W. Al-Nakib

The time course of the humoral immune response to rhinovirus infection

The specific humoral immune response of 17 volunteers to infection with human rhinovirus type 2 (HRV-2) has been measured both by neutralization and by ELISA. Six volunteers who had HRV-2-specific antibodies in either serum or nasal secretions before HRV-2 inoculation were resistant to infection and illness. Of the remaining 11 volunteers who had little pre-existing HRV-2-specific antibody, one was immune but 10 became infected and displayed increases in HRV-2-specific antibodies. These antibodies first increased 1-2 weeks after infection and reached a maximum at 5 weeks. All six resistant volunteers who had high pre-existing antibody and eight of the volunteers who became infected maintained their HRV-2-specific antibody for at least 1 year. At this time they were protected against reinfection. Two volunteers showed decreases in HRV-2-specific antibodies from either serum or nasal secretions. They became infected but not ill after HRV-2 inoculation 1 year later.

Detection of cytomegalovirus by DNA-DNA hybridization employing probes labelled with 32-phosphorus or biotin.

Various factors influencing the detection of human cytomegalovirus (HCMV) in infected cells by DNA-DNA hybridization have been investigated. Employing the Hind III O fragment of HCMV AD169 labelled with 32P, we found that detection sensitivity was highly influenced by the method employed for extraction of DNA from infected cells. Excision of the Hind III O fragment from the vector by restriction endonuclease digestion prior to 32P-labelling further improved the detection capability of the probe. Similarly, cytomegalovirus (CMV) DNA detection employing biotin-labelled probes and streptavidin/alkaline phosphatase in the hybridot assay was also highly dependent on the method of DNA extraction prior to hybridization. Finally, we describe an in situ assay employing a biotin-labelled probe and fluorescein-conjugated avidin to detect CMV DNA in cultured cells.

The effect of intranasal nedocromil sodium on viral upper respiratory tract infections in human volunteers.

Two studies involving double‐blind group comparative trials in human volunteers compared the effects of intranasal nedocromil sodium (2·6 mg active drug per nostril, q.i.d.) with placebo on clinical symptoms and performance impairment associated with the common cold. In the first study volunteers were challenged with rhinoviruses (RV9 and RV14), and in the second study with respiratory coronavirus. In both studies, active and placebo groups of volunteers were demographically similar. Infection rates in both groups were also similar. There were no withdrawals resulting from unusual symptoms related to either treatment. In the rhinovirus study (19, placebo; 20, nedocromil sodium) daily symptom scores and daily mean nasal secretion weights were significantly lower in the nedocromil sodium‐treated group. In the coronavirus study (26, placebo; 27, nedocromil sodium) there was little difference in the severity of colds between the active and placebo‐treated groups, but trends favoured nedocromil sodium. In both studies the impairment of performance in volunteers who developed a cold was significantly less in those treated with nedocromil sodium than in those treated with placebo.

Use of synthetic oligonucleotide probes to detect rhinovirus RNA

SummaryCurrent methods of detecting a human rhinovirus (HRV) infection are either based on isolation of virus in appropriate susceptible cell lines, which is time-consuming and requires considerable expertise, or are dependent on knowing the serotype. The extistence of over 100 immunologically distinct serotypes makes serotype specific assays, such as ELISA, unsuitable for general diagnostic assays. In this study a general rhinovirus assay is described which utilises synthetic oligonucleotides as probes in a filter hybridization assay. The probes are designed to bind to short but highly conserved regions of the rhinovirus genome. Indeed, the probes successfully detected all 57 rhinovirus serotypes tested. Furthermore, the test was used to demonstrate rhinovirus infection in clinical samples from 57 volunteers, inoculated with HRV, collected on six consecutive days. Clinical samples were taken prior to inoculation and on days 2–7 after inoculation. The filter hybridization assay gave results comparable to virus culture on days 2 and 3 post-inoculation, but was more sensitive on subsequent days.

