virology blog
About viruses and viral disease
On episode #223 of the science show This Week in Virology, Vincent, Alan, and Kathy discuss new influenza virus NA inhibitors, detection of EEEV antibody and RNA in snakes, and replication of the coronavirus EMC in human airway epithelial cells.
You can find TWiV #223 at www.microbe.tv/twiv.
Frederick Hayden, Professor of Medicine and Pathology, University of Virginia School of Medicine, U.K., has focused on the use of antiviral agents to prevent and treat respiratory viral infections. His interests range from the use of in vitro assays to study viral susceptibility and antiviral mechanisms of action, to clinical trials utilizing experimentally induced and naturally occurring infections. Work from his laboratory includes the demonstration that intranasal administration of interferons can prevent transmission of rhinovirus colds, studies of transmission of drug-resistant influenza A viruses in families, and the antiviral activity and clinical use of influenza neuraminidase inhibitors. His laboratory currently focuses on the application of nucleic acid hybridization to study rhinovirus pathogenesis, elucidating the phenotypic and genotypic basis of antiviral drug resistance in rhinovirus and influenza viruses, and clinical testing candidate antiviral agents for influenza and rhinovirus infections.
I discussed the use of antiviral drugs to treat influenza with Dr. Hayden during ICAAC Boston 2010, as part of TWiV 99. View the video below, or at YouTube.
There are two glycoproteins embedded in the influenza viral membrane: the hemagglutinin (HA) and neuraminidase (NA). The NA, shown in yellow in the illustration, is an enzyme that removes sialic acids from the surface of the cell, so that newly formed virions can be released. The NA protein is composed of a box-like head attached to the viral membrane via a stalk. The length of the stalk may be an important determinant of the virulence of avian influenza H5N1 viruses.
Examination of the sequence of all known influenza N1 NAs reveals that the proteins can be grouped into six classes depending on the length of the stalk region. The stalk regions of some NAs are intact, while others lack from 15 to 22 amino acids. In 2000, influenza H5N1 isolates from humans were identified with a deletion of stalk amino acids 49-68. The percent of human H5N1 isolates with this deletion has steadily increased from 15.8% in 2000 to 100% in 2007. This observation led to the question: does stalk length influence H5N1 pathogenesis in animals?
The authors produced a series of H5N1 reassortant viruses with NA stalks of different lengths. Six of the 8 viral RNAs were derived from the 1933 H1N1 isolate WSN. The pathogenicity of the reassortant viruses was determined in chickens and in mice. Three of the viruses were highly pathogenic in chickens: one with the NA from a 2004 H5N1 isolate containing the deletion of amino acids 49-68, a second with the NA from WSN virus, and a third with the N1 from a 1997 H5N1 isolate. The NA stalks from the latter two viruses have deletions, but they are different from the one in the 2004 H5N1 NA. The other viruses tested were found to be of low pathogenicity in chickens. These included a virus with an N1 from an 1996 H5N1 virus with an intact stalk, as well as viruses with NAs harboring different deletions of various lengths. The findings in mice paralleled those obtained in chickens.
These results show that the NA stalk plays a critical role in virulence of H5N1 avian influenza virus in chickens and in mice. The 20 amino acid deletion from amino acids 49-68 is associated with high virulence, although similar pathogenicity was observed for viruses with different stalk deletions. These results do not answer the important question: why viruses with this particular NA deletion have become prevalent among human H5N1 influenza virus isolates. H5N1 viruses with the same stalk deletion have been isolated from aquatic birds and terrestrial poultry since 2002. It has been suggested that the stalk deletion is associated with adaptation of influenza viruses to land-based poultry, but the advantages conferred by this particular NA are unknown.
Zhou, H., Yu, Z., Hu, Y., Tu, J., Zou, W., Peng, Y., Zhu, J., Li, Y., Zhang, A., Yu, Z., Ye, Z., Chen, H., & Jin, M. (2009). The Special Neuraminidase Stalk-Motif Responsible for Increased Virulence and Pathogenesis of H5N1 Influenza A Virus PLoS ONE, 4 (7) DOI: 10.1371/journal.pone.0006277
Tamiflu (Oseltamivir) is one of the few antiviral drugs available for treatment of influenza. Use of the drug has increased substantially because of the emergence of the 2009 H1N1 pandemic strain, against which no vaccine is yet available. A recent study has shown that low levels of oseltamivir can be detected in the aquatic environment. This finding raises the possibility that aquatic birds which harbor influenza virus could be exposed to the antiviral, leading to selection of drug resistant viruses.
