.5 cells that were pulsed with titrating doses of HCV epitope peptides or infected with HCV epitope mutants. HCV-specific CD8 T-cell activation (CD107a, gamma interferon, macrophage inflammatory protein 1β, tumor necrosis factor alpha) was dependent on the peptide concentrations and the relative percentages of HCV-infected Huh7.5A2 cells. HCV-infected Huh7.5A2 cells activated HCV-specific CD8 T cells at levels comparable to those achieved with 0.1 to 2 μM pulsed peptides, providing a novel estimate of the level at which endogenously processed HCV epitopes are presented on HCV-infected cells. While HCV-specific CD8 T-cell activation with cytolytic and antiviral effects was blunted by PD-L1 expression on HCV-infected Huh7.5A2 cells, resulting in the improved viability of Huh7.5A2 cells, PD-1 blockade reversed this effect, producing enhanced cytolytic elimination of HCV-infected Huh7.5A2 cells. Our findings, obtained using an infectious HCVcc system, show that the HCV-specific CD8 T-cell function is modulated by antigen expression levels, the percentage of HCV-infected cells, and the PD-1/PD-L1 pathways and has antiviral and cytotoxic effects. IMPORTANCE We developed several novel molecular and immunological tools to study the interactions among HCV, HCV-infected hepatocytes, and HCV-specific CD8 T cells. Using these tools, we show the level at which HCV-infected hepatoma cells present endogenously processed HCV epitopes to HCV-specific CD8 T cells with antiviral and cytotoxic effects. We also show the marked protective effect of PD-L1 expression on HCV-infected hepatoma cells against HCV-specific CD8 T cells.

Hepatitis B virus/hepatitis C virus (HBV/HCV) dual infection is common among high-risk individuals. To characterize the virological and immunological features of patients with HBV/HCV dual infection, we enrolled 1,049 individuals who have been identified as injection drug users. Patients were divided into single and dual infection groups according to the serological markers. We found the average HCV RNA level was significantly lower; however, HBV viral load was significantly higher in HBV/HCV dual-infected patients (n = 42) comparing HCV single infection (n = 340) or HBV single infection (n = 136). The level of anti-HBs in patients who experienced spontaneous HBV clearance was higher than that in HCV single-infected patients with HBV spontaneous clearance. The level of anti-HCV E2 in HBV/HCV dual infection was lower than that detected in HCV single infection. Serum levels of IL-6, IL-8, and TNF-α were significantly lower in HBV/HCV dual-infected patients than in patients infected with HBV or HCV alone. Taken together, two viral replications are imbalanced in dual infected patients. The anti-HBs and anti-HCV E2 antibody production were impaired and proinflammatory IL-6, IL-8, and TNF-α also downregulated due to dual infection. These findings will help further understanding the pathogenesis of HBV/HCV dual infection.

Hepatitis B, C, and D virus (HBV, HCV, and HDV) infections are known to be prevalent in injection drug users (IDUs); however, the relationship between the molecular epidemiologic features of hepatitis virus infection in high-risk individuals and the general population has not yet been established.In total, 1049 IDUs and 672 individuals who underwent physical examinations at Chenzhou hospital, Hunan Province, China, were enrolled. HBV, HCV, and HDV infections were screened with serologic tests in both populations. HBsAg-positive, anti-HCV IgG-positive, and anti-HDV IgG-positive samples were further confirmed by polymerase chain reaction, quantitative polymerase chain reaction, and DNA sequencing.Significantly higher HBV (21.54 vs 16.52%, P = 0.01), HCV (45.95% vs 1.34%, P 0.001), and HDV (5.62% vs 0.30%, P 0.001) infections were detected in IDUs compared with the general population. The dual infection of HBV/HCV or HBV/HDV was also significantly higher in IDUs than in the general population. HBV genotype B and HDV genotype II were dominants in both populations. HCV infection showed genotype 6a (49.52%) dominant in IDUs, but genotype 1b accounted for 50% infection, which was followed by genotype 6a (33.33%) in the general population. Higher viral loads were associated with HBV genotype B and HCV genotype 6a compared with non-dominant genotypic infections.HBV and HDV infections shared similar patterns by IDUs and the general populations, and HCV infection exhibited distinct features between two populations. Our results suggest different molecular epidemiologic characteristics of HBV, HCV, and HDV infection in two populations.

