Localization of rhinovirus replication in vitro with in situ hybridization.

To facilitate understanding of human rhinovirus (HRV) pathogenesis, methods were developed for detection of HRV infection in vitro using in situ hybridization (ISH). HRV-14 RNA probes and oligonucleotide probes representing conserved sequences in the 5'-non-translated region were labeled with 35S and used to detect infected HeLa or WI-38 strain human embryonic lung cells in cytological preparations. ISH was shown to be specific for detection of HRV on a single-cell basis. Subsequently, in human nasal polyps infected in vitro, both oligonucleotide- and ribo-probes produced a strong signal in association with ciliated epithelial cells. In human adenoids infected in vitro, a signal was observed in non-ciliated epithelial cells. This study shows that HRV replicates in ciliated cells in the epithelium of human nasal polyps infected in vitro, and the presence of viral RNA in non-ciliated cells of the human adenoid infected in vitro suggests that other cell types may also support rhinovirus replication.     

Performance of virus isolation and Directigen Flu A to detect influenza A virus in experimental human infection.

BACKGROUND: Few data exist to assess the sensitivity of different specimen types for viral detection during the course of influenza virus infection. OBJECTIVES: This study assessed the relationships between quantitative influenza A virus replication and antigen detectability by the enzyme immunosorbent assay (EIA) Directigen Flu A in different type of samples during experimental human infection. STUDY DESIGN: Fourteen volunteers were inoculated with influenza A virus A/Texas/36/91 (H1N1). Four specimens types were collected in sequence for quantitative isolation in cell culture and antigen testing from days 1 to 8 after inoculation. RESULTS: Seventy-one (63%) of nasopharyngeal wash specimens were culture positive, compared to 51 (46%) of throat gargles, 51 (46%) of nasal swabs, and 27 (24%) of throat swabs. All subjects shed virus in their nasopharyngeal wash at least one day and 86% of subjects had a positive nasopharyngeal wash culture on day 2 after inoculation. The mean viral titers were highest on day 2 post inoculation for all specimen types and averaged 3.6 log10 TCID50/ml for nasal washes, 1.2 log10 TCID50/ml for throat gargles, 1.8 log10 TCID50/ml for the nasopharyngeal swabs, and 0.6 log10 TCID50/ml for the throat swabs. Mean viral titers in the nasal washes were significantly different (P<0.05) compared to other specimen types. The peak of sensitivity of EIA (compared to culture) was the second day after inoculation. Nasopharyngeal and throat swab results were combined for this analysis and considered positive by culture if positive in either or both samples. Thus, on day 2 the number of EIA positive samples relative to the number culture positive was 9/12 (75%) for nasopharyngeal wash specimens, 2/9 (22%) for throat gargles, and 7/11 (64%) for the combined throat and nasal swabs specimens. CONCLUSIONS: Nasopharyngeal washes are the most sensitive sample type detecting influenza A virus in adults. For rapid diagnosis the Directigen Flu A is an alternative with a sensitivity compared to culture ranging between 64 and 78% if performed on nasopharyngeal specimens on day two or three after experimental infection in adults. However, if performed on other specimens or later in the course of infection the sensitivity is lower.     

Application of Directigen FLU-A for the detection of influenza A virus in human and nonhuman specimens.

Directigen FLU-A, a new enzyme immunoassay membrane test, rapidly detects influenza A virus antigen in specimens from patients. Nasopharyngeal washes and pharyngeal gargles were used to determine the effectiveness of the assay as applied to different types of routinely collected clinical samples. All specimens had been previously shown to contain influenza A virus by virus isolation in tissue culture. Directigen FLU-A was 90% sensitive (95% confidence interval, 56 to 99.7%) with nasopharyngeal washes but only 39% sensitive (95% confidence interval, 17 to 64%) with pharyngeal gargles (P = 0.018) when used with samples containing similar amounts of infectious virus (50% tissue culture infective dose, 1.0 to 4.5). The intensity of the positive reaction with Directigen FLU-A did not correlate with the amount of virus in the specimens. Directigen FLU-A was found to detect cell-associated antigen more readily than free virus; only 20 infected cells were required to identify cell-associated influenza A virus antigen, whereas the limit of detection for free virus was 1.63 x 10(3) infectious virus particles. These findings suggest that Directigen FLU-A detects the cell-associated antigen present in clinical specimens rather than free virus. In addition, Directigen FLU-A detected avian and swine influenza A viruses in both cloacal swabs (75% sensitivity) and swine lung homogenates (86% sensitivity), indicating its potential usefulness in the surveillance of nonhuman influenza A viruses.     

