Tolerance of one-month intranasal interferon

Under double-blind conditions, groups of volunteers (68 in total) were allocated at random to take intranasal solutions of placebo or one of three doses of highly purified leucocyte interferon by intranasal spray twice a day for 28 days. The highest dose would have been expected to protect against experimental colds. Treatment was discontinued because of upper respiratory symptoms as often in each of the interferon groups as in the placebo group. However, it was possible to distinguish clinically between "colds" on placebo and low-dose interferon and "reactions to treatment" on high-dose interferon. The features of the reactions to treatment were a protracted build-up of local symptoms and minor epistaxis. None of the volunteers on the high-dose interferon were thought to have a definite cold, but viruses were isolated from four out of six volunteers on low-dose interferon who had definite colds. Previous experiments had also shown this dose to be insufficient to protect against experimental rhinovirus challenge. The dose of interferon that appeared to protect against virus infection caused significant unwanted effects. It is essential to find interferon preparations with less inflammatory activity before interferon can be considered for use as a long-term prophylactic against the common cold.     

Complete sequence of the RNA genome of human rhinovirus 16, a clinically useful common cold virus belonging to the ICAM-1 receptor group

We report here the complete nucleotide sequence and predicted polyprotein sequence of HeLa cell-adapted human rhinovirus 16 (HRV16). This virus is more suitable than human rhinovirus 14 (HRV14) for clinical studies, and its growth and physical properties are favorable for biochemical and crystallographic analysis. The complete message-sense RNA genome of HRV16 is composed of 7124 bases, not including the poly(A) tail. An open reading frame, extending from base 626 to 7084 predicts a polyprotein containing 2152 amino acid residues. Comparison with other rhinovirus sequences shows HRV16 is much more representative of human rhinoviruses than HRV14. No apparent relationship was found between receptor group and amino acid sequence in VP1, the capsid protein bearing the binding site for the intercullular adhesion molecule-1 (ICAM-1) in both HRV14 and HRV16.Genbank accession number: L24917.     

Effect of experimental rhinovirus 16 colds on airway hyperresponsiveness to histamine and interleukin-8 in nasal lavage in asthmatic subjects in vivo

Background Asthma exacerbations are closely associated with respiratory virus infections. However, the pathophysiological consequences of such infections in asthma are largely unclear. Objective To examine the effect of rhinovirus 16 (RV16) infection on airway hypersensitivity to histamine. and on interleukin-8 (IL-8) in nasal lavage. Objective Twenty-seven non-smoking atopic, mildly asthmatic subjects participated in a placebo-controlled, parallel study. A dose of 0.5–2.9 ± 10⁴ TCID50 RV16 or placebo was nasally administered. Cold symptoms were recorded by questionnaire throughout the study. Histamine challenges were performed at entry, and on days 4 and 11 after inoculation. Nasal lavages were obtained at entry, and on days 2 and 9. The response to histamine was measured by PC₂₀ (changes expressed as doubling doses: DD). IL-8 levels were obtained by ELISA, and were expressed in ng/ml. Results RV infection was confirmed by culture of nasal lavage and/or by antibody titre rise in each of the RV16-treated subjects. Among the 19 RV16-treated subjects, eight developed severe cold symptoms. Baseline FEV₁ did not change significantly during the study in either treatment group (P= 0.99). However, in the RV16-treated subjects there was a decrease in PC₂₀ at day 4, which was most pronounced in those with a severe cold (mean change ± SEM: –1.14 ± 0.28 DD, P= 0.01). In addition. IL-8 levels increased in tbe RV16 group at days 2 and 9 (P < 0.001). The increase in nasal IL-8 at day 2 correlated significantly with the change in PC₂₀ at day 4 (r=–0.48, P= 0.04). Conclusion We conclude that the severity of cold, as induced by experimental RV16 infection, is a determinant of the increase in airway hypersensitivity to histamine in patients with asthma. Our results suggest that this may be mediated by an infiammatory mechanism, involving the release of chemokines such as IL-8.     

