Tropism of influenza B viruses in human respiratory tract explants and airway organoids.

Despite causing regular seasonal epidemics with substantial morbidity, mortality and socioeconomic burden, there is still a lack of research into influenza B viruses (IBVs). In this study, we provide for the first time a systematic investigation on the tropism, replication kinetics and pathogenesis of IBVs in the human respiratory tract.Physiologically relevant ex vivo explant cultures of human bronchus and lung, human airway organoids, and in vitro cultures of differentiated primary human bronchial epithelial cells and type-I-like alveolar epithelial cells were used to study the cellular and tissue tropism, replication competence and induced innate immune response of 16 IBV strains isolated from 1940 to 2012 in comparison with human seasonal influenza A viruses (IAVs), H1N1 and H3N2. IBVs from the diverged Yamagata- and Victoria-like lineages and the earlier undiverged period were included.The majority of IBVs replicated productively in human bronchus and lung with similar competence to seasonal IAVs. IBVs infected a variety of cell types, including ciliated cells, club cells, goblet cells and basal cells, in human airway organoids. Like seasonal IAVs, IBVs are low inducers of pro-inflammatory cytokines and chemokines. Most results suggested a higher preference for the conducting airway than the lower lung and strain-specific rather than lineage-specific pathogenicity of IBVs.Our results highlighted the non-negligible virulence of IBVs which require more attention and further investigation to alleviate the disease burden, especially when treatment options are limited.

Tropism of avian influenza A (H5N1) in the upper and lower respiratory tract.

Poor human-to-human transmission of influenza A H5N1 virus has been attributed to the paucity of putative sialic acid alpha2-3 virus receptors in the epithelium of the human upper respiratory tract, and thus to the presumed inability of the virus to replicate efficiently at this site. We now demonstrate that ex vivo cultures of human nasopharyngeal, adenoid and tonsillar tissues can be infected with H5N1 viruses in spite of an apparent lack of these receptors.

The M, E, and N structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles.

The production of virus-like particles (VLPs) constitutes a relevant and safe model to study molecular determinants of virion egress. The minimal requirement for the assembly of VLPs for the coronavirus responsible for severe acute respiratory syndrome in humans (SARS-CoV) is still controversial. Recent studies have shown that SARS-CoV VLP formation depends on either M and E proteins or M and N proteins. Here we show that both E and N proteins must be coexpressed with M protein for the efficient production and release of VLPs by transfected Vero E6 cells. This suggests that the mechanism of SARS-CoV assembly differs from that of other studied coronaviruses, which only require M and E proteins for VLP formation. When coexpressed, the native envelope trimeric S glycoprotein is incorporated onto VLPs. Interestingly, when a fluorescent protein tag is added to the C-terminal end of N or S protein, but not M protein, the chimeric viral proteins can be assembled within VLPs and allow visualization of VLP production and trafficking in living cells by state-of-the-art imaging technologies. Fluorescent VLPs will be used further to investigate the role of cellular machineries during SARS-CoV egress.

Comparison of nasopharyngeal flocked swabs and aspirates for rapid diagnosis of respiratory viruses in children.

BACKGROUND: The quality of clinical specimens is a crucial determinant for virological diagnosis. OBJECTIVES: We compared the viral diagnostic yield for influenza A and respiratory syncytial virus (RSV) from the recently developed nasopharyngeal flocked swabs (NPFS) with nasopharyngeal aspirates (NPA) collected in parallel from 196 hospitalized children with acute respiratory infection during the peak period of influenza A and RSV activity in Hong Kong. Specimens were tested by RT-PCR for influenza A and RSV and viral load determined. They were also tested by direct immunofluorescence (DIF) for influenza A and B, RSV, parainfluenza types 1-3 and adenovirus. RESULTS: Both NPA and NPFS had excellent sensitivity (100%) for detecting influenza A by RT-PCR but NPA was slightly more sensitive than NPFS for detecting RSV by both RT-PCR (100% vs. 92.3%) and DIF (87.2% vs. 84.6%) and for detecting influenza A by DIF (90.2% vs. 82.9%). Viral load for influenza A in NPA and NPFS was not significantly different but that for RSV was higher in NPA. CONCLUSION: NPA remains the optimal specimen for diagnosis of respiratory infections by RT-PCR and DIF. However, collection of NPFS is easier to perform in an out-patient setting, was more acceptable to parents and less likely to generate aerosols than NPA engendering potentially less infection control hazard.

