TWiM 64: URI and UTI at ICAAC

This episode of TWiM was recorded at the 53rd ICAAC in Denver, Colorado, where Michael Schmidt and I spoke with James Gern about rhinoviruses, and James Johnson about extra-intestinal pathogenic E. coli.

You can find TWiM #64 at microbeworld.org, or view the video below.

Zinc inhibits rhinovirus replication

hrv1a_zincThe title of this post should not come as a surprise to readers of virology blog – it was shown in 1974 that zinc could interfere with replication of rhinoviruses (see “Zinc and the common cold“). I am referring to the result of my first experiment to study the mechanism of zinc inhibition – something I promised I would document on these pages.

I am interested in understanding how zinc inhibits rhinovirus replication. Answering this question could lead to new ways to prevent common colds caused by these viruses. The first step was to reproduce the effect of zinc in my laboratory with my stocks of rhinovirus. I selected rhinovirus type 1a for my initial experiments because we’ve worked with this serotype in the past: we know the genome sequence and how the virus behaves in a mouse model. I started by doing a plaque assay with and without zinc in the medium. I prepared tenfold dilutions of virus and inoculated separate monolayers of HeLa cells with 2000, 200, and 20 plaque forming units. After allowing the virus to attach to cells for 45 minutes, I added an agar overlay to the cells with or without zinc chloride (ZnCl2). I selected 0.1 millimolar ZnCl2 because that is the concentration which had been reported to effectively inhibit plaque formation by rhinovirus type 1a. The plates were incubated for four days at 32°C and then stained. The results are shown in the photo. Plaque assays are typically done in duplicate but for simplicity only one plate of each dilution is shown.

Twenty plaques were observed on the highest dilution of virus plated in the absence of ZnCl2. Ten-fold lower dilutions produced increases in plaque number, although the plaques are too numerous to count. In the presence of ZnCl2, no plaques were observed on cells inoculated with 20 PFU. A few plaques are observed on the intermediate dilution and many more on the lowest dilution. Plaques observed in the presence of ZnCl2 are smaller than those observed in the absence of the metal.

What do you think is going on here, and what should I do next? If you’ve kept up with virology 101 you have all the tools to answer these questions. Please post your thoughts in the comments section.

KORANT, B., KAUER, J., & BUTTERWORTH, B. (1974). Zinc ions inhibit replication of rhinoviruses Nature, 248 (5449), 588-590 DOI: 10.1038/248588a0

Zinc and the common cold

cold-eezeShortly after I developed sore throat, cough, and congestion last week, a package of ‘Cold – Eeze’ materialized on my kitchen counter. The writing on the package of zinc-laden lozenges promised to ‘shorten your cold’, and noted that they were ‘clinically proven to reduce the duration of the common cold’. Do zinc lozenges have any effect on the common cold?

The common cold is the primary cause of doctor visits in the United States, leading to 189 million lost school days each year. But it’s important to point out that the common cold can be caused by a number of different viruses, including rhinovirus, coronavirus, influenza virus, adenovirus, and paramyxovirus. Rhinoviruses are responsible for over half of all common colds.

The idea that zinc could be used to treat the common cold originated from a 1974 paper in Nature which showed that zinc blocks the replication of rhinoviruses in cell culture. Viral plaque formation was inhibited over 99% when 0.1 millimolar zinc chloride was included in the agar overlay. However, this concentration of zinc is too high for therapeutic use, and subsequent studies showed that levels compatible with use of the metal ion in humans minimally inhibited rhinovirus replication in cell culture. Zinc does not readily pass through the cell membrane, explaining why high concentrations are required to produce an antiviral effect.

Many trials have been conducted to determine if zinc – taken as a lozenge, nasal spray, or ointment – has any effect on the common cold in humans. In one study, 200 children were given 15 mg zinc daily by mouth for seven months. The mean number of colds in the treated children was 1.2 compared with 1.7 in the untreated children – statistically significant but not therapeutically useful. Many studies have evaluated the effectiveness of zinc containing lozenges. In one, 65 people took one lozenge containing 23 mg zinc every two hours while awake. After one week, 86% of the treated group were free of cold symptoms, compared with 46% of the placebo group. In another similar study, the duration of cold symptoms was reduced in the zinc group versus the placebo group – 4.5 days compared with 8.1 days. However, as many studies have lead to the conclusion that zinc lozenges – as well as zinc administered intransally or in a gel – have no effect on severity or duration of the common cold. A good summary of many of these trials can be found in the Alternative Medicine Review cited below.

It’s important to note that in these studies the virus responsible for the colds is not identified. Given the prevalence of rhinoviruses it is appropriate to assume that these viruses are involved in over half of the colds observed. Nevertheless, the extreme variability in the trial results may in part reflect the fact that various etiologic agents are involved, some of which might not be susceptible to inhibition by zinc. Other possible explanations for the inconsistent results include differences in the zinc preparations used (zinc gluconate and zinc acetate), the quantity of zinc administered, and the composition of the lozenge.

Although inhibition of rhinovirus replication by zinc was reported in 1974, the mechanism is not understood. It is believed that zinc enters the cell and binds to the rhinovirus protein that will form the capsid. This interaction blocks cleavage of the protein, thereby inhibiting production of infectious virus. Consistent with this proposed mechanism is the observation that zinc ionophores – compounds that allow the uptake of zinc into cells – have recently been shown to inhibit rhinovirus replication. The effectiveness of such compounds, which include pyrithione and hinokitiol, for treating the common cold is currently being investigated.

Although zinc does inhibit rhinovirus replication, this activity might not account for the effect on the common cold. It has been suggested that zinc reduces inflammation in the respiratory tract, which would explain the observed decrease in symptoms observed in some trials.

