.. All, en mis cuadros, formas pequeas en espacios enormes y vacos..

(Joan Mir, 1958 - Bleu II)

Virologa Animal - MCR-0603 Ciencias Biolgicas y Ambientales Universidad San Francisco de Quito

Lecture 7: Influenza A virus

Influenza viruses
Segmented Negative Strand RNA Viruses Orthomyxoviridae family: Three genera of influenza virus:
Genus Influenzavirus A 8 genome segments Genus Influenzavirus B - 8 genome segments Genus Influenzavirus C - 7 genome segments, no neuraminidase

Influenza A virus
Orthomyxoviridae
Influenza virus most important Helical (-) stranded segmented RNA genome 100 nm, 13 kb genome divided into 8 segments Highly variable natural biology well-studied Replication in nucleus, assembly in cytoplasm

Influenza A virus components

Envelope proteins HA hemagglutinin: trimer of glycoproteins involved in attachment NA neuraminidase: tetramer involved in release from endosome M1 membrane matrix protein M2 membrane ion channel protein absent in type B and C Ribonucleoproteins (RNPs) 8 RNP segments per genome Each of 8 (-) strand RNA segments wrapped in NP (RNA+NP = RNP) One copy of polymerase complex, (PB1, PB2, PA, products of segments 1-3) at 3 -end of each segment Nonstructural proteins NS1 and NS2 localized to nucleus, encoded from overlapping ORFs Overlapping ORFs from spliced RNAs; NS2 is fusion from NS1

Influenza A virus life cycle

HA / Sialic acid attachment pH mediated release of RNA Entry to nucleus via nuclear pore Copied using viral polymerase (attached to each segment) mRNAs are capped Move out to the cytoplasm & are translated Translated proteins move back into nucleus for polyadenylation Switch between mRNA production & genome replication

Influenza A virus genome: segmented

Helical Protein coated (VSV) 8 segments Most encode 1 protein 3 segments encode 2 Splicing allows >1 ORF >1 protein

Influenza A virus RNA synthesis

Genome is protein coated Viral RNA polymerase in virion Primer-dependent polymerase Capped Primer is stolen from the cell (10-13nts) Sequence is random but has a cap ( Cap-snatching ) This is why genome must go to cell nucleus Viral RNA polymerase makes an mRNA Switch ensures genome replication (NP) NPs bind to genomic RNA RNA moves into new particles

Influenza A virus polyadenylation

RNA dependent RNA polymerase 3 subunits & active sites (red) 5 end of (-) strand (template) is bound to polymerase (top red spot) Template threads through stationary polymerase through 2nd active site (bottom red spot) New positive strand is generated (bright green strand) Eventually template gets stuck as it is sill attached at 5 end to 1st active site (top red spot) Series of U s in 5 end of sequence are complementary to A s at 3 end of new (+) strand Chatter occurs for about 150 A s until mRNA is released

Influenza virus: RNA Segment 1

Segment 1, encodes two proteins: PB1 and PB1-F2.

PB1 & PB1-F2

Nucleotide sequence of the first 180 bases of this mRNA. The top line is the nucleotide sequence of the viral mRNA. During translation this sequence is read in triplets, each of which specifies an amino acid. (M, D, V ..) Translation usually begins with an ATG which specifies the amino acid methionine (M); the next triplet, gat, specifies aspartic acid (D), and so on. Only the first 60 amino acids of the PB1 protein are shown; the protein contains a total of 758 amino acids.

Influenza virus RNA: Overlapping Reading Frame

Most influenza viral RNAs code for one protein. Segment 1 (7 & 8) code for two proteins, the second protein is made by translation of an overlapping reading frame. On the second line of the RNA sequence is another atg. This atg is not in the reading frame of the PB1 protein. It is the start codon for the second protein encoded in (Seg 1)RNA 2, the PB1-F2 protein F2 stands for frame 2, as protein is translated from the 2nd ORF.