Radiation-induced rhabdomyosarcomatous transformation of a recurrent meningeal haemangiopericytoma.

A 53-year-old woman presented in 1979 with a posterior fossa meningeal haemangiopericytoma (HPC) for which she underwent surgical resection and post-operative radiotherapy. Repeated tumour recurrences occurred 18 years afterwards which were treated with resections and stereotactic radiotherapy. Surgery for tumour recurrence in 2005 revealed features of rhabdomyosarcomatous transformation. To our knowledge, this is the first reported case of rhabdomyosarcomatous transformation within a HPC which was likely to be radiation-induced, and was associated with relentless disease progression more than 20 years after the initial presentation.

Avian influenza H5N1 in viverrids: implications for wildlife health and conservation.

The Asian countries chronically infected with avian influenza A H5N1 are 'global hotspots' for biodiversity conservation in terms of species diversity, endemism and levels of threat. Since 2003, avian influenza A H5N1 viruses have naturally infected and killed a range of wild bird species, four felid species and a mustelid. Here, we report fatal disseminated H5N1 infection in a globally threatened viverrid, the Owston's civet, in Vietnam, highlighting the risk that avian influenza H5N1 poses to mammalian and avian biodiversity across its expanding geographic range.

Molecular characterization of antigen-processing function in nasopharyngeal carcinoma (NPC): evidence for efficient presentation of Epstein-Barr virus cytotoxic T-cell epitopes by NPC cells.

Potentiation of the EBV-specific CTL response by immunization with CTL epitopes has been proposed as a logical approach for immune-targeting nasopharyngeal carcinoma (NPC) cells in vivo. This approach will undoubtedly be influenced by the ability of these malignant cells to endogenously process and present target epitopes on their cell surface for immune recognition by CTLs. Analysis of NPC cells in fresh tumor biopsies and long-term, established NPC tumors in nude mice revealed normal expression of the MHC-encoded putative peptide transporters TAP1 and TAP2, as well as the proteasome components LMP2 and LMP7, which have been shown previously to be essential components of the class I processing pathway. Moreover, these tumor cells also showed high levels of HLA class I alleles on the cell surface, suggesting that peptides are available for binding to nascent MHC molecules in the endoplasmic reticulum. Using a recombinant vaccinia virus to transiently express the EBV nuclear antigens, we studied the antigen-processing efficiency of NPC cells. Our findings demonstrate that, in contrast to cells from another EBV-associated malignancy, Burkitt's lymphoma, NPC cells display normal antigen-processing function and are efficiently recognized by HLA class I-restricted, virus-specific CTLs. These studies also provide a rationale for focusing on strategies designed to activate CTLs specific for EBV antigens that are expressed in NPC cells in vivo.

Evidence of cisplatin-induced senescent-like growth arrest in nasopharyngeal carcinoma cells.

Cellular senescence is a programmed cell response leading to growth arrest in human diploid fibroblasts. We have shown that a nasopharyngeal carcinoma cell line, CNE1, following treatment by the DNA-damaging agent cisplatin, can undergo cellular senescent-like growth arrest, similar to fibroblasts, judged by cellular morphological changes and the expression of senescence-associated beta-galactosidase (SA-beta-gal). This senescent-like change was dose related; at 0.5 microgram/ml, the percentage of cisplatin-induced SA-beta-gal-positive cells was high (40-96%), and the staining was intense. Higher doses (1.0 and 2.0 micrograms/ml) of cisplatin induced lower SA-beta-gal expression (30-70%), and the process was irreversible. This cisplatin-induced cellular senescent-like response was not due to the inhibition of telomerase activity. Our results indicate that cellular senescent-like pathways exist in nasopharyngeal carcinoma cells and can be induced by cisplatin. Our evidence suggests that cellular senescent-like responses may be a cellular protection mechanism that acts differently in response to different degrees of cellular damage.