Evaluation of BioStar FLU OIA assay for rapid detection of influenza A and B viruses in respiratory specimens.

BACKGROUND: Demand for the rapid diagnosis of influenza infections has increased with the advent of the availability of neuraminidase antiviral therapy for influenza A and B. Several rapid assays that detect both influenza A and B are now available. OBJECTIVES: In this study we compared the performance of the BioStar FLU OIA assay to Bartels Viral Respiratory Screening and Identification Kit (Bartels Inc., Issaquah, WA), and cell culture. STUDY DESIGN: A total of 145 patient specimens for influenza virus detection submitted in either viral transport medium or in sterile containers were evaluated by the three methods. Specimen types included nasal washings, nasal swabs, sputum, throat swabs, and bronchial alveolar lavage (BAL) fluids. RESULTS: Fifty six positive specimens were identified based on culture and/or DFA. Of these, 30 specimens were positive by the OIA assay for an overall sensitivity of 54%. The OIA assay detected 48% (n = 21) of the 44 culture positive specimens and 81% (n = 29) of the 36 DFA positive specimens. Eighty six of the 89 culture/DFA negative samples were negative by the OIA assay (97% specificity). Analysis of the OIA assay sensitivity from samples submitted in M4 transport medium or in sterile containers revealed that M4 transport medium does not reduce the sensitivity of the OIA assay. Fifteen of the 27 positive samples submitted in M4 transport medium were positive by the OIA assay (56% sensitivity) compared to 15 of 29 positive samples transported in sterile containers (52% sensitivity). Twelve specimens were either culture and/or DFA positive for viruses other than influenza, but negative by the OIA assay, suggesting that there was no cross reactivity of the OIA assay with the other virus types recovered in this study. CONCLUSIONS: The overall excellent specificity of the BioStar FLU OIA allows for treatment of positive patients for influenza, however, a negative result should be confirmed by DFA and culture.     

Improved detection of rhinoviruses in nasal and throat swabs by seminested RT-PCR

A seminested RT-PCR (nRT-PCR) was used to detect picornavirus (PV) RNA in cell cultures inoculated with rhinoviruses (HRVs) and enteroviruses (EVs). PCR tests in which a primary "touchdown" PCR was followed by secondary reactions using PV or HRV specific primers were able to differentiate HRVs of 48 serotypes from EVs. PVnRT-PCR and HRVnRT-PCR were then used to test nasal and throat swabs from adult subjects with naturally acquired respiratory virus infections. The swabs were also analysed for respiratory viruses by cell culture techniques and the rates of PV identification by the two methods were compared. PVnRT-PCR was found to be at least five times more sensitive than cell culture for the detection of PVs in these clinical specimens. Paired acute and convalescent serum samples were tested for complement fixing antibodies to adenovirus, influenza A and B, respiratory syncytial virus, parainfluenza viruses 1, 2, and 3, Myco plasma pneumoniae, and Chlamydia psittaci. An enzyme-linked immunosorbent assay (ELISA) was used to detect rises in antibody level to coronavirus types 229E and OC43. The overall rate of pathogen identification in 159 swabs from adult asthmatics increased from 28% when only cell culture and serology were used to 57% when these methods were supplemented by PVnRT-PCR. © 1993 Wiley-Liss, Inc.     

Isolation of a peptide inhibitor of human rhinovirus

Cell culture-based transdominant genetic techniques provide new methods for discovering peptide/RNA modulators of cellular pathways. We applied this technology to isolate a peptide inhibitor of human rhinovirus. A green fluorescent protein (GFP)-scaffolded library of cDNA fragments was expressed in HeLa cells from a retroviral vector and screened for inhibitors of rhinovirus-mediated cell killing. A DNA clone, I421, increased cell survival in an HRV14 challenge assay from less than 0.5% to greater than 60%. It encodes a 53-amino-acid C-terminal extension of the GFP scaffold. Particular subclones of Hela cells expressing I421 (exemplified by I421dp3) show a delay in virus production and a 50-fold decrease in viral RNA levels at 6-8 h postinfection. HRV2, HRV14, and HRV16 show a dramatic decrease in plaque-forming ability on I421dp3 while Coxsackievirus B3 showed a small reduction. Levels of ICAM-1, the receptor for the main rhinovirus serotype, are not altered in I421dp3.     

