Yet another avian influenza virus, H10N8, infects humans

chicken market

To the collection of avian influenza viruses known to sporadically infect humans – H5N1, H7N9, H7N2, H7N3, H7N7, H9N2, and H10N7 – we can now add H10N8, recently found in two individuals in China.

Avian influenza virus H10N8 was first detected in tracheal aspirates from a 73 year old woman who was hospitalized in November 2013 for severe respiratory illness. The patient, who died, had previously visited a live poultry market. A second infection with this virus was detected in January 2014.

Virus isolated from tracheal aspirates on day 7 of illness was named A/Jiangxi-Donghu/346/2013(H10N8). Nucleotide sequence analysis of the viral genome reveals that it is a reassortant. The HA gene most closely resembles that of a virus isolated from a duck in Hunan in 2012, while the NA gene resembles that of a virus isolated from a mallard in Korea in 2010. All six other RNA segments resemble those from circulating H9N2 viruses in China. These viruses have also provided genes for H7N9 and H5N1 viruses.

Examination of the viral protein sequences provides some clues about virulence of the virus. The HA protein sequence reveals a single basic amino acid at the cleavage site, indicating that the virus is of low pathogenicity in poultry, like H7N9 virus. The sequence in the sialic acid binding pocket of the HA protein indicates a preference for alpha-2,3 linked sialic acids, typical  for avian influenza viruses (human influenza viruses prefer alpha-2,6 linked sialic acids). A lysine at amino acid 627 in the PB2 protein is known to enhance the ability of the virus to replicate at mammalian temperatures; the H10N8 virus has a mixture of lysine and glutamic acid, the residue associated with less efficient replication. The sequence of the M2 protein indicates that the virus is resistant to the antiviral adamantanes. In vitro testing indicated sensitivity to NA inhibitors Tamiflu and Relenza.

It is not known if this novel H10N8 virus will spread further in the human population. A novel influenza H7N9 virus was first detected in humans in early 2013 and has since caused 250 human infections with 70 deaths. Similar incursions of avian influenza viruses into humans have probably taken place for as long as humans have had contact with poultry. We are now adept at detecting viruses and therefore we are noticing these infections more frequently.

Live poultry markets are clearly a risk factor for humans to acquire infections with avian influenza viruses, as noted by Perez and Garcia-Sastre:

Live bird markets in Asia are undoubtedly the major contributor in the evolution of avian influenza viruses with zoonotic potential, a fact for which we seem to remain oblivious.

Given their role in transmitting new viruses from animals to humans, I wonder why live poultry markets are not permanently closed.

Update: George Gao agrees that the live poultry markets in China should be closed.

Cutting through mucus with the influenza virus neuraminidase

influenza virusNeuraminidase is one of three different viral proteins embedded in the lipid membrane of influenza virus (NA is blue in the illustration at left). This enzyme has a clear and proven role in virus release from cells. NA is also believed to be important during virus entry, by degrading the mucus barrier of the respiratory tract and allowing virus to reach cells. This role is supported by the finding that treatment of mucus-covered human airway epithelial cells with the NA inhibitor Tamiflu substantially suppresses the initiation of infection.  Further evidence comes from the recent finding that influenza virus binds to sialic acids in mucus and that NA cleaves these sugars to allow infection.

The mucus layer of the respiratory tract has a defensive role because it contains soluble glycoproteins that are rich in sialic acids, which are cell receptors for some viruses. When influenza virions enter the respiratory tract, they are thought to be trapped in mucus where they bind sialic acids, preventing infection of the underlying cells. The role of NA in penetration of the mucus layer was studied in frozen sections of human tracheal and bronchial tissues, in which the mucus layer is preserved. Influenza A virions bind to this mucus layer; the interaction is blocked when the tissues are first treated with a bacterial neuramindase, which removes sialic acids from glycoproteins. These observations indicate that the mucus layer of human airway epithelial cells contain sialic acid-containing decoys that bind influenza A viruses.

Human salivary mucins, which approximate the mucus of the human respiratory tract, can protect cultured cells from influenza virus infection. The protective effect can be achieved by simply adding the mucins to cells. The inhibitory effect is dependent on the sialic acid content of the mucins: fewer cells are infected when higher concentrations are used. In contrast, porcine salivary mucins do not substantially reduce influenza virus infection. The type of sialic acids and the virus strain also determine the extent of protection.