Nasal mucosa in natural colds: effects of allergic rhinitis and susceptibility to recurrent sinusitis

The mechanisms of virus-induced airway hyperresponsiveness in asthma and allergy and the failure of host defence in patients suffering from secondary airway infections are still largely unknown. The aim of this study was to examine whether the presence of allergic rhinitis or susceptibility to recurrent sinusitis affects the structural and cellular changes in nasal mucosa during natural colds and convalescence. We compared the mucosal changes in biopsy samples during acute natural colds (days 2-4 of illness) and convalescence (3 weeks later) in patients with allergic rhinitis (n = 9), patients with susceptibility to sinusitis (n = 19) and healthy controls (n = 20). We saw similarly increased numbers of mucosal T and B lymphocytes and mast cells and increased vascular density during the acute colds compared to convalescence in all the three groups. The allergic subjects had elevated levels of eosinophils in the acute phase (P = 0.03), and the allergic and sinusitis-prone subjects had elevated levels of epithelial T cells (P = 0.04) and low levels of mast cells (P = 0.005) in convalescence compared to the control group. The sinusitis-prone subjects lacked intraepithelial cytotoxic cells in convalescence. In the allergic subjects, the reticular basement membrane was thicker in the acute phase compared to the convalescence (P = 0.05). These results suggest that various cells of the airways, including inflammatory and structural cells, are involved during viral respiratory infections in subjects with allergic rhinitis. The small numbers of mast cells and cytotoxic lymphocytes in the sinusitis-prone subjects may be related to their susceptibility to bacterial complications.     

The behaviour of recent isolates of human respiratory coronavirus in vitro and in volunteers: evidence of heterogeneity among 229E-related strains

Strains of human coronavirus (HCV) isolated between 1974 and 1976 have been studied in vitro and in volunteers. All strains caused colds in volunteers, and those cultivable in tissue culture (TC) produced significantly more coryza and less sore throat than strains growing only in organ culture (OC). The TC strains were serologically related to 229E, but these isolates produced colds with a frequency and severity that contrasted with the effects of 229E itself. Tests on volunteers' preinfection sera showed that the prevalence of antibody to 229E had increased during the period 1961-1979 and that during 1977-1979 only 11% of subjects had no neutralising antibody against 229E. Susceptibility to the 229E-related isolates PR and TO was associated with low preinfection serum neutralising antibody against the homologous virus, and paired sera frequently showed fourfold or greater antibody rises, most commonly against the homologous strain. Volunteers infected with TO were immune when reinoculated with the same strain approximately 1 year later, but other similar volunteers were at least partly susceptible to infection with a heterologous 229E-related virus after similar time intervals. Although the strains of HCV that were grown in tissue culture were all related to the prototype 229E, they appeared not to be identical with it, and this heterogeneity is probably a significant factor in the epidemiology of HCV infections.     

Evaluation of an enzyme-linked immunosorbent assay that measures rhinovirus-specific antibodies in human sera and nasal secretions

Rhinovirus-specific antibodies have traditionally been detected by their ability to neutralise the homologous rhinovirus serotype in tissue culture. Recently, however, we have described an enzyme-linked immunosorbent assay that detects rhinovirus-specific antibodies in sera and nasal secretions [Barclay and Al-Nakib, 1987]. Here we describe an evaluation of the ELISA in a study involving 71 adult volunteers inoculated intranasally with human rhinovirus type 2 (HRV-2). Pre- and post-inoculation serum samples and pre-inoculation nasal washings were tested for the presence of HRV-2-specific antibodies by ELISA. Such antibodies were associated with protection against infection when present locally in nasal secretions, but when also present in the serum they were associated with protection against both infection and the development of illness. The antibody concentrations showed strong correlation with each other and with that of antibodies detected by the neutralisation test. Following HRV-2 infection, rises in HRV-2-specific IgA in sera detected by ELISA occurred more frequently than rises in neutralising antibody. These results suggest that the ELISA is a sensitive and reliable indicator of recent infection, as well as a predictor of homologous immune status.     

An adult model of exclusive viral wheeze: inflammation in the upper and lower respiratory tracts