Comparison of rhinovirus-sensitive HeLa cells and human embryo fibroblasts for isolation of rhinoviruses from patients with respiratory disease.

Rhinovirus-sensitive HeLa cells (HeLa "R") and human embryo lung fibroblasts (HEL) were compared for the isolation of rhinoviruses from patients with respiratory disease. In the period May 1970 to December 1974, 526 rhinoviruses were isolated from 517 patients, 32% in both cell types, 59% in HeLa "R" only, and 9% in HEL only. The annual isolations in HeLa "R" were between 1.7 and 4 times greater than in HEL cells and may have been due to changes in the sensitivity of the cells or the prevalence of serotypes favoring the HeLa "R" cells. Acid lability was more easily demonstrated in HeLa "R" than in HEL cells, because the infectivity titers obtained were 10- to 100-fold higher.     

Entry of human rhinovirus 89 via ICAM-1 into HeLa epithelial cells is inhibited by actin skeleton disruption and by bafilomycin

HRV89, a major-group rhinovirus, uses intercellular adhesion molecule 1 (ICAM-1) for cell entry, while minor-group HRV2 uses the LDL receptor for clathrin-mediated endocytosis. Entry of HRV89 into HeLa epithelial cells was found to be inefficient, and infectious virus was still detected on the plasma membrane after 3 h of incubation with the cells. Endocytosis, and consequently infection, of HRV89 but not of HRV2, was almost completely blocked by the actin-polymerization inhibitor cytochalasin D, while the phosphatidylinositol 3-kinase inhibitor LY294002 had no effect on infection with either virus. Cytochalasin D also inhibited major-group HRV infection of rhabdomyosarcoma cells expressing ICAM-1 when the time available for uncoating was limited to 30 min. Although cholesterol depletion strongly inhibited HRV89 infection of HeLa cells, it only slightly affected HRV89 endocytosis, indicating that a lipid raft environment was not essential for virus uptake. The sodium-proton exchange inhibitor 5-(N-ethyl-N-isopropyl) amiloride (EIPA) significantly reduced cell entry and infection by HRV89 only at a concentration that also inhibited HRV2 infection and Alexa 488-transferrin entry. These data rule out classical macropinocytosis as an infectious entry pathway of HRV89 in HeLa cells. Notably, the proton ATPase inhibitor bafilomycin strongly affected cell entry of both viruses, suggesting a role for submembraneous pH in rhinovirus endocytosis.     

unr, a cellular cytoplasmic RNA-binding protein with five cold-shock domains, is required for internal initiation of translation of human rhinovirus RNA

Initiation of translation of the animal picornavirus RNAs occurs via a mechanism of direct ribosome entry, which requires a segment of the 5' UTR of the RNA, known as the internal ribosome entry site (IRES). In addition, translation of the enterovirus and rhinovirus (HRV) subgroups requires cellular trans-acting factors that are absent from, or limiting in rabbit reticulocytes, but are more abundant in HeLa cell extracts. It has been shown previously that HeLa cells contain two separable activities, each of which independently stimulates HRV IRES-dependent translation when used to supplement reticulocyte lysate; one of these activities was identified as polypyrimidine tract-binding protein (PTB). Here, the purification of the second activity is achieved by use of an RNA-affinity column based on the HRV 5' UTR. It comprises two components: a 38-kD protein (p38), which is a novel member of the GH-WD repeat protein family and has no intrinsic RNA-binding activity; and a 96- to 97-kD protein doublet, which was identified as unr, an RNA-binding protein with five cold-shock domains. Coimmunoprecipitation with antibodies against either protein shows that the two proteins interact with each other, and thus p38 is named unrip (unr-interacting protein). Recombinant unr acts synergistically with recombinant PTB to stimulate translation dependent on the rhinovirus IRES. In contrast, unr did not significantly augment the PTB-dependent stimulation of poliovirus IRES activity.     