Detection of enteroviruses using cDNA and synthetic oligonucleotide probes

n Abstractn n This study compares the detection of enterovirus RNA by cDNA probes prepared from both the 5 and 3 end of the genome of coxsackíe A21 and B4 with the use of synthetic oligonucleotides prepared from short but highly conserved sequences in the 5 end non-coding region of the picornavirus genome.n The cDNA probes detected enteroviruses with a variable level of sensitivity which presumably depended on the degree of genomic homology with the detecting probes. Generally probes from coxsackievirus A21 detected more enteroviruses than did similar probes from coxsackievirus B4. Probes from the 5 end of the genome of both viruses were more sensitive than 3 end probes. In contrast, synthetic oligonucleotides detected all enteroviruses efficiently suggesting that these probes could be useful as ‘universal’ probes to detect any enterovirus. This paper discusses the application of these probes in the diagnosis and differentiation of enteroviruses.n n

Factors affecting the detection of cytomegalovirus in urine by sandwich enzyme immunoassays

Some factors influencing the detection of human cytomegalovirus (HCMV) in urine were investigated employing 2 enzyme-linked immunosorbent assays (ELISAs); one utilised anti-CMV DNA polymerase while the other anti-CMV mouse monoclonals as the detecting antibodies. The use of anti-CMV DNA polymerase was found to be superior in detecting HCMV in both urine and tissue culture fluids than anti-CMV monoclonals. Furthermore, alkaline phosphatase conjugates produced much lower background than did peroxidase conjugates. In reconstruction experiments, the extremes of pH in the urine clearly had an adverse effect on the detection rate of extracellular virus. pH correction of urines to neutrality improved the detection rate considerably. On the other hand, pH correction had little effect on the detection rate of intracellular HCMV in urine, although it was improved when specimens were subjected to repeated cycles of freeze-thawing, ultrasonication, and storage at 4 degrees C. It was concluded that, in addition to the factors investigated which all appear to affect virus detection rate, there may well be additional factors that interfere with CMV detection in the urine by ELISA particularly with intracellular virus.

An ELISA for the detection of rhinovirus specific antibody in serum and nasal secretion.

n Abstractn n An enzyme-linked immunosorbent assay (ELISA) which detects rhinovirus specific antibody in human sera and nasal secretions, has been developed. This sandwich ELISA utilizes a rabbit antirhinovirus hyperimmune serum as the capture antibody and was found to be very sensitive, detecting rhinovirus specific antibody in the serum at dilutions of 1:106 and 1:103.5 and IgA immunoglobulins, respectively. Thus, this new assay is 102–104 times more sensitive than our standard neutralization test. Furthermore, this increase in sensitivity has enabled us to reliably detect rhinovirus specific immunoglobulins in unconcentrated nasal washings, which are thought to be particularly important for protection against rhinovirus reinfection. A preliminary study of the immune response in human volunteers challenged with rhinovirus using this new ELISA system is presented and further applications and potential of the method are also discussed.n n

A new generation of more potent synthetic antirhinovirus compounds: comparison of their MICs and their synergistic interactions

A new generation of synthetic antirhinovirus compounds has recently become available for in vitro evaluation. Thus a new group of compounds from Janssen was found to be 10-fold more active than enviroxime or 57-fold more active than dichloroflavan (DCF), against human rhinovirus 9 (HRV-9). In addition, they were also some 5- and 10-fold more potent than enviroxime and DCF, respectively, against HRV-2. Similarly, a new series of antirhinovirus compounds from Roche, although as active as enviroxime against HRV-9, were found to be 4-fold more potent than DCF against the same virus. Moreover, they were 45- and 90-fold more active than enviroxime and DCF, respectively, when tested against HRV-2. We found that generally HRV-2 was more sensitive to these new compounds than HRV-9. In this study we also report on the synergistic interaction between these new synthetic substances and also with some of the earlier compounds such as DCF and enviroxime and we discuss the possible implication of this synergistic activity regarding the future prevention and treatment of common colds caused by rhinoviruses.

Effects of zinc gluconate and nedocromil sodium on performance deficits produced by the common cold

Two studies are reported on the effects of drugs on the performance impairments induced by experimentally- produced colds. The first study examined the effects of zinc gluconate on choice reaction time, and showed that the zinc removed the cold-induced performance impairment. The second experiment used nedocromil sodium and, again, the drug was effective in reducing the drop in performance observed in volunteers with colds. While the precise mode of action of these compounds is unclear it is speculated that one mechanism involves changes in trigeminal stimulation, and this could be responsible for both the clinical efficacy and CNS effects of these drugs.

Failure to demonstrate synergy between interferon-α and a synthetic antiviral, enviroxime, in rhinovirus infections in volunteers

Marked synergy between the antirhinoviral effect of rHuIFN alpha and enviroxime has been observed in vitro but an attempt to demonstrate it in volunteers was unsuccessful. The sub-optimal intranasal dose of rHuIFN alpha (0.18 Mu four times daily for 4 1/4 days) used prophylactically in the trial did reduce the severity of colds induced by RV9 and 14, but the difference did not reach statistical significance and was not enhanced by the administration of enviroxime (0.28 mg six times daily for six days). The main reason for failure is thought to be the rapid removal of enviroxime from the nose when given intranasally.