High level expression of deltaN-p63: a mechanism for the inactivation of p53 in undifferentiated nasopharyngeal carcinoma (NPC)?

Undifferentiated nasopharyngeal carcinoma (NPC) is an epithelial malignancy that is consistently associated with Epstein-Barr virus (EBV) but which very rarely has p53 gene mutations in primary tumours. Since the tumour suppressor p53 is mutated in most human cancers or the wild type protein is inactivated in a significant number of the remainder, here we have investigated cellular factors that could compromise p53 function in primary NPC. Twenty-five primary tumours were judged to carry only wild type p53 by SSCP analysis of all exons and sequence determination of exons 4-9. Only one tumour was found to express significant levels of hMdm2 and in 24/25 there were no detectable mutations or deletions in exons 1beta and 2 of the p14(ARF) gene. However, immunohistochemistry consistently revealed that all the tumour cells express substantial amounts of the p53-related protein p63. Semi-quantitative RT-PCR analysis of mRNA from tumour biopsies showed that the dominant species expressed was invariably the truncated deltaN-isotype. Since this can block p53-mediated transactivation, it is potentially a dominant-negative isoform. In normal nasopharyngeal epithelium the distribution of p63 was restricted to the proliferating basal and suprabasal layers. We suggest that deltaN-p63 is a good candidate as a suppressor of wild type p53 function in these tumours and also that it may prove to be a valuable diagnostic marker for undifferentiated NPC.

Re-emergence of fatal human influenza A subtype H5N1 disease.

Human disease associated with influenza A subtype H5N1 re-emerged in January, 2003, for the first time since an outbreak in Hong Kong in 1997. Patients with H5N1 disease had unusually high serum concentrations of chemokines (eg, interferon induced protein-10 [IP-10] and monokine induced by interferon gamma [MIG]). Taken together with a previous report that H5N1 influenza viruses induce large amounts of proinflammatory cytokines from macrophage cultures in vitro, our findings suggest that cytokine dysfunction contributes to the pathogenesis of H5N1 disease. Development of vaccines against influenza A (H5N1) virus should be made a priority.

Proinflammatory cytokine responses induced by influenza A (H5N1) viruses in primary human alveolar and bronchial epithelial cells.

BACKGROUND: Fatal human respiratory disease associated with influenza A subtype H5N1 has been documented in Hong Kong, and more recently in Vietnam, Thailand and Cambodia. We previously demonstrated that patients with H5N1 disease had unusually high serum levels of IP-10 (interferon-gamma-inducible protein-10). Furthermore, when compared with human influenza virus subtype H1N1, the H5N1 viruses in 1997 (A/Hong Kong/483/97) (H5N1/97) were more potent inducers of pro-inflammatory cytokines (e.g. tumor necrosis factor-a) and chemokines (e.g. IP-10) from primary human macrophages in vitro, which suggests that cytokines dysregulation may play a role in pathogenesis of H5N1 disease. Since respiratory epithelial cells are the primary target cell for replication of influenza viruses, it is pertinent to investigate the cytokine induction profile of H5N1 viruses in these cells. METHODS: We used quantitative RT-PCR and ELISA to compare the profile of cytokine and chemokine gene expression induced by H5N1 viruses A/HK/483/97 (H5N1/97), A/Vietnam/1194/04 and A/Vietnam/3046/04 (both H5N1/04) with that of human H1N1 virus in human primary alveolar and bronchial epithelial cells in vitro. RESULTS: We demonstrated that in comparison to human H1N1 viruses, H5N1/97 and H5N1/04 viruses were more potent inducers of IP-10, interferon beta, RANTES (regulated on activation, normal T cell expressed and secreted) and interleukin 6 (IL-6) in primary human alveolar and bronchial epithelial cells in vitro. Recent H5N1 viruses from Vietnam (H5N1/04) appeared to be even more potent at inducing IP-10 than H5N1/97 virus. CONCLUSION: The H5N1/97 and H5N1/04 subtype influenza A viruses are more potent inducers of proinflammatory cytokines and chemokines in primary human respiratory epithelial cells than subtype H1N1 virus. We suggest that this hyper-induction of cytokines may be relevant to the pathogenesis of human H5N1 disease.

