Tafenoquine and its derivatives as inhibitors for the severe acute respiratory syndrome coronavirus 2.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has severely affected human lives around the world as well as the global economy. Therefore, effective treatments against COVID-19 are urgently needed. Here, we screened a library containing Food and Drug Administration (FDA)-approved compounds to identify drugs that could target the SARS-CoV-2 main protease (Mpro), which is indispensable for viral protein maturation and regard as an important therapeutic target. We identified antimalarial drug tafenoquine (TFQ), which is approved for radical cure of Plasmodium vivax and malaria prophylaxis, as a top candidate to inhibit Mpro protease activity. The crystal structure of SARS-CoV-2 Mpro in complex with TFQ revealed that TFQ noncovalently bound to and reshaped the substrate-binding pocket of Mpro by altering the loop region (residues 139-144) near the catalytic Cys145, which could block the catalysis of its peptide substrates. We also found that TFQ inhibited human transmembrane protease serine 2 (TMPRSS2). Furthermore, one TFQ derivative, compound 7, showed a better therapeutic index than TFQ on TMPRSS2 and may therefore inhibit the infectibility of SARS-CoV-2, including that of several mutant variants. These results suggest new potential strategies to block infection of SARS-CoV-2 and rising variants.

TLR-induced PAI-2 expression suppresses IL-1ß processing via increasing autophagy and NLRP3 degradation.

The NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome, a multiprotein complex, triggers caspase-1 activation and maturation of the proinflammatory cytokines IL-1ß and IL-18 upon sensing a wide range of pathogen- and damage-associated molecules. Dysregulation of NLRP3 inflammasome activity contributes to the pathogenesis of many diseases, but its regulation remains poorly defined. Here we show that depletion of plasminogen activator inhibitor type 2 (PAI-2), a serine protease inhibitor, resulted in NLRP3- and ASC (apoptosis-associated Speck-like protein containing a C-terminal caspase recruitment domain)-dependent caspase-1 activation and IL-1ß secretion in macrophages upon Toll-like receptor 2 (TLR2) and TLR4 engagement. TLR2 or TLR4 agonist induced PAI-2 expression, which subsequently stabilized the autophagic protein Beclin 1 to promote autophagy, resulting in decreases in mitochondrial reactive oxygen species, NLRP3 protein level, and pro-IL-1ß processing. Likewise, overexpressing Beclin 1 in PAI-2-deficient cells rescued the suppression of NLRP3 activation in response to LPS. Together, our data identify a tier of TLR signaling in controlling NLRP3 inflammasome activation and reveal a cell-autonomous mechanism which inversely regulates TLR- or Escherichia coli-induced mitochondrial dysfunction, oxidative stress, and IL-1ß-driven inflammation.

Interference of DNAJB6/MRJ Isoform Switch by Morpholino Inhibits Replication of HIV-1 and RSV.

The molecular chaperon MRJ (DNAJB6) exhibits two splice isoforms that have different roles in human viral infection, but the regulatory mechanism of MRJ isoform expression is yet unclear. In this study, we show that reduction of the polyadenylation factor CstF64 was correlated with the increase of the MRJ large isoform (MRJ-L) in human macrophages and elucidate the mechanism underlying CstF64-modulated MRJ isoform expression. Moreover, we exploited an antisense strategy targeting MRJ-L for virus replication. A morpholino oligonucleotide complementary to the 5' splice site of MRJ intron 8 downregulated MRJ-L expression and suppressed the replication of not only HIV-1 but also respiratory syncytial virus (RSV). We demonstrated that downregulation of the MRJ-L level reduced HIV-1 replication as well as the subgenomic mRNA and viral production of RSV. The present findings that two human health-threatening viruses take advantage of MRJ-L for infection suggest MRJ-L as a potential target for broad-spectrum antiviral strategy.

Detection of human metapneumovirus in hospitalized children with acute respiratory tract infection using real-time RT-PCR in a hospital in northern Taiwan.

