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Home , Antigenic shift

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Influenza

Introduction:

The name Myxovirus is a group of enveloped characterized by their ability to adsorb onto mucoprotein receptors on erythrocytes, causing hemagglutination. They are spherical and filamentous RNA viruses. They are now classified into two separate families:

1. - consisting of the viruses 2. - consisting of Newcastle disease virus, virus, parainfluenza viruses, measles and respiratory syncytial viruses. Influenza viruses are classic respiratory viruses.

Morphology:  The influenza virus is typically spherical or filamentous with a diameter of 80-120 nm but pleomorphism is common.  The virus consists of ribonucleoprotein in helical symmetry.  The negative sense single-stranded RNA genome is segmented and exists as eight pieces. These segments code for different which are NS1, NS2, NP, M1, M2, M3, HA and NA.  The genome consists of an RNA-dependent RNA polymerase, which transcribes the negative-polarity genome into mRNA. The genome, therefore, is not infectious.  The nucleocapsid is surrounded by an envelope, which has an inner membrane layer and an outer layer.  The membrane protein is also known as the matrix or ‘M protein’ composed of two components, M1 and M2.  The protein part of the envelope is virus coded but the lipid layer is derived from the modified cell membrane, during the process of replication by budding.  Projecting from the envelope are two types of spikes (peplomers): Hemagglutinins (HA) spikes which are triangular in cross-section and 2 | P a g e

the mushroom-shaped (NA) peplomers which are less numerous.

Viability characteristics:  The virus is inactivated by heating at 50 ͦC for 30 minutes.  It remains viable at 0-4 ͦ C for about a week.  It can be preserved for years at -70 ͦC or by freeze drying.  Influenza viruses are resistant to slow drying.  The virus survives slow drying and may remain viable on fomites such as blankets for about two weeks.  They remain viable in dust up to 2 weeks.  Ether, formaldehyde, phenol, salts of heavy metal and many other chemical disinfectants destroy infectivity.  Iodine is particularly effective.  Hemagglutinating, enzymic and complement-fixing activities of the virus are more stable than infectivity.

Antigenic and genomic properties:

The of the influenza virus can be classified into two types:

1) Internal Antigens: . The internal is the ribonucleoprotein and is hence called the RNP antigen. Because it is found free in infected tissues and occurs in the supernatant when the virus containing fluidis centrifuged, it was also called as ‘soluble’ (S) antigen. The RNP antigen is type specific and based on its nature, influenza viruses are classified into types A, B and C. The RNP antigens of types A, B and C are distinct but all strains of any one type possess the same antigen. The RNP antigen is stable and does not exhibit any significant . . M protein antigen, like the RNP antigen is also type specific and distinct for A, B and C types of influenza viruses. The envelope lipid antigen is 3 | P a g e

host specific and is determined by the in which virus replication takes place. 2) Surface antigen: The term ‘viral’ or V antigen was used to describe the surface antigen of the influenza virus. The V antigen is actually composed of at least two virus-coded proteins, hemagglutinins and neuraminidase. . Hemagglutinin is a glycoprotein. It is a trimer. It is specific. It undergoes antigenic variation. It is composed of two polypeptides, HA 1 and HA 2. It is responsible for hemagglutination and hemadsorption. It enables the virus to adsorb to mucoprotein receptors on red cells as well as on respiratory epithelial cells. Anti-hemagglutinin antibodies are produced following infection and immunization. This antibody is protective by preventing adsorption of the virus to cells. Hemagglutinins is a strain- specific antigen and is capable of great variation. Fifteen distinct HA subtypes, H1-H15, have been identified in viruses, but only four of them have been found in human isolates so far. The hemagglutinin consists of 500 spikes, each measuring 12 nm in length. The triangular shaped HA is inserted into the virus membrane by its tail end. The distal end, which contains five antigenic sites (designated HA1-HA5), is responsible for binding of virion to host cells. . Neuraminidase is a glycoprotein enzyme which destroys cell receptors by hydrolytic cleavage. It is a tetramer. It consists of 100 mushroom-shaped spikes. The mushroom-shaped NA is inserted into the virus membrane by its hydrophobic tail end. The distal end contains antigenic as well as enzymatically active sites. The anti-neuraminidase antibody is formed following infection and immunization. It is not as effective in protection as the anti-hemagglutinin antibody. It does not prevent the adsorption of virus onto cells but can inhibit the release and spread of progeny virions and may thus contribute to limiting the infection. It is a strain specific antigen and exhibits variation. Nine different subtypes have been identified (N1-N9). Neuraminidase is an isoenzyme. The function of neuraminidase is to cleave the neuraminic acid and to release progeny virions from the infected host cells. The neuraminidse also degrades the 4 | P a g e

