Iris H. Y. Ng

Cytokine Responses in Severe Acute Respiratory Syndrome Coronavirus-Infected Macrophages In Vitro: Possible Relevance to Pathogenesis

ABSTRACT The pathogenesis of severe acute respiratory syndrome (SARS) remains unclear. Macrophages are key sentinel cells in the respiratory system, and it is therefore relevant to compare the responses of human macrophages to infections with the SARS coronavirus (SARS-CoV) and other respiratory viruses. Primary human monocyte-derived macrophages were infected with SARS-CoV in vitro. Virus replication was monitored by measuring the levels of positive- and negative-strand RNA, by immunofluorescence detection of the SARS-CoV nucleoprotein, and by titration of the infectious virus. The gene expression profiles of macrophages infected with SARS-CoV, human coronavirus 229E, and influenza A (H1N1) virus were compared by using microarrays and real-time quantitative reverse transcriptase PCR. Secreted cytokines were measured with an enzyme-linked immunosorbent assay. SARS-CoV initiated viral gene transcription and protein synthesis in macrophages, but replication was abortive and no infectious virus was produced. In contrast to the case with human coronavirus 229E and influenza A virus, there was little or no induction of beta interferon (IFN-β) in SARS-CoV-infected macrophages. Furthermore, SARS-CoV induced the expression of chemokines such as CXCL10/IFN-γ-inducible protein 10 and CCL2/monocyte chemotactic protein 1. The poor induction of IFN-β, a key component of innate immunity, and the ability of the virus to induce chemokines could explain aspects of the pathogenesis of SARS.

Functional Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Production by Avian Influenza Virus–Infected Macrophages

Abstract Severe human disease associated with influenza A H5N1 virus was first detected in Hong Kong in 1997. Its recent reemergence in Asia and high associated mortality highlight the need to understand its pathogenesis. We investigated the roles of death receptor ligands (DRLs) in H5N1 infection. Significant up-regulation of tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) and TNF-α, but not Fas ligand (FasL) mRNA, was detected in human monocyte–derived macrophages (MDMs) infected with avian influenza viruses A/Hong Kong/483/97 (H5N1/97) or its precursor, A/Quail/Hong Kong/G1/97. H5N1/97-infected MDMs exhibited the strongest induction of apoptosis in Jurkat T cells, and it could be reduced by TRAIL–receptor 2 blocking antibody. Furthermore, influenza virus infection enhanced the sensitivity of Jurkat T cells to apoptosis induced by TNF-α, TRAIL, and FasL. Our data suggested that functional TRAIL produced by influenza virus–infected MDMs was related to their cytotoxicity and that the enhanced sensitization to DRL-induced apoptosis detected in avian influenza may contribute to disease pathogenesis

Differential Expression of Chemokines and Their Receptors in Adult and Neonatal Macrophages Infected with Human or Avian Influenza Viruses

Abstract In 1997, avian influenza virus H5N1 was transmitted directly from chicken to human and resulted in a severe disease that had a higher mortality rate in adults than in children. The characteristic mononuclear leukocyte infiltration in the lung and the high inflammatory response in H5N1 infection prompted us to compare the chemokine responses between influenza virus–infected adult and neonatal monocyte-derived macrophages (MDMs). The effects of avian influenza virus A/Hong Kong/483/97 (H5N1) (H5N1/97), its precursor A/Quail/Hong Kong/G1/97 (H9N2) (H9N2/G1), and human influenza virus A/Hong Kong/54/98 (H1N1) (H1N1/98) were compared. Significantly higher expression of CCL2, CCL3, CCL5, and CXCL10 was induced by avian influenza viruses than by human influenza virus. Moreover, the increase in CCL3 expression in H5N1/97-infected adult MDMs was significantly higher than that in neonatal MDMs. Enhanced expression of CCR1 and CCR5 was found in avian virus–infected adult MDMs. The strong induction of chemokines and their receptors by avian influenza viruses, particularly in adult MDMs, may account for the severity of H5N1 disease

Detection of SARS Coronavirus in Patients with Severe Acute Respiratory Syndrome by Conventional and Real-Time Quantitative Reverse Transcription-PCR Assays