BACKGROUND: We have previously reported an experimental infection of young adults with a history of episodic and exclusive viral wheeze (EVW) using human coronavirus, in which 16 of 24 with EVW (15 atopic) and 11 of 19 healthy controls (seven atopic) developed a symptomatic cold with evidence of infection, but only those with EVW developed lower respiratory tract symptoms and increased airway responsiveness. OBJECTIVE: The aim of this study was to compare the EVW and control groups from this study for inflammatory changes occurring in the upper and lower respiratory tracts during the experimental infection, in particular, to determine whether eosinophil-driven inflammation was associated with EVW. METHODS: Nasal lavage and induced sputum were collected prior to inoculation (day 0) and 2, 4 and 17 days later. Differential cell counts were performed and supernatant was assayed for IL-8, IL-5, IFN-gamma, and eosinophilic cationic protein (ECP). RESULTS: There was no difference between the two groups in any measurement at baseline. In both groups, during colds the volume of nasal secretion increased as did leucocyte counts in both upper and lower respiratory tracts. A modest increase in nasal neutrophil count was seen in both EVW and control groups with symptomatic colds on day 2 (median (quartile) difference from baseline 5.4 (0.0, 11.0) and 1.8 (-1.1, 2.2)x10(4)/mL of secretions, respectively). The change in nasal neutrophil counts in all subjects correlated with nasal symptom scores. A significant relative increase in sputum differential neutrophil count was seen on day 4 in the EVW group with a cold but not in controls (mean difference (95% confidence interval) 20.4 (9.6, 31.1)% and 3.1 (-8.2, 14.5)%, respectively, P<0.01); however, this increase did not correlate with lower respiratory tract symptom scores. IL-8 increased in both the upper and lower respiratory tracts in both EVW and control subjects with colds, the largest change being seen on day 4 in the sputum of those with EVW (mean difference from baseline (95% confidence interval) 2.5 (0.55-4.46) ng/mL). Only modest changes were seen in IFN-gamma and no changes were seen in IL-5 or ECP. None of the results was influenced by the atopic status of the subjects in either group. CONCLUSIONS: EVW wheeze is characterized by neutrophilic inflammation in both the upper and lower respiratory tracts without eosinophilia (even in atopic subjects). IL-8 is likely to be an important chemokine in this process. Symptoms and airway responsiveness were correlated with change in neutrophils.     

Subjects with allergic rhinitis show signs of more severely impaired paranasal sinus functioning during viral colds than nonallergic subjects

BACKGROUND: Viral cold is thought to be the major contributing factor in the pathogenesis of sinusitis, as it causes ostiomeatal obstruction. The aim was to evaluate whether paranasal sinus functioning during viral colds is similar in subjects with and without allergic rhinitis. METHODS: Forty-eight volunteers were examined during an early (2-4 days) natural cold and again 3 weeks later. The examinations included computed tomography (CT) scans, nasal mucosal biopsies, and viral and bacterial specimens. Subjects with positive skin prick tests and persistent or intermittent rhinitis were considered to have allergic immunoglobulin E (IgE)-mediated rhinitis. In addition, specific IgE antibodies to staphylococcal enterotoxin B (SEB) were measured. RESULTS: Nine subjects (19%) had allergic rhinitis. The allergic subjects were significantly more often IgE sensitized to SEB than the nonallergic subjects (33%vs 3%, P = 0.02). Viral etiology of the cold was identified in 32 (67%) subjects. The subjects with allergic rhinitis had significantly higher CT scores compared with nonallergic subjects during the colds (median (range) scores 16 (6-22) vs 6 (0-17), P = 0.004). In both groups, the median scores declined markedly during convalescence, but the difference remained significant (P = 0.009). Among the allergic subjects, those who were IgE sensitized to SEB tended to have the highest CT scores [median (range) 16 (16-22)]. Total serum IgE and the nasal subepithelial eosinophil counts correlated with the CT scores during the cold (rs = 0.38, P = 0.008 and rs = 0.46, P = 0.001, respectively). CONCLUSIONS: Subjects with allergic IgE-mediated rhinitis had more severe paranasal sinus changes in CT scans than nonallergic subjects during viral colds. These changes indicate impaired sinus functioning and may increase the risk of bacterial sinusitis.     

Replication of respiratory viruses, particularly influenza virus, rhinovirus, and coronavirus in HuH7 hepatocarcinoma cell line