Oseltamivir is an inhibitor of the influenza neuraminidase (NA) glycoprotein. This enzyme removes sialic acids from the surface of the cell, so that newly formed virions can be released. Neuraminidase inhibitors such as Tamiflu and Relenza function by preventing cleavage of sialic acid from the cell surface. These inhibitors act by binding to the sialic-acid binding pocket of the NA protein.
Tamiflu is s prodrug, oseltamivir phosphate, which is converted to the active form, oseltamivir carboxylate (OC) in the liver. The active form of oseltamivir is excreted in the urine, and is not removed by sewage treatment plants. It has therefore been suggested that OC may present in the aquatic environment, and could expose natural reservoirs of influenza virus to low levels of the antiviral.
Because Japan is the largest consumer of Tamiflu, the levels of OC were determined in the Yodo River system in the Kyoto and Osaka prefectures. This river was selected because it is distant from the sea and located in a densely populated area. Surface water was collected before (June 2007) and during (December 2007 and February 2008) the flu season. No OC was detected in water samples from June 2007. At the onset of the flu season, December 2007, the antiviral was found at levels between 2 and 7 nanograms per liter (ng/L). At the peak of the flu season, in February, levels increased to 12 – 58 ng/L. Levels of OC were higher in water samples taken near sewage treatment plants, compared with those obtained farther away. The amounts detected are close to the concentration of drug that causes 50% inhibition of virus replication (the IC50) in cell cultures, reported to be between 80–230 ng/L.
The authors suggest that dabbling ducks, a natural reservoir of influenza virus, could ingest OC. As influenza in dabbling ducks is a gastrointestinal infection, the virus would encounter oseltamivir in the gut, which could promote selection of viruses resistant to the drug.
It is not known whether OC in aquatic environments leads to influenza virus resistance to Tamiflu. Clearly additional studies must be done to determine whether the antiviral drug can be found in other waters around the world. Influenza viruses should be isolated from aquatic birds living in OC contaminated environments to monitor resistance to Tamiflu.
Söderström, H., Järhult, J., Olsen, B., Lindberg, R., Tanaka, H., & Fick, J. (2009). Detection of the Antiviral Drug Oseltamivir in Aquatic Environments PLoS ONE, 4 (6) DOI: 10.1371/journal.pone.0006064
Our discussion of influenza virus replication has so far brought us to the stage of viral RNA synthesis. Last time we discussed the formation of viral RNAs, an event which takes place in the cell nucleus. Now we’ll consider how these RNAs participate in the assembly of new infectious viral particles, as illustrated in the following figure.
For simplicity, the nucleus is not shown. But remember that the viral RNAs have to be exported from the nucleus to the cytoplasm, where viral assembly occurs. First, the viral mRNAs are translated to produce all the proteins needed to synthesize a new virus particle. The mRNAs encoding the HA and NA glycoproteins are translated by ribosomes that are bound the the endoplasmic reticulum – the membranous organelle that assists in transporting certain proteins to the plasma membrane. As the HA and NA proteins are produced, they are inserted into the membrane of the endoplasmic reticulum as shown. These proteins are then transported to the cell surface via small vesicles that eventually fuse with the plasma membrane. As a result, the HA and NA are inserted in the correct direction in the lipid membrane of the cell. The M2 protein is sent to this location in a similar way.
The (-) strand viral RNAs that will be packaged into new virus particles are produced in the cell nucleus, then exported to the cytoplasm. These RNAs are joined with the viral proteins PA, PB1, PB2, and NP. Viral proteins other than HA, NA, and M2 are produced by translation on free ribosomes, as shown for M1. The latter protein binds to the membrane where HA, NA, and M2 have been inserted. The assembly consisting of viral RNAs and viral proteins – called a ribonucleoprotein complex or RNP -Â travels to the site of assembly. The virion then forms by a process called budding, during which the membrane bulges from the cell and is eventually pinched off to form a free particle.
As new virions are produced by budding, they would immediately bind to sialic acid receptors on the cell surface, were it not for the action of the viral NA glycoprotein. This enzyme removes sialic acids from the surface of the cell, so that newly formed virions can be released. This requirement explains how the neuraminidase inhibitors Tamiflu and Relenza function: they prevent cleavage of sialic acid from the cell surface. In the presence of these inhibitors, virions bud from the cell surface, but they remain firmly attached. Therefore Tamiflu and Relenza block infection by preventing the spread of newly synthesized virus particles to other cells.