The aetiology, origins, and diagnosis of severe acute respiratory syndrome.

Severe acute respiratory syndrome (SARS) is a new infectious disease that first emerged in Guangdong province, China, in November, 2002. A novel coronavirus was later identified in patients with SARS. The detection of the virus in these patients, its absence in healthy controls or other patients with atypical pneumonia, and the reproduction of a similar disease in a relevant animal model fulfilled Koch's postulates for implicating this coronavirus as the causal agent of SARS. The full genome sequence was determined within weeks of the virus's identification. The rapid progress in the aetiology, the development of laboratory diagnostic tests, and the defining of routes of viral transmission were facilitated through a unique WHO-coordinated virtual network of laboratories, which shared information on a real-time basis through daily teleconferences. Subsequent studies have indicated that the SARS coronavirus is of animal origin, that its precursor is still present in animal populations within the region, and that live-animal markets in southern China may have provided the animal-human interphase that allowed this precursor virus to adapt to human-human transmission. These findings underscore the potential for the re-emergence of SARS and the need for laboratory tests for early diagnosis. However, the low viral load in the respiratory tract makes early diagnosis of SARS a diagnostic challenge, although improvements in the sensitivity of molecular diagnostic methods continue to be made.

Effect of p16INK4a on chemosensitivity in nasopharyngeal carcinoma cells.

The p16INK4a tumor suppressor gene is frequently inactivated in nasopharyngeal carcinoma (NPC) and hence it may play an important role in the suppression of this tumor. In order to study the effect of p16INK4a restoration in NPC cells, full-length human p16INK4a gene was transfected into a NPC cell line, CNE1. Four individual clones with differential levels of p16INK4a protein expression, were selected for further studies. The introduction of p16INK4a into CNE1 cells induced growth suppression through G0/G1 cell cycle arrest; however, the cell growth rate was not correlated to the levels of p16INK4a protein expression. To study whether transfection of p16INK4a could protect NPC cells from radiation, cisplatin and 5-fluorouracil (5FU), the cellular sensitivity of p16INK4a transfectants and vector control were investigated. An increase in sensitivity to 5FU was observed (2-fold compared to IC50) in all 4 clones compared to vector-transfected control. P16INK4a transfection also resulted in increased sensitivity to cisplatin (1.5-1.8-fold) in 3 out of 4 cell lines. However, no difference in radiosensitivity was found between the p16INK4a transfectants and the control. These findings indicate that p16INK4a suppresses NPC cell growth through G0/G1 arrest and modulating cellular response to chemotherapeutic drugs in NPC cells. Therefore, restoration of p16INK4a may have a therapeutic purpose in the treatment of NPC.

Cisplatin-induced p53-independent growth arrest and cell death in cancer cells.

In a recent study, it was shown that DNA damaging agent cisplatin-induced growth arrest and cell death in cancer cells by a pathway sharing some of the characteristics of replicative senescence. The aim of this study was to determine the role of p53, p21WAF1 and p16INK4A proteins in this alternative route to cancer cell death in additional human cancer cell lines. After exposure to cisplatin, all the cell lines underwent growth arrest and expressed the senescence marker senescence-associated beta-galactosidase, but showed none of the features of apoptosis. However, there was no change in p53 protein expression, and neither p21WAF1 nor p16INK4A was expressed before or up to 4 days after cisplatin exposure. These findings provide further evidence that cells carrying mutations resulting in loss of function in the p53 gene can be killed by cisplatin via a p53-independent route with some similarities to replicative senescence, but not apoptosis.

Radiation therapy for nasopharyngeal carcinoma: histologic appearances and patterns of tumor regression.