BACKGROUND/PURPOSE: Human metapneumovirus (hMPV) is a newly discovered respiratory pathogen. This prospective hospital-based study investigated the clinical role and features of hMPV in Taiwan. METHODS: Respiratory specimens collected from hospitalized children with acute respiratory tract infection between September 1, 2003 and April 10, 2005 were screened for metapneumovirus using real-time reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: During the study period, 930 specimens were obtained from 926 hospitalized children. After exclusion of 200 cases due to lack of clinical evidence of airway infection or diseases with known etiology, 726 were included in the analysis. Among these, 33 children had a positive result for hMPV infection. The majority of these patients were admitted during spring and early summer. Twenty-one (63.6%) were younger than 2 years of age. hMPV accounted for 13.3% of respiratory infections occurring between the ages of 18 and 24 months and was as common a respiratory pathogen as respiratory syncytial virus (RSV) in that age group. The 11 patients (33.3%) with underlying diseases had a similar disease course to those without underlying diseases. A co-pathogen was found in 11 patients (33.3%). Infected children between 2 and 5 years of age had significantly higher titers of hMPV in their respiratory specimens (103.88 copies/microL) than children younger than 2 years (102.26 copies/microL) (p = 0.013) and children older than 5 years (102.25 copies/microL) (p = 0.005). hMPV positive cases were significantly older than those with RSV infection (p = 0.002) and had a shorter duration of hospitalization (p = 0.001), fewer days of oxygen use (p = 0.001) and higher levels of C-reactive protein (p = 0.004). CONCLUSION: Metapneumovirus circulates in children in northern Taiwan during spring and early summer. hMPV was the most common respiratory pathogen in children aged between 18 and 24 months hospitalized with acute respiratory tract infection. Real-time RT-PCR is a sensitive method for investigating the epidemiology and diseases associated with hMPV.

Large Isoform of Mammalian Relative of DnaJ is a Major Determinant of Human Susceptibility to HIV-1 Infection.

Individual differences in susceptibility to human immunodeficiency virus type 1 (HIV-1) infection have been of interest for decades. We aimed to determine the contribution of large isoform of Mammalian DnaJ (MRJ-L), a HIV-1 Vpr-interacting cellular protein, to this natural variation. Expression of MRJ-L in monocyte-derived macrophages was significantly higher in HIV-infected individuals (n = 31) than their uninfected counterparts (n = 27) (p = 0.009). Fifty male homosexual subjects (20 of them are HIV-1 positive) were further recruited to examine the association between MRJ-L levels and occurrence of HIV infection. Bayesian multiple logistic regression revealed that playing a receptive role and increased levels of MRJ-L in macrophages were two risk factors for HIV-1 infection. A 1% rise in MRJ-L expression was associated with a 1.13 fold (95% CrI 1.06-1.29) increase in odds of contracting HIV-1 infection. Ex vivo experiments revealed that MRJ-L facilitated Vpr-dependent nuclear localization of virus. Infection of macrophage-tropic strain is a critical step in HIV-1 transmission. MRJ-L is a critical factor in this process; hence, subjects with higher macrophage MRJ-L levels are more vulnerable to HIV-1 infection.

Status of cellular rather than humoral immunity is correlated with clinical outcome of enterovirus 71.

We valuated specific cellular and humoral immune response of cases of enterovirus 71 (EV71) infection and correlated immune response with clinical outcome. After obtaining informed consent, we enrolled 30 EV71 cases including 7 cases with brainstem encephalitis plus pulmonary edema, 12 cases of CNS (CNS) involvement and 11 uncomplicated cases. We measured antibodies specific to EV71, lymphocyte proliferation response and EV71-stimulated cellular response of Th1/Th2 cytokines and chemokines. The 7 EV71 cases involving brainstem encephalitis plus pulmonary edema had a significantly lower phytohemagglutinin stimulation index than other cases (p = 0.04). After EV71 stimulation of peripheral mononuclear cells, there was a significant increase in cellular Th1 cytokine (gamma-interferon) and proinflammatroy cytokines. However, cases with pulmonary edema had significantly lower cellular gamma-interferon (p = 0.04), lower cellular IL-1beta (p = 0.04), lower cellular IL-6 (p = 0.04), lower cellular tumor necrosis factor-alpha response (p = 0.04), and lower cellular macrophage inflammatory protein-1alpha (p = 0.04) response compared with other cases. Their titers of EV71 neutralizing antibodies demonstrated no difference among cases. These results suggest lower EV71-specific cellular response may be associated with immunopathogenesis of EV71-related pulmonary edema.