Abstract Background: A novel coronavirus (CoV) was recently identified as the agent for severe acute respiratory syndrome (SARS). We compared the abilities of conventional and real-time reverse transcription-PCR (RT-PCR) assays to detect SARS CoV in clinical specimens. Methods: RNA samples isolated from nasopharyngeal aspirate (NPA; n = 170) and stool (n = 44) were reverse-transcribed and tested by our in-house conventional RT-PCR assay. We selected 98 NPA and 37 stool samples collected at different times after the onset of disease and tested them in a real-time quantitative RT-PCR specific for the open reading frame (ORF) 1b region of SARS CoV. Detection rates for the conventional and real-time quantitative RT-PCR assays were compared. To investigate the nature of viral RNA molecules in these clinical samples, we determined copy numbers of ORF 1b and nucleocapsid (N) gene sequences of SARS CoV. Results: The quantitative real-time RT-PCR assay was more sensitive than the conventional RT-PCR assay for detecting SARS CoV in samples collected early in the course of the disease. Real-time assays targeted at the ORF 1b region and the N gene revealed that copy numbers of ORF 1b and N gene sequences in clinical samples were similar. Conclusions: NPA and stool samples can be used for early diagnosis of SARS. The real-time quantitative RT-PCR assay for SARS CoV is potentially useful for early detection of SARS CoV. Our results suggest that genomic RNA is the predominant viral RNA species in clinical samples.

Resistance to Neuraminidase Inhibitors Conferred by an R292K Mutation in a Human Influenza Virus H7N9 Isolate Can Be Masked by a Mixed R/K Viral Population

ABSTRACT We characterized the A/Shanghai/1/2013 virus isolated from the first confirmed human case of A/H7N9 disease in China. The A/Shanghai/1/2013 isolate contained a mixed population of R (65%; 15/23 clones) and K (35%; 8/23 clones) at neuraminidase (NA) residue 292, as determined by clonal sequencing. A/Shanghai/1/2013 with mixed R/K at residue 292 exhibited a phenotype that is sensitive to zanamivir and oseltamivir carboxylate by the enzyme-based NA inhibition assay. The plaque-purified A/Shanghai/1/2013 with dominant K292 (94%; 15/16 clones) showed sensitivity to zanamivir that had decreased by >30-fold and to oseltamivir carboxylate that had decreased by >100-fold compared to its plaque-purified wild-type counterpart possessing dominant R292 (93%, 14/15 clones). In Madin-Darby canine kidney (MDCK) cells, the plaque-purified A/Shanghai/1/2013-NAK292 virus exhibited no reduction in viral titer under conditions of increasing concentrations of oseltamivir carboxylate (range, 0 to 1,000 µM) whereas the replication of the plaque-purified A/Shanghai/1/2013-NAR292 and the A/Shanghai/2/2013 viruses was completely inhibited at 250 µM and 31.25 µM of oseltamivir carboxylate, respectively. Although the plaque-purified A/Shanghai/1/2013-NAK292 virus exhibited lower NA enzyme activity and a higher Km for 2′-(4-methylumbelliferryl)-α-d-N-acetylneuraminic acid than the wild-type A/Shanghai/1/2013-NAR292 virus, the A/Shanghai/1/2013-NAK292 virus formed large plaques and replicated efficiently in vitro. Our results confirmed that the NA R292K mutation confers resistance to oseltamivir, peramivir, and zanamivir in the novel human H7N9 viruses. Importantly, detection of the resistance phenotype may be masked in the clinical samples containing a mixed population of R/K at NA residue 292 in the enzyme-based NA inhibition assay. IMPORTANCE The neuraminidase (NA) inhibitors oseltamivir and zanamivir are currently the front-line therapeutic options against the novel H7N9 influenza viruses, which possess an S31N mutation that confers resistance to the M2 ion channel blockers. It is therefore important to evaluate the sensitivity of the clinical isolates to NA inhibitors and to monitor for the emergence of resistant variants. We characterized the A/Shanghai/1/2013 (H7N9) isolate which contained a mixed population of R/K at NA residue 292. While the clinical isolate exhibited a phenotype of sensitivity to NA inhibitors using the enzyme-based NA inhibition assay, the plaque-purified A/Shanghai/1/2013 virus with dominant K292 was resistant to zanamivir, peramivir, and oseltamivir. Resistance to NA inhibitors conferred by the R292K mutation in a human influenza virus H7N9 isolate can be masked by a mixed R/K viral population, and this should be taken into consideration while monitoring antiviral resistance in patients with H7N9 infection. The neuraminidase (NA) inhibitors oseltamivir and zanamivir are currently the front-line therapeutic options against the novel H7N9 influenza viruses, which possess an S31N mutation that confers resistance to the M2 ion channel blockers. It is therefore important to evaluate the sensitivity of the clinical isolates to NA inhibitors and to monitor for the emergence of resistant variants. We characterized the A/Shanghai/1/2013 (H7N9) isolate which contained a mixed population of R/K at NA residue 292. While the clinical isolate exhibited a phenotype of sensitivity to NA inhibitors using the enzyme-based NA inhibition assay, the plaque-purified A/Shanghai/1/2013 virus with dominant K292 was resistant to zanamivir, peramivir, and oseltamivir. Resistance to NA inhibitors conferred by the R292K mutation in a human influenza virus H7N9 isolate can be masked by a mixed R/K viral population, and this should be taken into consideration while monitoring antiviral resistance in patients with H7N9 infection.