We recently described the discovery of oxygenated N-alkyl deoxynojirimycin (DNJ) derivative 7 (CM-10-18) with antiviral activity against dengue virus (DENV) infection both in vitro and in vivo. This imino sugar was promising but had an EC(50) against DENV in BHK cells of 6.5 μM, which limited its use in in vivo. Compound 7 presented structural opportunities for activity relationship analysis, which we exploited and report here. These structure-activity relationship studies led to analogues 2h, 2l, 3j, 3l, 3v, and 4b-4c with nanomolar antiviral activity (EC(50) = 0.3-0.5 μM) against DENV infection, while maintaining low cytotoxicity (CC(50) > 500 μM, SI > 1000). In male Sprague-Dawley rats, compound 3l was well tolerated at a dose up to 200 mg/kg and displayed desirable PK profiles, with significantly improved bioavailability (F = 92 ± 4%).

Cellular α-glucosidases I and II are enzymes that sequentially trim the three terminal glucoses in the N-linked oligosaccharides of viral envelope glycoproteins. This process is essential for the proper folding of viral glycoproteins and subsequent assembly of many enveloped viruses, including dengue virus (DENV). Imino sugars are substrate mimics of α-glucosidases I and II. In this report, we show that two oxygenated alkyl imino sugar derivatives, CM-9-78 and CM-10-18, are potent inhibitors of both α-glucosidases I and II in vitro and in treated animals, and efficiently inhibit DENV infection of cultured human cells. Pharmacokinetic studies reveal that both compounds are well tolerated at doses up to 100mg/kg in rats and have favorable pharmacokinetic properties and bioavailability in mice. Moreover, we showed that oral administration of either CM-9-78 or CM-10-18 reduces the peak viremia of DENV in mice. Interestingly, while treatment of DENV infected mice with ribavirin alone did not reduce the viremia, combination therapy of ribavirin with sub-effective dose of CM-10-18 demonstrated a significantly enhanced antiviral activity, as indicated by a profound reduction of the viremia. Our findings thus suggest that combination therapy of two broad-spectrum antiviral agents may provide a practically useful approach for the treatment of DENV infection.

α-Glucosidases I and II are endoplasmic reticulum-resident enzymes that are essential for N-linked glycan processing and subsequent proper folding of glycoproteins. In this report, we first demonstrate that downregulation of the expression of α-glucosidase I, II, or both in Huh7.5 cells by small hairpin RNA technology inhibited the production of hepatitis C virus (HCV). In agreement with the essential role of α-glucosidases in HCV envelope glycoprotein processing and folding, treatment of HCV-infected cells with a panel of imino sugar derivatives, which are competitive inhibitors of α-glucosidases, did not affect intracellular HCV RNA replication and nonstructural protein expression but resulted in the inhibition of glycan processing and subsequent degradation of HCV E2 glycoprotein. As a consequence, HCV virion assembly and secretion were inhibited. In searching for imino sugars with better antiviral activity, we found that a novel imino sugar, PBDNJ0804, had a superior ability to inhibit HCV virion assembly and secretion. In summary, we demonstrated that glucosidases are important host factor-based antiviral targets for HCV infection. The low likelihood of drug-resistant virus emergence and potent antiviral efficacy of the novel glucosidase inhibitor hold promise for its development as a therapeutic agent for the treatment of chronic hepatitis C.

Type III interferons (IFN), IFN-lambda or IL-28/29, are new members of the IFN super-family. Except for using distinct receptors, type I and type III IFNs share the same major post receptor signaling components to activate the transcription of a similar set of IFN-stimulated genes (ISGs). To examine the antiviral effects of the new type IFNs against West Nile virus (WNV), we compared the antiviral effects of IFN-alpha and IFN-lambda on WNV virus-like particle (VLP) infection and replicon replication in Huh7.5 and Hela cells. The results revealed that (i) both types of IFNs could efficiently prevent the WNV infection, but IFN-alpha demonstrated a stronger antiviral efficacy; (ii) WNV genome replication in VLP-infected cells and replicon-containing cell lines could only be inhibited by IFN-alpha, but not IFN-lambda; (iii) in agreement with the observed antiviral effects, only IFN-lambda-induced activation of JAK-STAT signaling pathway and induction of ISG expression were completely inhibited in WNV replicon-containing cell lines, but IFN-alpha signal transduction was either unaffected or only partially inhibited in Huh7.5 or Hela cells by the virus. Hence, the differential inhibition of WNV on IFN-alpha and IFN-lambda signal transduction implies that the receptors of the two types of IFNs, but not the common post receptor signaling components, could be selectively targeted either directly by WNV nonstructural proteins or indirectly by the cellular responses induced by the virus infection to inhibit the signal transduction of the cytokines.