As for the Cold-eeze on my kitchen counter – the package was never opened. The unimpressive results of clinical trials made the idea of taking 6-8 lozenges a day for several days less appealing than enduring sore throat, cough, and congestion for less than a week. But the lozenges served a different purpose – I am now very interested in the revealing the mechanism of zinc inhibition of rhinovirus replication. I have begun experiments in my lab to solve this problem, and I’ll write about what I discover.

Korant BD, Kauer JC, & Butterworth BE (1974). Zinc ions inhibit replication of rhinoviruses. Nature, 248 (449), 588-90 PMID: 4363085

Geist FC, Bateman JA, & Hayden FG (1987). In vitro activity of zinc salts against human rhinoviruses. Antimicrobial agents and chemotherapy, 31 (4), 622-4 PMID: 3038000

Krenn, B., Gaudernak, E., Holzer, B., Lanke, K., Van Kuppeveld, F., & Seipelt, J. (2008). Antiviral Activity of the Zinc Ionophores Pyrithione and Hinokitiol against Picornavirus Infections Journal of Virology, 83 (1), 58-64 DOI: 10.1128/JVI.01543-08

Roxas M, & Jurenka J (2007). Colds and influenza: a review of diagnosis and conventional, botanical, and nutritional considerations. Alternative medicine review : a journal of clinical therapeutic, 12 (1), 25-48 PMID: 17397266

Is an effective treatment for the common cold at hand?

2289334120_c27ed36206_mThe sequence of all known rhinovirus genomes reported in Science last week is an important advance for the field. Analyses of the sequences have revealed new relationships among the viruses, evidence for recombination, a new viral species, and conserved regions of the genome. These findings will be extremely valuable for those studying the biology, pathogenesis, and epidemiology of the common cold. But the press has over reacted to this work –  it was reported on the front page of the New York Times with the headline “Cure for the Common Cold? Not Yet, but Possible“.  Does the work deserve such fanfare?

The Times quoted Stephen Liggett, an asthma expert, as saying “We are now quite certain that we see the Achilles’ heel, and that a very effective treatment for the common cold is at hand.” He was apparently referring to the observation that a sequence within the 5′-noncoding region of the viral genome is highly conserved among the 99 rhinovirus sequences, in comparison with other regions of the viral RNA. He suggested that all 99 rhinovirus serotypes would therefore be susceptible to the same drug. But what kind of drug, and what function would it inhibit? The very 5′-end of the genome of enteroviruses and rhinoviruses binds viral and cellular proteins, and these interactions are essential for viral replication. So it might be possible to identify small molecules that block these protein-RNA interactions. But such drugs are very difficult to identify. Furthermore, if such a drug were identified, its efficacy would have to be tested against all rhinovirus serotypes. Therefore it is not clear that knowing that this sequence of the genome is conserved helps to identify drug targets and more readily than did the observations made years ago about the importance of RNA-protein interactions in this region. Clearly, many years of research are needed before such drugs are developed – not consistent with Dr. Liggett’s a treatment is ‘at hand’.

An even more crucial aspect of the problem was omitted from the Times article. Even if an antiviral drug could be identified that blocks essential RNA-protein interactions, it probably would not be useful in treating the common cold. As we discussed last week, rhinoviruses cause acute infections – characterized by rapid onset of disease, a relatively brief period of symptoms, and resolution within days. Most are complete by the time the patient feels ill, and the virus has already spread to another host. Antiviral therapy  must be given early in infection to be effective. There is little hope of treating most acute viral infections with antiviral drugs until rapid diagnostic tests are become available.

To be fair, some of the scientists quoted in the Times article were more realistic about the possibilities for rhinovirus treatments. One antiviral drug expert noted that it costs about $700 million to bring a drug to market. Because most rhinovirus infections are benign, who would pay for such an expensive drug, and would the Food and Drug Administration ever approve it? Ann Palmenberg, the lead author on the study, was even more realistic, admitting that a rhinovirus vaccine would not likely be made.

I cannot see how this new study identified a new or better target for therapeutic intervention. So why get the public excited by running a front page headline in the New York Times? It’s great to keep the public informed about scientific progress – but the press should not cry wolf. If this advance does not soon lead to a treatment for the common cold, the public will shake their heads and lose a bit more trust in science.

I’m not blaming the scientists for this over reaction to their study. I am sure that the journal Science engaged in strong pre-publication promotion: more publicity is better for their advertising revenues. And the newspapers are equally at fault: they should speak to a broader range of scientists to obtain a more balanced view. I particularly blame the author of the Times article, Nicholas Wade, for not sufficiently researching his article.

Perhaps Dr. Liggett and his colleagues would benefit from the lessons of history – specifically, the history of poliomyelitis and its conquest. On March 9, 1911, three years after the isolation of poliovirus, The Rockefeller Institute issued a press release, saying that it believed “that its search for a cure for infantile paralysis is about to be rewarded. Within six months, according to Dr. Simon Flexner, definite announcement of a specific remedy may be expected.” They quoted Dr. Flexner:  “We have already discovered how to prevent the disease, and the achievement of a cure, I may conservatively say, is not now far distant.” Dr. Flexner’s imminent ‘cure’ was a failure, and a successful poliovirus vaccine required another 44 years of research. Last week’s Times article seemed to have a similar overdose of hubris.

A. C. Palmenberg, D. Spiro, R. Kuzmickas, S. Wang, A. Djikeng, J. A. Rathe, C. M. Fraser-Liggett, S. B. Liggett (2009). Sequencing and Analyses of All Known Human Rhinovirus Genomes Reveals Structure and Evolution Science DOI: 10.1126/science.1165557