The aminobisphosphonate pamidronate controls influenza pathogenesis by expanding a γδ T cell population in humanized mice

There are few antiviral drugs for treating influenza, and the emergence of antiviral resistance has further limited the available therapeutic options. Furthermore, antivirals are not invariably effective in severe influenza, such as that caused by H5N1 viruses. Thus, there is an urgent need to develop alternative therapeutic strategies. Here, we show that human Vγ9Vδ2 T cells expanded by the aminobisphosphonate pamidronate (PAM) kill influenza virus-infected cells and inhibit viral replication in vitro. In Rag2(-/-)γc(-/-) immunodeficient mice reconstituted with human peripheral mononuclear cells (huPBMCs), PAM reduces disease severity and mortality caused by human seasonal H1N1 and avian H5N1 influenza virus, and controls the lung inflammation and viral replication. PAM has no such effects in influenza virus-infected Rag2(-/-)γc(-/-) mice reconstituted with Vγ9Vδ2 T cell-depleted huPBMCs. Our study provides proof-of-concept of a novel therapeutic strategy for treating influenza by targeting the host rather than the virus, thereby reducing the opportunity for the emergence of drug-resistant viruses. As PAM has been commonly used to treat osteoporosis and Pagets disease, this new application of an old drug potentially offers a safe and readily available option for treating influenza.

Type 1 Responses of Human Vγ9Vδ2 T Cells to Influenza A Viruses

ABSTRACT γδ T cells are essential constituents of antimicrobial and antitumor defenses. We have recently reported that phosphoantigen isopentenyl pyrophosphate (IPP)-expanded human Vγ9Vδ2 T cells participated in anti-influenza virus immunity by efficiently killing both human and avian influenza virus-infected monocyte-derived macrophages (MDMs) in vitro. However, little is known about the noncytolytic responses and trafficking program of γδ T cells to influenza virus. In this study, we found that Vγ9Vδ2 T cells expressed both type 1 cytokines and chemokine receptors during influenza virus infection, and IPP-expanded cells had a higher capacity to produce gamma interferon (IFN-γ). Besides their potent cytolytic activity against pandemic H1N1 virus-infected cells, IPP-activated γδ T cells also had noncytolytic inhibitory effects on seasonal and pandemic H1N1 viruses via IFN-γ but had no such effects on avian H5N1 or H9N2 virus. Avian H5N1 and H9N2 viruses induced significantly higher CCL3, CCL4, and CCL5 production in Vγ9Vδ2 T cells than human seasonal H1N1 virus. CCR5 mediated the migration of Vγ9Vδ2 T cells toward influenza virus-infected cells. Our findings suggest a novel therapeutic strategy of using phosphoantigens to boost the antiviral activities of human Vγ9Vδ2 T cells against influenza virus infection.