Detection of viral antigens and isolation methods has long been used for the diagnosis of respiratory virus infections. The objective was to determine the ability of HuH7 cells to support the replication of prototype and wild strains of respiratory viruses. The cell culture-adapted strains of influenza viruses A and B, parainfluenza viruses 1-4, respiratory syncytial viruses A and B, both strains of the human metapneumoviruses, numerous rhinoviruses, most of the adenoviruses, coronaviruses 229E and OC43, and a number of enteroviruses (poliovirus type 3, coxsackie virus B1, echovirus type 30) replicate in HuH7. The kinetic study of the replication of influenza A and B viruses showed that there were infected cells in HuH7 and MDCK lines as early as 24 hr post-infection. However, the replication of influenza A and B viruses was more rapid and intense on MDCK cells than on HuH7 cells. During the three winters of 1999-2000, 2000-2001, and 2001-2002, of the 1,226 (23.3%) direct fluorescent assay-positive nasal aspirates from children admitted to hospital, 788 were positive for respiratory syncytial virus, 228 for influenza virus, 133 for parainfluenza virus, and 77 for adenovirus. Of the 4,032 direct fluorescent assay-negative nasal aspirates, 571 virus isolates were identified by using HuH7 cell culture; 272 rhinoviruses, 100 influenza viruses A and B, 85 enteroviruses, 40 adenoviruses, 35 coronaviruses, 31 parainfluenza viruses, and 10 respiratory syncytial viruses. Interestingly, 100/328 (30.5%) influenza viruses A and B, 40/189 (21.1%) adenoviruses, and 31/164 (19%) parainfluenza viruses type 1-3, not detected by direct fluorescent assay, were identified by isolation in HuH7 cell culture.     

Development and characterization of DNAzyme candidates demonstrating significant efficiency against human rhinoviruses

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Phylogenetic analysis of a highly conserved region of the polymerase gene from 11 coronaviruses and development of a consensus polymerase chain reaction assay

Viruses in the genus Coronavirus are currently placed in three groups based on antigenic cross-reactivity and sequence analysis of structural protein genes. Consensus polymerase chain reaction (PCR) primers were used to obtain cDNA, then cloned and sequenced a highly conserved 922 nucleotide region in open reading frame (ORF) 1b of the polymerase (pol) gene from eight coronaviruses. These sequences were compared with published sequences for three additional coronaviruses. In this comparison, it was found that nucleotide substitution frequencies (per 100 nucleotides) varied from 46.40 to 50.13 when viruses were compared among the traditional coronavirus groups and, with one exception (the human coronavirus (HCV) 229E), varied from 2.54 to 15.89 when compared within these groups. (The substitution frequency for 229E, as compared to other members of the same group, varied from 35.37 to 35.72.) Phylogenetic analysis of these pol gene sequences resulted in groupings which correspond closely with the previously described groupings, including recent data which places the two avian coronaviruses—infectious bronchitis virus (IBV) of chickens and turkey coronavirus (TCV)—in the same group [Guy, J.S., Barnes, H.J., Smith L.G., Breslin, J., 1997. Avian Dis. 41:583–590]. A single pair of degenerate primers was identified which amplify a 251 bp region from coronaviruses of all three groups using the same reaction conditions. This consensus PCR assay for the genus Coronavirus may be useful in identifying as yet unknown coronaviruses.     

体外培养新生小鼠胸腺的超微结构

目的 :观察器官培养的新生小鼠胸腺内细胞的生长状况。方法 :将新生BALB/c小鼠胸腺分为 4组 ,分别在体外培养 1d、3d、5d和 7d ,然后用透射电镜观察其各部位的超微结构。结果 :从 1d组开始 ,胸腺细胞的生长状况便呈现出明显的区域性差别 :胸腺周边部的细胞排列紧密 ,形态结构正常 ,与对照组无差别 ;胸腺中间部的细胞排列较松散 ,少部分形态结构正常 ,大部分呈死亡状态。结论 :胸腺周边部的细胞生长良好 ;中间部的细胞少量生长良好 ,大部分死亡     

SARS病毒N蛋白特异性单链抗体的筛选

目的 利用大容量人源性SARS单链抗体库筛选N蛋白的特异性单链抗体。方法利用RT-PCR的方法克隆了SARS病毒的N蛋白基因，克隆入原核表达载体pBV220，在大肠杆菌DH5a中进行表达并经离子交换色谱纯化后得到了纯度达95％以上的N蛋白，然后以此蛋白包被25cm^2组织培养瓶，对SARS单链抗体库进行先松弛，后严谨的筛选、富集，进行4轮筛选后对所筛的抗体进行ELISA鉴定。结果经过4轮的筛选、富集，得到了1株高亲和力的N蛋白单链抗体N18。结论在对SAILS疾病的早期检测或筛查、SARS病毒的生活周期及发病机理的研究中N18将起到重要的作用。     

Sequence analysis of the leader RNA of two porcine coronaviruses: Transmissible gastroenteritis virus and porcine respiratory coronavirus