Development of a high-throughput human rhinovirus infectivity cell-based assay for identifying antiviral compounds

Asthma and chronic obstructive pulmonary disease exacerbations are associated with human rhinovirus (HRV) lung infections for which there are no current effective antiviral therapies. To date, HRV infectivity of cells in vitro has been measured by a variety of biochemical and immunological methods. This paper describes the development of a high-throughput HRV infectivity assay using HeLa OHIO cells and a chemiluminescent-based ATP cell viability system, CellTiter-Glo from Promega, to measure HRV-induced cytopathic effect (CPE). This CellTiter-Glo assay was validated with standard antiviral agents and employed to screen AstraZeneca compounds for potential antiviral activity. Compound potency values in this assay correlated well with the quantitative RT-PCR assay measuring HRV infectivity and replication in human primary airway epithelial cells. In order to improve pan-HRV screening capability, compound potency was also measured in the CellTiter-Glo assay with a combination of 3 different HRV serotypes. This HRV serotype combination assay could be used to identify quickly compounds with desirable broad spectrum antiviral activity.     

Demonstration of dual rhinovirus infection in humans by isolation of different serotypes in human heteroploid (HeLa) and human diploid fibroblast cell cultures.

The ability to isolate rhinoviruses in human heteroploid cell cultures was investigated by inoculating HeLa cells (HeLa M) with specimens previously shown to be positive in human diploid cell cultures. The 135 positive specimens selected were representative of 22 different rhinovirus types, and 4 to 9 specimens were available for each serotype. Specimens were inoculated into human diploid fetal tonsil fibroblasts (FT), HeLa cells with 30 mM Mg2+, and HeLa cells without increased Mg2+. One hundred twelve rhinovirus strains (83%) were reisolated in FT cells, whereas 76 rhinovirus strains (56%) were recovered in HeLa cells with 30 mM Mg2+. All strains recovered in FT were the same serotype as that originally recovered in diploid cells, but five of the HeLa cell isolates (3.7% of total specimens) were different serotypes, indicating dual rhinovirus infections. Four rhinovirus serotypes, (3, 42, 48, and 70) were recovered in HeLa but not in diploid cells; these serotypes were rare in our previous studies. Isolation of rhinovirus in FT cells was usually accomplished at first passage, whereas rhinovirus cytopathic effects in HeLa cells were not observed at first passage, but required one, two, or (rarely) three blind passages. Only 28 rhinoviruses (21%) were recovered in HeLa cells without increased Mg2+; however, three serotypes, types 16, 36, and 58, were recovered as effectively in HeLa cells, with or without added Mg2+, as they were in FT cells. In general, rhinoviruses were less efficiently recovered in HeLa cells; however, certain serotypes may be detected better by HeLa cells.     

Epidemiological analysis and follow‐up of human rhinovirus infection in children with asthma exacerbation

To determine the prevalence of human rhinovirus (HRV) infection in children with acute asthma exacerbations, investigation of HRV viral load and severity of asthma exacerbations is also required. Nasopharyngeal aspirates and swabs were collected and assessed for respiratory viruses. HRV‐positive samples were sequenced to identify types and determine viral load. Outpatients with asthma exacerbations underwent follow‐up evaluations, their swabs were collected and clinical outcomes were recorded at their next clinic visit 4 weeks later. One hundred forty‐three inpatients and 131 outpatients, including 88 patients with asthma exacerbations and 43 controls with stable asthma were recruited. HRV‐A was mainly detected in September and February (45.5% and 33.3%, respectively), while HRV‐C was mainly detected in November and April (70.0% and 55.6%, respectively). HRV‐C was the primary type and was primarily found in inpatients with severe asthma exacerbations. HRV‐A viral load in the group of inpatients with severe exacerbations was higher than in the mild and moderate groups (P < 0.001 and P = 0.022). The HRV‐A viral load of both inpatients and outpatients was higher than that of HRV‐C (P < 0.001 and P = 0.036). The main genotypes were HRV‐C53 and HRV‐A20 among inpatients, and this genotype caused more severe clinical manifestations. HRV persisted for no more than 4 weeks, and their symptoms or signs of disease were well‐controlled well. HRV‐C was most frequently detected in asthma exacerbations. HRV‐A with high viral load led to severe asthma exacerbations.