Development of immunoglobulin G enzyme-linked immunosorbent assay for the serodiagnosis of severe acute respiratory syndrome.

Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel SARS-associated coronavirus (SARS-CoV). The clinical characteristics are high fever, rapidly progressive diffuse pneumonitis and respiratory distress. It is highly infectious through intimate contact or direct contact with infectious body fluids. Outbreaks within communities and hospitals have been reported. Development of rapid and reliable diagnostic tools is urgently needed. We developed an immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA), using whole virus antigen of SARS-CoV. Eighty-six serum samples collected from patients who were hospitalized for other causes were examined to determine the cut-off O.D. value. The cut-off O.D. value was defined as 0.175 by calculating the mean O.D. value of the 86 sera plus 3 standard deviations. To determine the sensitivity and specificity of the ELISA, 56 positive sera and 204 negative sera were tested. The sensitivity was 96.4% and the specificity was 100%. The results suggest that the IgG ELISA using whole virus antigen of SARS-CoV has a high sensitivity and specificity in detecting SARS IgG antibodies. This IgG ELISA is a powerful tool for serodiagnosis of SARS.

Immunofluorescence assay for detection of the nucleocapsid antigen of the severe acute respiratory syndrome (SARS)-associated coronavirus in cells derived from throat wash samples of patients with SARS.

An antigen detection assay for severe acute respiratory syndrome (SARS) coronavirus was established in this study by an indirect immunofluorescence test, which utilized cells derived from throat wash samples of patients with SARS and a rabbit serum that recognized the nucleocapsid protein of SARS-associated coronavirus (SARS-CoV) but not that of other human coronavirus tested. It detected SARS-CoV in 11 of 17 (65%) samples from SARS patients as early as day 2 of illness but in none of the 10 samples from healthy controls. Compared with other diagnostic modalities for detecting SARS-CoV, this assay is simpler, more convenient, and economical. It could be an alternative for early and rapid diagnosis, should SARS return in the future.

Human immunodeficiency virus type 1 Vpr interacts with antiapoptotic mitochondrial protein HAX-1.

Human immunodeficiency virus type 1 viral protein R (Vpr) is required for viral pathogenesis and has been implicated in T-cell apoptosis through its activation of caspase 3 and caspase 9 and perturbation of mitochondrial membrane potential. To understand better Vpr-mitochondria interaction, we report here the identification of antiapoptotic mitochondrial protein HAX-1 as a novel Vpr target. We show that Vpr and HAX-1 physically associate with each other. Overexpression of Vpr in cells dislocates HAX-1 from its normal residence in mitochondria and creates mitochondrion instability and cell death. Conversely, overexpression of HAX-1 suppressed the proapoptotic activity of Vpr.

Detection of SARS-associated coronavirus in throat wash and saliva in early diagnosis.

The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is thought to be transmitted primarily through dispersal of droplets, but little is known about the load of SARS-CoV in oral droplets. We examined oral specimens, including throat wash and saliva, and found large amounts of SARS-CoV RNA in both throat wash (9.58 x 10(2) to 5.93 x 10(6) copies/mL) and saliva (7.08 x 10(3) to 6.38 x 10(8) copies/mL) from all specimens of 17 consecutive probable SARS case-patients, supporting the possibility of transmission through oral droplets. Immunofluorescence study showed replication of SARS-CoV in the cells derived from throat wash, demonstrating the possibility of developing a convenient antigen detection assay. This finding, with the high detection rate a median of 4 days after disease onset and before the development of lung lesions in four patients, suggests that throat wash and saliva should be included in sample collection guidelines for SARS diagnosis.