The leader RNA sequence was determined for two pig coronaviruses, tranmissible gastroenteritis virus (TGEV), and porcine respiratory coronavirus (PRCV). Primer extension, of a synthetic oligonucleotide complementary to the 5 end of the nucleoprotein gene of TGEV was used to produce a single-stranded DNA copy of the leader RNA from the nucleoprotein mRNA species from TGEV and PRCV, the sequences of which were determined by Maxam and Gilbert cleavage. Northern blot analysis, using a synthetic oligonucleotide complementary to the leader RNA, showed that the leader RNA sequence was present on all of the subgenomic mRNA species. The porcine coronavirus leader RNA sequences were compared to each other and to published coronavirus leader RNA sequences. Sequence homologies and secondary structure similarities were identified that may play a role in the biological function of these RNA sequences.The nucleotide sequence data reported in this paper have been submitted to the EMBL/Genbank/DDBJ nucleotide sequence databases and have been assigned the accession numbers X52157, X52668.     

Comparison of multiplex PCR assays and conventional techniques for the diagnostic of respiratory virus infections in children admitted to hospital with an acute respiratory illness

The performances of four multiplex PCR (m-PCR) were compared to direct immunofluorescence assay (DFA) and HuH7 cell culture for the detection of viruses in 263 children admitted to hospital with an acute respiratory illness. One hundred fifty (57.6%) nasal aspirates were found DFA-positive; 188 (72.3%) were found positive by both DFA and HuH7 cell culture, and 242 (92%) were PCR-positive. The m-PCR detected 124 viruses which were not found by conventional methods: 68 rhinovirus, 17 human metapneumovirus, 15 respiratory syncytial virus (RSV), 8 parainfluenza virus (PIV), 5 coronavirus 229E, 3 OC43 and 3 NL63, 4 enterovirus, 2 influenza virus B and C virus. The m-PCR were more sensitive, had the advantages of a shorter delay in specific diagnosis, and a lower cost than DFA and culture. Using these m-PCR, the prevalence of each virus was compared between in-patient and out-patient groups of children attending the emergency unit of the hospital. Nasal aspirates from 411 (91.5%) children were found positive by the PCRs. RSV, rhinovirus, and influenza virus were the most frequent viruses detected in this population, representing 43.6%, 31.8%, and 8.8% of the virus found, respectively, followed by human metapneumovirus (4.4%), coronavirus (3.4%), parainfluenza virus (3.2%), adenovirus (2.3%), and enterovirus (2.1%). RSVs were detected more significantly in the in-patient group than in the out-patient group, and influenza viruses were detected more frequently in the out-patient group than in the in-patient group. Moreover, the use of m-PCR pointed out the frequency of rhinovirus and mixed viral detections in these patients. In conclusion, according to the requirements of speed and low cost of the methods, and to achieve the highest rate of detection of respiratory viruses, the combined use of DFA and m-PCR is today likely to be the best way to improve diagnosis of respiratory illnesses in children.     

Prospective genotyping of human rhinoviruses in children and adults during the winter of 2009-2010

BackgroundAbout 100 serotypes of human rhinovirus (HRV), classified into two species, have been identified by 1990. Uncultivable HRV variants have recently been identified and designated a new species. Recent improved diagnosis has led to a re-appraisal of the clinical impact of HRV infections in lower respiratory diseases.ObjectivesTo characterise clinical features in hospitalised patients with positive HRV RNA detection and to determine the distribution of HRV species in respiratory infections diagnosed during the winter of 2009–2010.Study designProspective virus typing was conducted by sequencing the VP4/VP2 genomic regions, and clinical data were collected.ResultsFifty-eight patients (for 63 respiratory specimens) were included. Phylogenetic analysis identified 52% of HRV species A, 6% of species B and 40% of species C, and revealed the co-circulation of 34 different HRV types during the study period. Three infants had successive infections with two or three different types. Five patients were admitted to an intensive care unit, four of them on arrival. Bronchiolitis, pneumonia and exacerbation of asthma were observed in 34/45 children. Pneumonia and severe exacerbation of chronic lung disease were observed in 8/13 adults, of whom 1, with immunocompromised status, died of multivisceral failure.ConclusionsThis study underlines the diversity of co-circulating strains and the potential severity of clinical presentations associated with HRV infections.     

Isolation and culture of beating cells from human fetal heart

Summary A method is described for culture of myocardial cells from cardiac tissue of 8 to 12 wk-old human fetuses. Modification of the existing procedures for cell isolation and growth has helped to improve the yield of viable cells. Cultured myocardial cells can be used for experimental studies.