Influenza virus RNA: Translation into protein

The sequence of the viral RNA is shown from the beginning not using reading frame 1 Translation instead initiates with the internal atg, which is in the second reading frame. This open reading frame encodes the PB1-F2 protein which, in this case, is 90 amino acids in length (its length varies in different isolates). The protein is much shorter than PB1 because translation stops at a termination codon (tga) long before the end of the RNA. Because PB1-F2 is encoded in reading frame 2, its amino acid sequence is completely different from that of PB1. The sequences used for this example are from the 1918 H1N1 strain of influenza.

Influenza virus attachment to cells

You can see the individual hemagglutinin spikes (HA)on the virion binding to the cell receptor on the cell. The cell receptor is sialic acid a small sugar that is attached to many different proteins on the cell surface.

Influenza virions attach to cells when the HA attached onto the very small sialic acid.

Role of different sialic acids

Attachment of all influenza A virus strains to cells requires sialic acids. A number of chemically different forms of sialic acids. Influenza virus strains vary in their affinity for them. These differences may determine which animal species can be infected. Below, sialic acid is linked to the sugar galactose by what is called an alpha(2,3) linkage. This means that the carbon atom at position number 2 of the sialic acid hexose is joined, via an oxygen atom, to the carbon at position 3 of the hexose of galactose.

Sialic acid: epithelial cell locations

Avian influenza virus strains preferentially bind to sialic acids attached to galactose via an alpha(2,3) linkage. This is the major sialic acid on epithelial cells of the duck gut. In contrast, human influenza virus strains preferentially attach to sialic acids attached to galactose by an alpha(2,6) linkage. This is the major type of sialic acid present on human respiratory epithelial cells. Alpha(2,3) linked sialic acids are found on ciliated epithelial cells, which are a minor population within the human respiratory tract, and also on some epithelial cells in the lower tract.

Receptor Specificity : alpha(2,3) vs alpha(2,6)

Receptor specificity has implications for human infection with avian influenza virus strains.
For example, highly pathogenic avian H5N1 influenza viruses undergo limited replication in the human respiratory tract due to the presence of some cells with alpha(2,3) linked sialic acids. However, efficient human to human transmission requires that the avian viruses recognize sialic acids with alpha(2,6) linkages. Consistent with this hypothesis, the results of studies of early influenza virus isolates from the 1918, 1957, and 1968 pandemics suggest that these viruses preferentially recognized alpha(2,6) linked sialic acids.

Epithelial cells of the pig trachea produce both alpha(2,3) and alpha(2,6) linked sialic acids. This is believed to be the reason why pigs can be infected with both avian and human influenza virus strains and serve as a melting pot for the emergence of new viruses.

Influenza HA cleavage is required for infectivity

The influenza virus hemagglutinin (HA) is the viral protein that attaches to cell receptors. The HA also plays an important role in the release of the viral RNA into the cell, by causing fusion of viral and cellular membranes. HA must be cleaved by cellular proteases to be active as a fusion protein. The HA on the influenza virion is a trimer: it is made up of three copies of the HA polypeptide. The cleavage site for cell proteases on the HA protein is located near the viral membrane.

HA Fusion
The globular head of the HA protein, which attaches to cell receptors, is at the top, and the viral membrane is at the bottom. For clarity, only one HA cleavage site is labeled. The uncleaved form of the protein is called HA0; after cleavage by a cellular enzyme, two proteins are produced, called HA1 (blue) and HA2 (red). HA2 contains a sequence of hydrophobic amino acids called a fusion peptide. During entry of influenza virus into cells, the fusion peptide inserts into the endosomal membrane and causes fusion of the viral and cell membranes. Consequently, the influenza viral RNAs can enter the cytoplasm.