mucus layer, thereby exposing the epithelial membrane of the respiratory tract for infection by the virus.

Difference between HA and NA:

Sr.No. Hemagglutinin (HA) Neuraminidase (NA) 1. It is a trimer. It is a tetramer. 2. It consists of 500 spikes. It consists of 100 spikes. 3. HA is triangular in shape. NA is mushroom-shaped. 4. There are 15 distinct subtypes of There are 9 distinct subtypes of NA HA designated as H1-H15. designated as (N1-N9). 5. Anti-hemagglutinin antibodies are The anti-neuraminidase antibody is produced following infection and formed following infection and immunization and this antibody is immunization which is not as protective by preventing effective in protection as the anti- adsorption of the virus to cells. hemagglutinin antibody. It does not prevent the adsorption of virus onto cells.

Antigenic variations:

Antigenic variation is a unique feature of influenza virus. The surface antigens HA and NA show variations and are primarily responsible for antigenic variations exhibited by influenza viruses. The internal RNP antigen and M protein are stable, hence do not contribute to the antigenic variations. Antigenic variations are of two types:

1) and 2) . 1) Antigenic shift: The abrupt, drastic, discontinuous change is called the antigenic shift. This occurs due to major antigenic changes in HA or NA antigens, and is caused by replacement of the gene for HA by one coding for a completely different amino acid sequence. The antigenic shift is 5 | P a g e

characterized by alteration of virtually all the antigenic sites of the HA. This occurs independently in the HA and NA. 2) Antigenic drift: The gradual, sequential, regular antigenic change in influenza virus is known as antigenic drift. This occurs due to minor antigenic changes in the HA or NA occurring at frequent intervals. This is caused by a single affecting HA glycoprotein. The antigenic drift is characterized by changes in certain epitopes in the HA, while others are being conserved.

Difference between antigenic shift and antigenic drift: Sr. Antigenic shift Antigenic drift no. 1. Abrupt, drastic and Gradual, sequential and regular discontinuous variation in the variation at periodic intervals antigenic structure 2. Results in a different strain Results in a new strain Related to predecessor strain Not related to predecessor strain 3. Antigenic drift is due to Antigenic shift is due to gene mutation and selection 4. Responsible for epidemics of Responsible for epidemics as well influenza as pandemics of influenza.

Gene Reassortment:

Because the influenza virus genome is segmented, genetic reassortment can occur when a host cell is infected simultaneously with viruses of two different parent strains. This process of genetic reassortment accounts for the periodic appearance of the novel types of influenza A strains that cause influenza pandemics.

Influenza viruses of animals, such as aquatic , chickens, swine, and horses show high host specificity. These animal viruses are the source of the RNA segments that encode the antigenic shift variants that cause epidemics among humans. For example, if a person is infected simultaneously by an avian and 6 | P a g e human influenza strains, then it is possible that a genetic reassortment could occur in infected cells in humans. The reassortment could lead to emergence of a new human , the progeny of which will contain a mixture of genome segments from the two strains (e.g. a new variant of human influenza A virus bearing the avian virus HA).