To determine the role of the viral NA in mucin-mediated inhibition, Tamiflu was mixed with virus before infection of mucus-coated cells. The presence of Tamiflu increased the inhibition of infection caused by mucins, indicating that the sialic acid-cleaving (sialidase) activity of NA is needed to overcome inhibition by human salivary mucins. When influenza virions are incubated with human salivary mucins linked to beads, sialic acids are cleaved from mucins, and this enzymatic activity is inhibited by Tamiflu. Human salivary mucins inhibit NA by binding to the active site of the enzyme.

These studies establish a clear role for the influenza viral NA in bypassing the defenses of the mucosal barrier. An important message is that not all strains of influenza virus are equally inhibited by mucus; presumably this property is one of many that determines viral virulence. The balance between how tightly the viral HA binds to sialic acids, and how well the NA cleaves them, is probably one predictor of how well we our protected by our mucus.

 

Changing influenza virus neuraminidase into a receptor binding protein

neuraminidaseThe hemagglutinin (HA) and neuraminidase (NA) glycoproteins of the influenza virus particle serve distinct functions during infection. The HA binds sialic acid-containing cellular receptors and mediates fusion of the viral and cell membranes, while the NA removes sialic acids from glycoproteins. Apparently this division of labor is not absolute: influenza viruses have been identified with NA molecules that serve as receptor binding proteins.

An influenza virus was created that could not bind sialic acid by introducing multiple mutations into the HA gene. This mutant virus was not expected to be infectious, but nevertheless did propagate to moderate titers in cell culture. A single amino acid change was identified in the NA protein of this virus: G147R, which is just above the active site of the enzyme (illustrated; active site marked with green spheres). Passage of the virus in cell culture produced a virus that multiplied to higher titers; improved growth was caused by a K62E change in the HA stalk. The results of site-directed mutagenesis showed that the G147R change allowed the NA protein to serve the receptor binding function normally provided by HA. It is not clear how the HA change leads to improved growth of the G147 virus.

Although the G147R NA can serve as receptor binding protein, the HA is still required for fusion: abolishing this activity by mutation or by treatment with a fusion-blocking antibody did not allow virus growth.

The influenza NA protein is an enzyme (sialidase) that cleaves sialic acids from cellular and viral proteins. The G147R NA is active as a sialidase, and this activity can be blocked by the antiviral compound oseltamivir, which is an NA inhibitor. Treatment of G147R-containing virus with oseltamivir also blocked virus binding to cells. Virus-like particles that contain G147R NA but not HA can attach to sialic acid-containing red blood cells. This attachment can be reversed by oseltamivir. After binding to red blood cells, these virus-like particles slowly fall off, a consequence of NA cleaving sialic acid receptors. These observations indicate that the G147R NA binds to sialic acids at the active site of the enzyme, and cleaves the same receptor that it binds.

Treatment of cells with a bacterial sialidase that removes a broad range of sialic acids only partially inhibits G147R NA-mediated binding to cells. In contrast, growth of wild type influenza virus is completely blocked by this treatment. Therefore the receptor recognized by G147R NA is not the same as that bound by wild type virus.

Changing the influenza virus NA to a receptor binding protein is not simply a laboratory curiosity: the G147R NA change was found in 31 of 19,528 NA protein sequences in the Influenza Virus Resource. They occur in seasonal H1N1 viruses that circulated before 2009, in the 2009 swine-origin pandemic H1N1 virus, and in avian H5N1 viruses. The presence of this change in phylogenetic clusters of seasonal H1N1 and chicken H5N1 sequences suggests that they are also found in circulating viruses, and are not simply sequence errors or the product of passage in the laboratory.

These observations emphasize the remarkable flexibility of the influenza viral glycoproteins in their ability to switch receptor binding function from HA to NA. They might also have implications for vaccines, whose effectiveness are thought to depend largely on the induction of antibodies that block the function of HA protein. The work underscores the importance of serendipity in science: the HA receptor binding mutant virus was originally produced as a negative control for a different experiment.

TWiV 236: Flu gets the VIP treatment

On epside #236 of the science show This Week in Virology, Vincent, Alan and Kathy review novel approaches to preventing influenza virus infection.

You can find TWiV #236 at www.microbe.tv/twiv.

Influenza neuraminidase and H5N1 pathogenicity

influenza_virion_250There 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