Influenza A strains, tropism & cleavage

If the HA protein is not cleaved to form HA1 and HA2, fusion cannot occur. In humans, influenza virus replication is restricted to the respiratory tract, because that is the only location where the protease that cleaves HA is produced. However, the HA protein of highly pathogenic H5 and H7 avian influenza virus strains can be cleaved by proteases that are produced in many different tissues. As a result, these viruses can replicate in many organs of the bird, including the spleen, liver, lungs, kidneys, and brain. This property may explain the ability of avian H5N1 influenza virus strains to replicate outside of the human respiratory tract. Like the HA proteins of highly pathogenic H5 and H7 viruses, the HA of the 1918 influenza virus strain can also be cleaved by ubiquitous cellular proteases. The H5 and H7 HA proteins have multiple basic amino acid residues at the HA1-HA2 cleavage site which allows cleavage by widely expressed proteases.

Packaging of the segmented influenza RNA genome

The RNA genome of influenza viruses is segmented . The virions of influenza A and B viruses contain 8 different RNAs. How is the correct number of RNA segments inserted into newly synthesized virus particles? During influenza virus assembly, viral RNAs and viral proteins called a ribonucleoprotein complex or RNP -travels to the plasma membrane. There the virion forms by a process called budding, during which the membrane bulges from the cell and is eventually pinched off to form a free particle.

Packaging of RNA segments

Production of an infectious virus particle requires incorporation of at least one copy of each of the eight RNA segments. Two different mechanisms proposed to explain how each virion receives a full complement of genomic RNA : 1. random 2. selective 1. Random: 1 infectious particle for every 400 particles assembled (8!/88). If more than 8 RNA segments could be packaged into each virion, then the fraction of infectious particles would be significantly increased. For example, if 12 RNA molecules could fit into each virion, then 10% of the particles would have the complete viral genome. In support of this mechanism, influenza viruses with more than 8 RNA segments have been observed.

2. Selective packaging:
Each of the eight genomic RNAs has a different signal that allows incorporation into virus particles. These signals are believed to be within the noncoding and coding sequences at the 5 - and 3 -ends of the viral RNAs. The sequences interact and form structures that are unique to each segment, and which have been shown to be essential for incorporation of each segment into virions. Consistent with this hypothesis, electron microscopy reveals that during budding, the viral RNPs are organized in a distinct pattern, as shown in the image.
This observation argues that RNPs are not randomly incorporated into virions The mechanisms by which these signals are recognized, and how they ensure incorporation of one copy of each RNA segment into the particle, are not known.

Reassortment of the influenza virus genome

Mutation is an important source of RNA virus diversity that is made possible by the error-prone nature of RNA synthesis. Viruses with segmented genomes, such as influenza virus, have another mechanism for generating diversity: reassortment. When an influenza virus infects a cell, the individual RNA segments enter the nucleus. There they are copied many times to form RNA genomes for new infectious virions. The new RNA segments are exported to the cytoplasm, and then are incorporated into new virus particles which bud from the cell. If a cell is infected with two different influenza viruses, the RNAs of both viruses are copied in the nucleus. When new virus particles are assembled at the plasma membrane, each of the 8 RNA segments may originate from either infecting virus. The progeny that inherit RNAs from both parents are called reassortants.

Re-assortment
The diagram below shows a cell that is co-infected with two influenza viruses L and M. The infected cell produces both parental viruses as well as a reassortant R3 which inherits one RNA segment from strain L and the remainder from strain M.

Exchange of RNA segments between mammalian and avian influenza viruses that give rise to pandemic influenza. For example, the 2009 H1N1 pandemic strain is a reassortant of avian, human, and swine influenza viruses.
Reassortment can only occur between influenza viruses of the same type.