The R292K Mutation That Confers Resistance to Neuraminidase Inhibitors Leads to Competitive Fitness Loss of A/Shanghai/1/2013 (H7N9) Influenza Virus in Ferrets

BACKGROUND Neuraminidase (NA) inhibitors are the only licensed therapeutic option for human zoonotic H7N9 infections. An NA-R292K mutation that confers broad-spectrum resistance to NA inhibitors has been documented in H7N9 patients after treatment. METHODS We evaluated the transmission potential of a human influenza A H7N9 isolate with a NA-R292K mutation in the ferret model followed by genotyping assay to monitor its competitive fitness in vivo. RESULTS Plaque-purified A/Shanghai/1/2013 wild-type and NA-R292K viruses transmitted at comparable efficiency to direct or respiratory droplet contact ferrets. In ferrets inoculated with the plaque-purified A/Shanghai/1/2013 NA-R292K virus with dominant K292 (94%), the resistant K292 genotype was outgrown by the wild-type R292 genotype during the course of infection. Transmission of the resistant K292 genotype was detected in 3/4 direct contact and 3/4 respiratory droplet contact ferrets at early time points but was gradually replaced by the wild-type genotype. In the respiratory tissues of inoculated or infected ferrets, the wild-type R292 genotype dominated in the nasal turbinate, whereas the resistant K292 genotype was more frequently detected in the lungs. CONCLUSIONS The NA inhibitor-resistant H7N9 virus with the NA-R292K mutation may transmit among ferrets but showed compromised fitness in vivo while in competition with the wild-type virus.

Evaluation of the ClearVoice Strategy in Adults Using HiResolution Fidelity 120 Sound Processing

Objectives This study aimed to evaluate the benefits of ClearVoice strategy on speech perception in noise and in everyday listening situations in Cantonese-speaking cochlear implant users. Methods Twelve experienced adult users of the Harmony implant and HiRes 120 sound processing participated in the study. The study employed a prospective within-subjects design wherein speech recognition in adults using HiRes 120 without ClearVoice turned on (control option) was compared to their performance with HiRes 120 with ClearVoice turned on. Each subject was evaluated with two different ClearVoice gain settings: -12 dB (ClearVoice medium) and -18 dB (ClearVoice high) after one-week of use. The Cantonese hearing in noise test and a questionnaire were used as the outcome measures. Results Subjects performed significantly better with ClearVoice medium than with control option in noise. No significant difference in performance was noted among the 3 settings in quiet. Most subjects reported high level of satisfaction with ClearVoice in daily listening situations and preferred to keep ClearVoice on. Conclusion ClearVoice can help cochlear implant recipients to hear better in noise.

Evaluation of a Subunit H5 Vaccine and an Inactivated H5N2 Avian Influenza Marker Vaccine in Ducks Challenged with Vietnamese H5N1 Highly Pathogenic Avian Influenza Virus

The protective efficacy of a subunit avian influenza virus H5 vaccine based on recombinant baculovirus expressed H5 haemagglutinin antigen and an inactivated H5N2 avian influenza vaccine combined with a marker antigen (tetanus toxoid) was compared with commercially available inactivated H5N2 avian influenza vaccine in young ducks. Antibody responses, morbidity, mortality, and virus shedding were evaluated after challenge with a Vietnamese clade 1 H5N1 HPAI virus [A/VN/1203/04 (H5N1)] that was known to cause a high mortality rate in ducks. All three vaccines, administered with water-in-oil adjuvant, provided significant protection and dramatically reduced the duration and titer of virus shedding in the vaccinated challenged ducks compared with unvaccinated controls. The H5 subunit vaccine was shown to provide equivalent protection to the other two vaccines despite the H5 antibody responses in subunit vaccinated ducks being significantly lower prior to challenge. Ducks vaccinated with the H5N2 marker vaccine consistently produced antitetanus toxoid antibody. The two novel vaccines have attributes that would enhance H5N1 avian influenza surveillance and control by vaccination in small scale and village poultry systems.