     

Internalization of human rhinovirus 14 into HeLa and ICAM-1-transfected BHK cells

Virus adsorption and uptake of human rhinovirus 14 (HRV14) were studied with HeLa cells and baby hamster kidney (BHK) cells which were transfected with the HRV14 receptor intercellular adhesion molecule-1 (ICAM-1). Transmission electron microscopy of HeLa cells revealed that HRV14 was internalized via clathrin-coated pits and -coated vesicles. A minority of virus particles also used uncoated vesicles for entry. The internalization showed the characteristics of receptor-mediated endocytosis. Presence of the carboxylic ionophore monensin inhibited viral uncoating, indicating a pH-dependent entry mechanism. The expression of ICAM-1 on the surface of the ICAM-1 transfected baby hamster kidney cells (BHK-ICAM cells) allowed extensive virus adsorption and internalization through membrane channels. Virus particles were lined up in these channels like pearls on a string, but did not induce a productive infection. Although ICAM-1 was expressed to the same degree on BHK-ICAM and HeLa cells, HRV14 induced neither viral protein and RNA syntheses nor infectious virus progeny in BHK-ICAM cells. ICAM-1 on the transfected BHK cells was a functional active receptor as it rendered these cells permissive to coxsackievirus A21. These results suggest that HRV14 uptake into BHK-ICAM cells is blocked directly in or shortly after its final step of internalization, the uncoating. Our findings underline that the receptor ICAM-1 determines virus uptake into cells, however, is not sufficient to confer susceptibility of BHK cells to HRV14 infection. Received: 11 October 1996     

The effects of cromolyn sodium on the nasal mast cells

To elucidate the characteristics of two different kinds of nasal mast cells, ie, the mucosal mast cells (MMC) and connective tissue mast cells, we examined the inhibitory effects of cromolyn sodium (CS) on in vitro allergen-induced histamine release from the nasal mast cells. The pharyngeal mast cells were also examined as control. Nasal scrapings (containing mainly MMC) were faintly sensitive to CS, whereas both the chopped nasal mucosas without epithelial layer and chopped pharyngeal mucosas (containing mostly connective tissue mast cells) were markedly sensitive in patients with allergic rhinitis. This result indicates that there are at least two distinct types of mast cells in allergic nasal mucosa, and the nasal MMC are insensitive to CS.     

Rapid detection of respiratory syncytial virus in nasopharyngeal secretions by enzyme-linked immunosorbent assay.

A new enzyme-linked immunosorbent assay (ELISA) respiratory syncytial virus antigen detection kit (Ortho Diagnostics, Inc., Raritan, N.J.) was compared with virus culture and with the indirect fluorescent antibody test (FAT) by using fresh nasal washings from children with suspected respiratory syncytial virus infection. Both uncentrifuged nasal washings and pellets from centrifuged split specimens were examined by ELISA. The ELISA was considered positive when the optical density was greater than the mean background optical density plus 0.15. Specimens positive by ELISA but negative by culture and FAT were reexamined in an ELISA blocking assay. Of 337 uncentrifuged specimens, 124 (37%) were positive by culture, 107 (32%) were positive by FAT, and 166 (49%) were positive by ELISA. Blocking assays showed that 21 of 30 (70%) of the seemingly false-positive ELISA tests were, in fact, true-positives and that the cultures and FATs were falsely negative. A patient specimen was considered positive when it was positive by virus culture, FAT, or blocking assay. The sensitivity, specificity, and positive predictive value of the ELISA test were 88, 94, and 95%, respectively. Centrifugation of nasal washings raised the sensitivity from 88 to 92% but reduced the specificity from 94 to 81%. We conclude that the Ortho ELISA test of uncentrifuged nasal washings is more sensitive than virus culture or indirect FAT and is highly specific.     