Nasopharyngeal carcinoma is a common malignancy in Hong Kong and is treated by external radiotherapy. After 6.5 weeks of radiotherapy, the nasopharynx of 100 patients was examined and biopsy specimens were taken. All patients had repeated examination and biopsies done every 2 weeks until exophytic tumor was not seen and biopsy samples were negative on more than one examination of the nasopharynx. The interval between the cessation of therapy and biopsy ranged from 1 day to 11 weeks. Twenty-three patients had atypical findings and five of these had residual tumor requiring gold grain implantation brachytherapy. We identified a number of distinct pathologic changes in the post-biopsy material. Most of these changes disappeared 8 weeks after the cessation of therapy, but the presence of residual tumor after this time was an indication for subsequent therapy. If the date of the first biopsy was delayed until 6 weeks after the completion of radiotherapy, the percentage of atypical biopsies containing residual tumor rose from 21% to 55%. The posttreatment biopsy should be performed twice, as four patients had negative biopsy findings due to sampling error.

Epstein-Barr virus is detected in undifferentiated nasopharyngeal carcinoma but not in lymphoepithelioma-like carcinoma of the urinary bladder.

The Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC) and with lymphoepithelioma-like carcinomas developing in certain anatomic sites. In this study, an in situ hybridization was used to identify EBV-encoded ribonucleic acid (RNA) (EBER1) transcripts in 32 of 45 cases of NPC but not in any of the 11 lymphoepithelioma-like carcinomas developing in the urinary bladder. EBER1 was most commonly detected in those NPCs having undifferentiated or nonkeratinizing squamous histology rather than the keratinizing squamous cell subtype of NPC. The EBV-encoded latent membrane protein 1 (LMP1) was expressed focally in only seven of 21 EBER1-positive NPCs by an immunohistochemical technique. These findings imply that EBER1 hybridization is more sensitive than LMP1 immunohistochemistry on paraffin sections in detecting carcinoma-associated virus. Previous in vitro studies have suggested that LMP1 expression might be a function of differentiation, but this study of naturally infected NPCs showed no strong correlation between LMP1 positivity and degree of tumor differentiation, albeit a limited spectrum of differentiation that could be examined. In two cases in which frozen tissue was available, the NPCs were monoclonal with respect to viral DNA structure, implying that the virus was present before malignant transformation. Unlike NPCs, the lymphoepithelioma-like carcinomas of the bladder were uniformly EBV negative, lending further evidence to the growing body of literature linking EBV with lymphoepithelial carcinomas of foregut-derived tissues but not with similar-appearing tumors developing in other anatomic sites.

Expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in undifferentiated nasopharyngeal carcinoma (lymphoepithelioma) and in malignant epithelial tumors.

Immunoreactivity for intercellular adhesion molecule-1 (ICAM-1) and for vascular cell adhesion molecule-1 (VCAM-1), two adhesion molecules of the immunoglobulin (Ig) superfamily, was tested and measured on tissue sections from 16 undifferentiated nasopharyngeal carcinomas (U-NPC), 12 keratinizing squamous cell carcinomas (SCCs) of the head and neck region, and 54 malignant epithelial tumors of various origin. Neoplastic cells of all cases of U-NPC were diffusely and intensely stained for ICAM-1 and VCAM-1. Moreover, ICAM-1 messenger RNA (mRNA) and VCAM-1 mRNA were detected by Northern blot analysis of RNA extracts from two tumors. In the other epithelial tumors focal or diffuse staining for ICAM-1 was observed in 40 cases (66%), whereas reactivity for VCAM-1 was detected in a single case of metastatic undifferentiated carcinoma of unknown origin. The biopsy specimens of U-NPC showed variable infiltration by leukocytes, which were positive for the integrins lymphocyte function antigen-1 (LFA-1) and alpha-4/beta-1, the corresponding ligands for ICAM-1 and VCAM-1. The possibility that ICAM-1 and VCAM-1 on neoplastic cells may favor the intratumoral recruitment of leukocytes in a way similar to that occurring in crypt epithelium of the palatine tonsil is discussed.