Timeline of Influenza A reassortment

Influenza A and Bioterrorism

Fouchier s group made mutations to H5N1 which doesn t transmit well between humans, and made H1N1 mutations that allowed it to transmit as well as swine flu. Serially infected ferrets with transmissible mutants US tried to block the publication of the results based on a Bioterrorism risk

Influenza etiology
Spread person-to-person by aerosol, direct or indirect contact, in water no vectors Incubation period 1-3 days Causes myalgia, sore throat, fever, headache, cough which may be protracted Symptoms typically last 2-7 days Intensity of symptoms differs greatly depending on virus strain

Influenza virus transmission

Influenza virus may be transmitted among humans in 3 ways: 1. Direct contact with infected individuals; 2. Contact with contaminated objects (called fomites, such as toys, doorknobs) 3. Inhalation of virus-laden aerosols. Coughing may produce several hundred droplets, a sneeze can generate between 20-40,000 aerosolized particles of different sizes. The largest droplets fall to the ground within a few meters and will transmit an infection only to those in the immediate vicinity. Other droplets travel a distance determined by their size. Those droplets 1-4 microns in diameter are called droplet nuclei ; these remain suspended in the air for very long periods and may not only travel long distances, but can reach the lower respiratory tract. Inhalation of droplets and droplet nuclei places virus in the upper respiratory tract, where it may initiate infection.

Case study of transmission

The importance of aerosol transmission was illustrated by an outbreak of influenza aboard a commercial airplane in the late 1970s. The plane, carrying 54 persons, was delayed on the ground for three hours, during which time the ventilation system was not functional. Most of the travelers remained on board. Within 72 hours, nearly 75% of the passengers developed influenza. The source of the infection was a single person on the airplane with influenza.

Total # cases: 605 Total # deaths: 357 Mortality rate = 59%

2003-2012 15 Countries

What is acceptable?

How many people die from influenza?

WHO reports that as of 15 June 2009, 76 countries have officially reported 35, 928 cases of influenza A(H1N1) infection, including 163 deaths. These numbers can be used to calculate a case fatality ratio (CFR) of 0.45%.

The seasonal baseline is calculated using statistical procedures using data from the previous five years, and the epidemic threshold is calculated as 1.645 standard deviations above the seasonal baseline. This is the point at which the observed proportion of deaths attributed to pneumonia or influenza becomes significantly higher than would be expected without substantial influenza-related mortality.

A game of statistics
For the 2007 08 influenza season, the percentage of deaths attributed to pneumonia and influenza exceeded the epidemic threshold for 8 consecutive weeks from January 12 May 17, 2008, with a peak at 9.1% at week 11. In contrast, pneumonia and influenza deaths remained below the epidemic threshold in the relatively mild 20082009 season:

Influenza epidemiology
Flu is spread worldwide by migratory birds Birds usually affected little by virus Virus is shed into cold water, and is stable there
Spread by shearwaters (migratory birds) from Australia to California turkeys in the same season Arctic Terns spread flu to chickens in Scotland, Alaska and Russia 1980 seal flu epidemic linked to duck/tern recombinant; also found in humans

Spanish flu pandemic, 1918

One of the greatest pandemics in the history of mankind Infected >200 million people; 20-40 million killed More died in a year than in the four year peak of the Black Plaque in the 14th century People between 15 and 34 years old most differentially affected death rate 20 times higher than previous years Approximately half of soldiers who died in WWI died of flu Some evidence strain first appeared in France in 1916 Some evidence recombinant HA led to this strain Infectious cDNA clones of complete 1918 strain recently finished many similarities to current H5N1 avian flu Could this happen again? Vaccine/theraputic availability and knowledge of epidemiology makes another flu pandemic of the same proportions unlikely, but possible

Influenza vaccine
Live attenuated and inactivated vaccine can be used Inactivated vaccine commonly used now 70-80% effective May be made against: 1) whole virus; 2) subviral particles; 3) surface antigen Multiple virus strains used in production of a given vaccine Prediction of strain or strains most likely to be epidemic in a given year is critical to vaccine production
WHO meets twice a year, makes recommendations Recommendations given to vaccine manufacturers, which make vaccines for northern and southern hemispheres

250 million doses made per year Effective H5N1 vaccine made recently, now being increased

Vaccines & Antiviral therapeutics Lecture 15 to follow