Yunwen Hu

Human Infection with a Novel Avian-Origin Influenza A (H7N9) Virus

BACKGROUND Infection of poultry with influenza A subtype H7 viruses occurs worldwide, but the introduction of this subtype to humans in Asia has not been observed previously. In March 2013, three urban residents of Shanghai or Anhui, China, presented with rapidly progressing lower respiratory tract infections and were found to be infected with a novel reassortant avian-origin influenza A (H7N9) virus. METHODS We obtained and analyzed clinical, epidemiologic, and virologic data from these patients. Respiratory specimens were tested for influenza and other respiratory viruses by means of real-time reverse-transcriptase-polymerase-chain-reaction assays, viral culturing, and sequence analyses. RESULTS A novel reassortant avian-origin influenza A (H7N9) virus was isolated from respiratory specimens obtained from all three patients and was identified as H7N9. Sequencing analyses revealed that all the genes from these three viruses were of avian origin, with six internal genes from avian influenza A (H9N2) viruses. Substitution Q226L (H3 numbering) at the 210-loop in the hemagglutinin (HA) gene was found in the A/Anhui/1/2013 and A/Shanghai/2/2013 virus but not in the A/Shanghai/1/2013 virus. A T160A mutation was identified at the 150-loop in the HA gene of all three viruses. A deletion of five amino acids in the neuraminidase (NA) stalk region was found in all three viruses. All three patients presented with fever, cough, and dyspnea. Two of the patients had a history of recent exposure to poultry. Chest radiography revealed diffuse opacities and consolidation. Complications included acute respiratory distress syndrome and multiorgan failure. All three patients died. CONCLUSIONS Novel reassortant H7N9 viruses were associated with severe and fatal respiratory disease in three patients. (Funded by the National Basic Research Program of China and others.).

Association between adverse clinical outcome in human disease caused by novel influenza A H7N9 virus and sustained viral shedding and emergence of antiviral resistance

BACKGROUND On March 30, a novel influenza A subtype H7N9 virus (A/H7N9) was detected in patients with severe respiratory disease in eastern China. Virological factors associated with a poor clinical outcome for this virus remain unclear. We quantified the viral load and analysed antiviral resistance mutations in specimens from patients with A/H7N9. METHODS We studied 14 patients with A/H7N9 disease admitted to the Shanghai Public Health Clinical Centre (SPHCC), China, between April 4, and April 20, 2013, who were given antiviral treatment (oseltamivir or peramivir) for less than 2 days before admission. We investigated the viral load in throat, stool, serum, and urine specimens obtained sequentially from these patients. We also sequenced viral RNA from these specimens to study the mutations associated with resistance to neuraminidase inhibitors and their association with disease outcome. FINDINGS All patients developed pneumonia, seven of them required mechanical ventilation, and three of them further deteriorated to become dependent on extracorporeal membrane oxygenation (ECMO), two of whom died. Antiviral treatment was associated with a reduction of viral load in throat swab specimens in 11 surviving patients. Three patients with persistently high viral load in the throat in spite of antiviral therapy became ECMO dependent. An Arg292Lys mutation in the virus neuraminidase (NA) gene known to confer resistance to both zanamivir and oseltamivir was identified in two of these patients, both also received corticosteroid treatment. In one of them, wild-type sequence Arg292 was noted 2 days after start of antiviral treatment, and the resistant mutant Lys292 dominated 9 days after start of treatment. INTERPRETATION Reduction of viral load following antiviral treatment correlated with improved outcome. Emergence of NA Arg292Lys mutation in two patients who also received corticosteroid treatment led to treatment failure and a poor clinical outcome. The emergence of antiviral resistance in A/H7N9 viruses, especially in patients receiving corticosteroid therapy, is concerning, needs to be closely monitored, and considered in pandemic preparedness planning. FUNDING National Megaprojects of China for Infectious Diseases, Shanghai Municipal Health and Family Planning Commission, the National Key Basic Research Program of China, Ministry of Science and Technology, and National Natural Science Foundation of China.

HBsAg inhibits TLR9-mediated activation and IFN-α production in plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs), the professional producers of type I interferons (IFN-alpha/beta), play a pivotal role in innate and adaptive immune responses against viral infections. Although functional impairment of circulating pDCs in chronic hepatitis B (CHB) patients has been reported previously, the mechanism responsible for these defects remains unclear. We hypothesize that HBsAg circulating in high amounts during HBV infection may interact with pDC and contribute to pDC dysfunction. In support of this hypothesis we show that pDCs treated with HBsAg secreted much less IFN-alpha than control pDCs. Furthermore, suppression is specific for TLR9, with no effects upon TLR7-mediated IFN-alpha secretion. HBsAg inhibited TLR9-mediated IRF-7 expression and nuclear translocation, which are important for induction of IFN-alpha gene transcription. HBsAg upregulated the SOCS-1 expression and bound to BDCA-2 receptors on the plasma membrane of pDCs, resulting in the inhibition of the IFN-alpha production. In conclusion, the above data suggested that HBsAg may directly interfere with the function of pDC through HBsAg-mediated upregulation of SOCS-1 expression and BDCA-2 ligation, which could partially explain how HBV evades the immune system to establish a persistent infection.