Molecular epidemiology and genetic diversity of human rhinovirus affecting hospitalized children in Rome

Human rhinoviruses (HRV) have been re-classified into three species (A–C), but the recently discovered HRV-C strains are not fully characterized yet. This study aimed to undertake a molecular and epidemiological characterization of HRV strains infecting children hospitalized over one year in two large research hospitals in Rome. Nasal washings from single HRV infections were retrospectively subjected to phylogenetic analysis on two genomic regions: the central part of the 5′Untranslated Region (5′UTR) and the Viral Protein (VP) 4 gene with the 5′ portion of the VP2 gene (VP4/2). Forty-five different strains were identified in 73 HRV-positive children: 55 % of the cases were HRV-A, 38 % HRV-C and only 7 % HRV-B. HRV-C cases were less frequent than HRV-A during summer months and more frequent in cases presenting wheezing with respect to HRV-A. Species distribution was similar with respect to patient age, and seasonality differed during summer months with fewer HRV-C than HRV-A cases. On admission, a significantly higher number of HRV-C cases presented with wheezing with respect to HRV-A. The inter- and intra-genotype variability in VP4/2 was higher than in 5′UTR; in particular, HRV-A patient VP4/2 sequences were highly divergent (8–14 %) at the nucleotide level from those of their reference strains, but VP4 amino acid sequence was highly conserved. In HRV-C isolates, the region preceding the initiator AUG, the amino acids involved in VP4 myristoylation, the VP4–VP2 cleavage site and the cis-acting replication element were highly conserved. Differently, VP4 amino acid conservation was significantly lower in HRV-C than in HRV-A strains, especially in the transiently exposed VP4 N-terminus. This study confirmed the high number of different HRV genotypes infecting hospitalized children over one year and reveals a greater than expected variability in HRV-C VP4 protein, potentially suggestive of differences in replication.     

Molecular detection and confirmation of Neisseria gonorrhoeae in urogenital and extragenital specimens using the Abbott CT/NG RealTime assay and an in-house assay targeting the porA pseudogene

Culture for detection of Neisseria gonorrhoeae (NG) is being replaced by molecular assays, but difficulties are observed with false positive and negatives results, especially for extragenital samples. This study evaluates the Abbott CT/NG Real-Time assay and a real-time porA pseudogene assay. Samples (n = 600) from a mixed prevalence Irish population include 164 male urines with corresponding urethral swabs, 58 endocervical swabs, 173 male pharyngeal swabs, 205 male rectal swabs, 36 NG clinical isolates and 26 commensal Neisseria species isolates. There was a 100% concordance between the Abbott CT/NG Real-Time and the porA assay. The positivity rate was 1.2%, 1.7%, 8.1% and 5.8% for FVU/urethral swabs, endocervical, pharyngeal and rectal swabs, respectively. These results were compared to culture and discrepancies were found with nine pharyngeal and three rectal swabs. Seven of the 12 discrepant positive samples were sequenced and were confirmed "true positives". The sensitivity and specificity of the molecular assays was 100%. The sensitivity of the culture-based testing was 100% for urogenital samples but 36% and 75% for pharyngeal and rectal swabs, respectively. The combined Abbott CT/NG and porA assays provide a valuable alternative to culture and also generate a significant increase in the diagnosis of pharyngeal and rectal NG infection.     

Practical recommendations for the detection of pediatric respiratory syncytial virus infections.

In our private clinic-hospital setting, respiratory syncytial virus (RSV) was isolated from infants more frequently and sooner from nasal washes (84%; 4.2 days) than from throat swabs (45%; 5.5 days) or nasopharyngeal swabs (39%; 5.7 days). Immunofluorescence of nasal wash cells identified 72% of the infants with virus isolations from nasal washes in less than one day. We therefore recommend the combination of isolation and immunofluorescence on nasal wash specimens for optimal detection of RSV-infected infants. Immunofluorescence of respiratory tract cells was also useful for monitoring the presence of RSV antigen in intubation secretions during ribavirin antiviral therapy. RSV infectivity was maintained in phosphate-buffered saline at room temperature for 6 h. Transport and inoculation of specimens in less than 6 h yielded RSV isolates from 50% of sampled infants during the two RSV seasons examined. For optimal RSV isolation, we recommend inoculation of HEp-2 tubes less than or equal to 4 days old. Replacing medium after 3 days as compared with 7 days did not increase recovery of RSV and provided little practical reduction in time to detection of cytopathology.