Many studies have conclusively demonstrated that the aquatic birds (such as water fowl) are a common source of these new genes. The pigs act as mixing vessels, where these virulent genes of water fowl mix with the genome of influenza virus giving rise to new variant of influenza virus.

Designation of influenza viruses:

Influenza virus type A can be classified into subtypes based on the variations in their surface antigens. The WHO proposed a new system of classification in 1971 and was later modified, which takes into account

Pathogenicity:

The route of entry is the respiratory tract. In experimental infection in volunteers, very small doses (approximately three viable particles) can initiate infection when given as aerosols. Larger doses are required when infection is by intranasal instillation. The facilitates infection by reducing the viscosity of the mucus film lining the respiratory tract and exposing the cell surface receptors for virus adsorption. The ciliated cells of the respiratory tract are the main sites of viral infection. These cells are damaged and shed laying bare the basal cells in the trachea and bronchi. This renders the respiratory tract highly vulnerable to bacterial invasion. Viral pneumonia, seen only in the more severe cases, is associated with hyperemia and thickening of the alveolar walls, interstitial infiltration with leucocytes, capillary thrombosis and leucocytic exudation. In some cases, a hyaline membrane is formed. Occupying the alveolar 7 | P a g e ducts and alveoli. In the late stages, there is infiltration with macrophages which engulf and remove desquamated alveolar cells.

The disease is ordinarily confined to the respiratory tract. Very rarely was the virus isolated from the spleen, liver, kidneys, and other organs during the 1957 pandemic.

Viral replication:

Influenza virus, hepatitis delta virus and are the only RNA viruses that have an important stage of their replication cycle in the nucleus. Infection of the host cell begins by adsorption of the cell by influenza virus, which is mediated through the HA. HA is first cleaved by an extracellular protease to a modified HA that actually mediates the attachment of the virus to the cell surface. Once inside the cell, the virus uncoats within the endosomes.

In the nucleus of the host cell, the virion RNA polymerase transcribes the eight- genome segments into eight viral mRNAs. Most of the viral mRNAs, however, move out of the nucleus into the cytoplasm, where they are translated into viral proteins. Some of the viral mRNAs continue to remain in the nucleus and serve as the templates for synthesis of the negative-strand RNA genome for the progeny virions. In the nucleus also, two proteins, namely, nucleoprotein (NP) and matrix protein are synthesized, which then bind with the RNA genome of the viral progeny and form a complex, which is subsequently transported to the cytoplasm.

Matrix protein mediates the interaction of the nucleocapsid with the envelope, and finally, the virion is released from the cell by budding from the cell membrane at the site where the HAs and NAs are present.

Pathogenesis of influenza virus:

 Influenza virus is transmitted from person to person primarily in droplets released by sneezing and coughing. 8 | P a g e

 Inhaled Influenza viruses reach lower respiratory tract, tracheobronchial tree, the primary site of the disease.  They attach to sialic acid receptors on epithelial cells by HA present on the .  Relatively few viruses are needed to infect lower respiratory tract than upper respiratory tract.  Neuraminidase of the viral envelope may act on the N- acetyl neuraminic acid residues in mucus to produce liquefaction.  In concert with mucociliary transport, this liquefied mucus may help spread the virus through the respiratory tract.  Infection of mucosal cells results in cellular destruction and desquamation of the superficial mucosa.  The resulting edema and mononuclear cell infiltration of the involved areas are accompanied by symptoms including nonproductive cough, sore throat and nasal discharge.  Although the cough may be striking, the most prominent symptoms of influenza are systemic: fever, muscle aches, and general prostration.  The virus remains localized to the respiratory tract; hence viremia does not occur.