Expression profiles and function of Toll-like receptors 2 and 4 in peripheral blood mononuclear cells of chronic hepatitis B patients.

Toll-like receptors (TLRs) play a central role in sensing and initiating innate antiviral response. In this study, we first investigated the expression of TLR1-10 mRNA transcripts in peripheral blood mononuclear cells (PBMCs) from chronic HBV-infected (CHB) patients and healthy donors by quantitative real-time PCR. The expression of TLR1, TLR2, TLR4 and TLR6 transcripts was significantly lower in PBMCs from CHB patients, and the down-regulation of TLR2 was related to HBV genotype C. Flow cytometric analysis showed that the expression of TLR2 on PBMCs was significantly decreased in CHB patients. Furthermore, impaired cytokine production was observed in PBMCs from CHB patients after challenged with TLR2 and TLR4 ligands and was correlated with the levels of plasma hepatitis B virus surface antigen (HBsAg). In conclusion, our study reveals a possible interaction between HBsAg, TLR signaling and the innate immune response, which may partially explain the mechanism of HBV infection induced immuno-tolerance.

Recovery from severe H7N9 disease is associated with diverse response mechanisms dominated by CD8+ T cells

The avian origin A/H7N9 influenza virus causes high admission rates (>99%) and mortality (>30%), with ultimately favourable outcomes ranging from rapid recovery to prolonged hospitalization. Using a multicolour assay for monitoring adaptive and innate immunity, here we dissect the kinetic emergence of different effector mechanisms across the spectrum of H7N9 disease and recovery. We find that a diversity of response mechanisms contribute to resolution and survival. Patients discharged within 2–3 weeks have early prominent H7N9-specific CD8+ T-cell responses, while individuals with prolonged hospital stays have late recruitment of CD8+/CD4+ T cells and antibodies simultaneously (recovery by week 4), augmented even later by prominent NK cell responses (recovery >30 days). In contrast, those who succumbed have minimal influenza-specific immunity and little evidence of T-cell activation. Our study illustrates the importance of robust CD8+ T-cell memory for protection against severe influenza disease caused by newly emerging influenza A viruses.

Hepatitis B Virus Surface Antigen Selectively Inhibits TLR2 Ligand–Induced IL-12 Production in Monocytes/Macrophages by Interfering with JNK Activation

It is widely accepted that chronic hepatitis B virus (HBV) infection is the result of an ineffective antiviral immune response against HBV infection. Our previous study found that the hepatitis B surface Ag (HBsAg) was related to decreased cytokine production induced by the TLR2 ligand (Pam3csk4) in PBMCs from chronic hepatitis B patients. In this study, we further explored the mechanism involved in the inhibitory effect of HBsAg on the TLR2 signaling pathway. The results showed that both Pam3csk4-triggered IL-12p40 mRNA expression and IL-12 production in PMA-differentiated THP-1 macrophage were inhibited by HBsAg in a dose-dependent manner, but the production of IL-1β, IL-6, IL-8, IL-10, and TNF-α was not influenced. The Pam3csk4-induced activation of NF-κB and MAPK signaling were further examined. The phosphorylation of JNK-1/2 and c-Jun was impaired in the presence of HBsAg, whereas the degradation of IκB-α, the nuclear translocation of p65, and the phosphorylation of p38 and ERK-1/2 were not affected. Moreover, the inhibition of JNK phosphorylation and IL-12 production in response to Pam3csk was observed in HBsAg-treated monocytes/macrophages (M/MΦs) from the healthy donors and the PBMCs and CD14-positive M/MΦs from chronic hepatitis B patients. Taken together, these results demonstrate that HBsAg selectively inhibits Pam3csk4- stimulated IL-12 production in M/MΦs by blocking the JNK–MAPK pathway and provide a mechanism by which HBV evades immunity and maintains its persistence.

Hepatitis B virus polymerase impairs interferon-α-induced STA T activation through inhibition of importin-α5 and protein kinase C-δ.