Inhalation of Influenza virus ↓ Virus reach lower respiratory tract, tracheobronchial tree ↓ Virus attach to sialic acid receptors on epithelial cells by HA present on the viral envelope ↓ Neuraminidase of the viral envelope act on the N-acetyl neuraminic acid residues in mucus ↓ Produce liquefaction ↓ 9 | P a g e

Helps to spread the virus through respiratory tract ↓ Infection of mucosal cells ↓ Cellular destruction and desquamation of the superficial mucosa ↓ Edema and mononuclear cell infiltration ↓ Nonproductive cough, sore throat and nasal discharge

Flowchart of pathogenesis of Influenza virus

Prophylaxis of Influenza virus:

Influenza A subtype H1N1 and H3N2 are most common prevailing human influenza viruses. The trivalent vaccine used worldwide contains influenza A strains from H1N1 and H3N2, along with an influenza B strain. Influenza virus vaccines have been used for about 40 years to prevent influenza, primarily influenza A and B .Following types of vaccines are used: 1) Inactivated vaccines: Initially inactivated vaccines were used. The viruses for these vaccines are grown in chick embryos, inactivated by formalin, purified to some extent, and adjusted to a dosage known to elicit an antibody response in most individuals. The vaccine contain the strain of type A and B viruses that believed most likely to produce epidemics during the following winter. The vaccine is administered parenterally. One or two doses are require, depending on the immune experience of the population with related antigens. The vaccines are recommended especially for persons at high risk, especially those over 65 years of age and those with chronic cardiopulmonary disease. 10 | P a g e

Local and systemic reactions to the vaccine are minor and occur in the first day or two after vaccination. In some persons, the vaccine may cause reactions allergic to egg proteins present in the vaccine.The killed vaccines do not induce the formation of secretory antibodies in the secretory mucosa, although they elicit production of specific protective antibodies in the serum. 2) Live attenuated vaccines: Live attenuated vaccines are now being developed as alternatives to inactivated vaccines. These vaccines induce production of specific secretory antibodies in respiratory mucosa. Earlier, the live vaccine used the viruses that were attenuated by repeated egg passage and was given by intranasal instillation. But these vaccines often failed to protect the children from clinical disease. Recently, temperature –sensitive (ts) mutunt Strains have been used in the live attenuated vaccine preparations. These avirulent mutaunts are able to grow at 32- 34 ° C in the nasopharyngal secretions, but not at 37° C in the lungs. These vaccines are useful to protect the children from clinical disease. Types of Advantage Disadvantage vaccine Whole virus This vaccine confers confers protection in 60- The subsequent vaccines 90 of vaccines and the protection lasts for 1 -5 infecting virus may years show slow antigenic drift and the vaccine – induced antibody will be less effective in conferring protection against the new strains. Split virus These vaccines have been shown to induce Just as immunogenic as vaccines fewer side effects in the vaccines whole virus vaccine. Subunit virus Fewer reactions than those if given whole No significant vaccines virus vaccines and absorbed subunit vaccine; disadvantage. therefore, the best vaccines available at present are the aqueous subunit vaccine. 11 | P a g e

Live attenuated Immunization with live attenuated influenza No significant vaccines virus vaccines induces a solid immunity than disadvantage. do inactivated vaccines. When given intranasally, few side effects are produced.

Chemoprophylaxis:

Chemoprophylaxis by amantadine and rimantadine hydrochloride has been shown to be more successful. These two drugs effectively prevent infection and illness caused by A, but not type B viruses( because they lack M2 components). The persons with high risk can be protected by administering in a dosage of 100 mg/day. The drugs interfere with virus uncoating and transport by blocking the transmemebrane M2 ions channel. These antiviral agents prevent about 50% of infections and about 67% of illnesses under natural conditions. When administered for 10 days to household contacts of a person with influenza, these drugs protect up to 80% of the persons from illness. Side effects are greater for amantadine but the limited to the central nervous system.

 Laboratory Diagnosis :

During an epidemic of influenza, the clinical diagnosis can be made, but definitive diagnosis on the laboratory methods.

 Specimens : Specimens include nasal or throat washings or sputum for viral antigen and viral RNA, throat gargles for isolation of viruses, and serum for viral antibodies.  Direct antigen detection A rapid, specific diagnosis of influenza is made by demonstrating viral antigens directly on cells obtained from the nasopharynx. Immunoflurorescence (IF) or enzyme –linked immunosorbent assay using specific monoclonal antibodies are used commercially to detect viral antigen. The results of the rapid tests are useful to start 12 | P a g e

start treatment with the NA inhibitors within 48 hours of the onset of symptoms.  Isolation of the virus Thorat gargles are the specimens of choice. The specimen is collected in saline broth or a buffered salt solution and is sent immediately to the laboratory, or if delayed is stored at -4 °C. The virus is isolated from the specimen by inoculation into embryonated eggs or into certain cell cultures.  Egg inoculation : The specimen is inoculated into the amniotic cavity of the chick embryo. After incubation at 35° C for 3 days, the amniotic fluid and allantonic fluid are harvested and tested for the presence of viral HA. This is carried out by using fowl and guinea pig red cells in parallel and incubating at room temperature and at 4°C. usually, influenza A viruses agglutinate only guinea pig cells, influenza B both fowl and guinea pig red cells, and influenza C agglutinate only fowl cells at 4°C. if the test is positive ,the isolate is then typed by a serological test(e.g.,hemagglutination inhibition test) using specific antisera to types A,B and c.  Cell culture : Influenza virus is usually isolated from respiratory secretions by growing in tissue cultures (monkey kidney or baboon kidney cell lines ).The cell cultures are incubated at 33 °C in the roller drums in the presence of trypsin, but without serum. Virus growth in tissue cultures is detected by direct demonstration of viral antigen in infected cell cultures by IF or by testing for hemadsorption with human O,fowl, and guinea pig red cells. In a positive hemadsorption test, red cells adhere to the virus budding from infected cells. Oif the cultures tests positive, serologic tests with specific antisera may be used to identify the virus.  serodiagnosis demonstration of a rise in serum antibody titer between acutephase and convalescent – phase sera by a serological test is diagnostic of 13 | P a g e

infection. The acute –phase sera are collected within a few days of illness and the convalescent sera 7-10 days after the illness. Complement fixation tests (CFTs) with RNP antigens of influenza types A,B, and C and also the CFTs with V antigens are employed for demonstration of rising antibody titer in the paired sera samples. Hemagglutination inhibition test, enzymes neutralization test, radial immunodiffusion test, and ELISA are the other tests also used for demonstration of antibodies, However, none of these techniques are useful to identify all infections. Various approaches followed for laboratory diagnosis of influenza are summarized in table no 61-6.  H5N1 Flu The H5N1 flu, caused by an avian subtype influenza virus, which has been associated with bird flu in the domesticated birds, can be transmitted from birds to humans. The H5N1 was first described in Hong Kong in 1997. Human infections caused by this virus were established in only 18 individuals of which six died. Since then, sporadic cases of H5N1 infection continued to be described in southern China. An epidemic of bird flu occurred in domesticated birds in Southeast Asia, primarily Vietnam , in January 2004. More than 240 human cases have been documented and more than 140 persons have died due to poultry outbreaks and bird-to-human transmission. Most deaths have been reported in Vietnam and Indonesia. Sporadic outbreaks have continued to occur since the 2004 outbreaks even outside Southeast Asia, including Turkey. Till now, no conclusive evidences are available to show the human –to- human scientists are concerned that a slight mutation could convert H5N1 to a strain that would be easily transferred from human-to- human. Such a strain , it is believed, could potentially spread rapidly and cause a catastrophic worldwide pandemic. Hence, efforts are currently underway to develop an effective vaccine against H5N1. In addition , the number of drugs that are effective against influenza are bring increasingly evaluated. Ribavirin has been shown activity when tested in animal models. 14 | P a g e

Another avian subtype, H9N2 , was described in two young children in march 1999. However, after that despite concern, no further outbreak of H9N2 infection has been documented. Experts are also concerned that a virulent strain of H9N2 influenza similar to H5N1 flu may mutate to allow human –to –human infection and that such a strain may possess the combination of transmissibility, infectivity and lethality.  H1N1