Treatment with exogenous interferon (IFN)‐α is not effective in the majority of patients with chronic hepatitis B virus (HBV) infection. Recent evidence suggests that HBV has evolved strategies to block the nuclear translocation of signal transducer and activator of transcription (STAT) 1 to limit IFN‐α–induced cellular antiviral responses. However, it remains unclear whether STAT1 translocation is impaired in chronic hepatitis B patients and what mechanisms are involved. Here we report that the expression of HBV polymerase (Pol) in human hepatic cell lines inhibited induction of IFN‐stimulated genes and resulted in a weakened antiviral activity of IFN‐α. Ectopic expression of Pol suppressed IFN‐α–induced STAT1 serine 727 phosphorylation and STAT1/2 nuclear accumulation, whereas STAT1 tyrosine 701 phosphorylation, and STAT1‐STAT2 heterodimer formation were not affected. Further studies demonstrated that Pol interacted with the catalytic domain of protein kinase C‐δ (PKC‐δ) and perturbed PKC‐δ phosphorylation and its association with STAT1, which resulted in the suppression of STAT1 Ser727 phosphorylation. Moreover, Pol was found to interfere with nuclear transportation of STAT1/2 by competitively binding to the region of importin‐α5 required for STAT1/2 recruitment. Truncation analysis suggested that the terminal protein and RNase H domains of Pol were able to bind to PKC‐δ and importin‐α5, respectively, and were responsible for the inhibition of IFN‐α signaling. More importantly, the inhibition of STAT1 and PKC‐δ phosphorylation were confirmed in a hydrodynamic‐based HBV mouse model, and the blockage of IFN‐α–induced STAT1/2 nuclear translocation was observed in HBV‐infected cells from liver biopsies of chronic HBV patients. Conclusions: These results demonstrate a role for Pol in HBV‐mediated antagonization of IFN‐α signaling and provide a possible molecular mechanism by which HBV resists the IFN therapy and maintains its persistence. (HEPATOLOGY 2013;)

Hepatitis C virus non-structural protein NS5A interacts with FKBP38 and inhibits apoptosis in Huh7 hepatoma cells

Hepatitis C virus non‐structural protein NS5A plays an important role in viral replication and various cellular events. To gain further insight into the function of NS5A, we screened a human fetal liver cDNA library for its interacting proteins using the yeast two‐hybrid system. FKBP38, a 38 kDa immunosuppressant FK506‐binding protein, was identified and its interaction with NS5A was confirmed by both in vitro and in vivo. The interaction was mapped to the amino acids 148–236 of NS5A containing a BH domain (Bcl‐2 homology domain). Besides, both NS5A and FKBP38 were found to localize in mitochondria and endoplasmic reticulum. Moreover, NS5A stably expressing Huh7 hepatoma cells showed more resistance to apoptosis and such inhibition of apoptosis could specifically be abrogated by depletion of FKBP38 using RNA interference. These results indicate that HCV NS5A inhibits apoptosis through interaction with FKBP38.

HBsAg inhibits IFN-α production in plasmacytoid dendritic cells through TNF-α and IL-10 induction in monocytes.

Type I Interferon (IFN) is one of the first lines of defense against viral infection. Plasmacytoid dendritic cells (pDCs) are professional IFN-α-producing cells that play an important role in the antiviral immune response. Previous studies have reported that IFN-α production is impaired in chronic hepatitis B (CHB) patients. However, the mechanisms underlying the impairment in IFN-α production are not fully understood. Here, we report that plasma-derived hepatitis B surface antigen (HBsAg) and HBsAg expressed in CHO cells can significantly inhibit toll like receptor (TLR) 9-mediated Interferon-α (IFN-α) production in peripheral blood mononuclear cells (PBMCs) from healthy donors. Further analysis indicated that monocytes participate in the inhibitory effect of HBsAg on pDCs through the secretion of TNF-α and IL-10. Furthermore, TLR9 expression on pDCs was down-regulated by TNF-α, IL-10 and HBsAg treatment. This down-regulation may partially explain the inhibition of IFN-α production in pDCs. In conclusion, we determined that HBsAg inhibited the production of IFN-α by pDCs through the induction of monocytes that secreted TNF-α and IL-10 and through the down-regulation of TLR9 expression on pDCs. These data may aid in the development of effective antiviral treatments and lead to the immune control of the viral infections.

Rupintrivir is a promising candidate for treating severe cases of enterovirus-71 infection: Evaluation of antiviral efficacy in a murine infection model

Enterovirus-71 (EV71) infections can cause life-threatening diseases with neurological symptoms. Currently, no direct targeting antivirals are available to combat severe EV71 infection. Rupintrivir (AG7088) is a compound originally designed for Rhinovirus 3C protease. Previous computational analyses by us and crystallography studies by others suggested that rupintrivir is also a high affinity inhibitor to EV71 3C. Thus, we aimed to further evaluate its anti-EV71 activity in vivo at clinically acceptable doses. It was observed that administration of rupintrivir in suckling mice largely protected them from limb paralysis and dramatically improved survival (38.5% DMSO vs. 90.9% at 0.1mg/kg, p=0.006). Histological, immunohistochemical and quantitative RT-PCR analyses confirmed that rupintrivir profoundly alleviated virus induced necrotizing myositis, suppressed viral RNA and blocked EV71 VP1 expression in various tissues. In conclusion, we established that rupintrivir can strongly contain the spread of EV71 infection in vivo at a clinically acceptable dose (as low as 0.1mg/kg). As its safety has been fully tested in previous clinical trials, rupintrivir is suitable for immediate evaluation of potential benefits in EV71-infected individuals with life-threatening neurological symptoms.