Orthomyxoviruses

1 Orthomyxovirusesﻓﺎﯾﺮوﺳﺎت / اﻟﻤﺮﺣﻠﮫ اﻟﺜﺎﻟﺜﮫ . ﺟﺎﻣﻌﮫ ذي ﻗﺎرم/ ﻛﻠﯿﮫ اﻟﻄﺐ / ﻓﺮع اﻻﺣﯿﺎء اﻟﻤﺠﮭﺮﯾﮫ / اﻧﺘﻈﺎر ﻋﻼوي ﺟﻌﻔﺮ . دPhD. M.Sc. Microbiology Introduction These viruses are classified under two families These are: • Orthomyxoviridae, consisting of influenza viruses. • Paramyxoviridae, consisting of parainfluenza, mumps, measles, respiratory syncytial. • The genus Orthomyxovirus includes influenza viruses, the causative agents of worldwide epidemics of influenza. Influenza Viruses Influenza viruses belong to the family of Orthomyxoviridae and are the causative agents of influenza, a respiratory disease in humans (Flu)with well-defined systemic symptoms that occurs in sporadic, epidemic, and pandemic forms. Influenza A and B viruses cause substantial morbidity and mortality in humans and a considerable financial burden worldwide, whereas influenza C viruses cause sporadic outbreaks of mild respiratory disease, mainly in children. N.B v Epidemic refers to an increase, often sudden, in the number of cases of a disease above what is normally expected in a population within a geographic area v Pandemic refers to an epidemic that has spread over several countries or continents, usually affecting a large number of people. Properties of the Virus Morphology Influenza viruses are spherical or filamentous, enveloped particles 80–120 nm in diameter. It is composed of a characteristic segmented negative sense, single-stranded RNA genome, a nucleocapsid, and an envelope (See-Fig). Source: ViralZone:www.expasy.org/viralzone, SIB Swiss Institute of Bioinformatics)

2 The viral genome is a single-stranded antisense RNA. The genome consists of an RNA-dependent RNA polymerase, which transcribes the negative-polarity genome into mRNA. • The RNA genome is segmented and consists of eight segments in Influenza A & B and seven segments in influenza C viruses. These segments code for different proteins, which are NS1, NS2, NP, M1, M2, M3, HA, and NA. The genome is present in a helically symmetric nucleocapsid surrounded by a lipid envelope. The envelope has an inner membrane protein layer and an outer lipid layer. The membrane proteins are known as matrix or M protein and are composed of two components M1 and M2. • Two types of spikes or peplomers project from the envelope: (a) The triangular hemagglutinin (HA) peplomers and (b) The mushroom-shaped neuraminidase (NA) peplomers. Classification and Nomenclature Ø Genus Influenza virus A contains human and animal strains of influenza type A Ø Influenza virus B contains human strains of type B, Ø Influenza virus C contains influenza type C viruses of humans and swine. Ø Antigenic differences exhibited by two of the internal structural proteins, the nucleocapsid (NP) and matrix (M) proteins, are used to divide influenza viruses into types A, B, and C. Ø Antigenic differences exhibited by two of the internal structural proteins, the nucleocapsid (NP) and matrix (M) proteins, are used to divide influenza viruses into types A, B, and C. Ø Antigenic variations in the surface glycoproteins, HA and NA, are used to subtype type A viruses Ø So far, 18 subtypes of HA (H1–H18) , 11 subtypes of NA (N1–N11), in many different combinations, have been recovered from humans and animals. e.g of current subtypes of Influenza A viruses H1N1 , H2N3 • The standard nomenclature system for influenza virus isolates includes the following information: type, host of origin, geographic origin, strain number, and year of isolation. • Antigenic descriptions of the HA and the NA are given in parentheses for type A. e.g A/Hong Kong/03/68(H3N2) Hemagglutinin (HA) Ø The HA protein of influenza virus binds virus particles to susceptible cells and is the major antigen against which neutralizing (protective) antibodies are directed. Ø Variability in HA is primarily responsible for the continual evolution of new strains and subsequent influenza epidemics.

3 Ø Hemagglutinin binds with the sialic acid cell receptor, and initiates the infection in the host cell. Ø HA derives its name from its ability to agglutinate erythrocytes under certain conditions. Ø The HA agglutinates certain RBC , which is inhibited by the neutralizing Abs. This forms the basis of the hemagglutination inhibition test used in the serodiagnosis of influenza. Ø Hemagglutinin has potency to undergo antigenic variations. Neuraminidase (NA) The NA is a glycoprotein and tetramer. It consists of 100 mushroom-shaped spikes. v The NA functions at the end of the viral replication cycle. It cleaves the neuraminic acid and to release progeny virions from the infected host cells. v It is a sialidase enzyme that removes sialic acid from glycoconjugates. v NA facilitates release of virus particles from infected cell surfaces during the budding process and helps prevent self-aggregation of virions by removing sialic acid residues from viral glycoproteins. v It is possible that NA helps the virus negotiate through the mucin layer in the respiratory tract to reach the target epithelial 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: antigenic shift and antigenic drift. Antigenic shift ² Antigenic shift: Occurs due to major antigenic changes in HA or NA antigens ² Caused by replacement of the gene for HA by one coding for a completely different amino acid sequence. ² The antigenic shift is characterized by alteration of virtually all the antigenic sites of the HA. ² Demonstrated in type A influenza virus only. ² Antigenic shift variants appear less frequently, about every 10 or 11 years. • Antigenic shift reflects drastic changes in the sequence of a viral surface protein, caused by genetic reassortment between human, swine, and avian influenza viruses. • The completely novel antigens that appear during antigenic shift are acquired by genetic reassortment. • Influenza B and C viruses do not exhibit antigenic shift because few related viruses exist in animals.

4 Antigenic drift • Minor antigenic changes are termed antigenic drift • Occurs due to minor antigenic changes in the HA or NA occurring at frequent intervals. • Antigenic drift is caused by the accumulation of point mutations in the gene, resulting in amino acid changes in the protein. Sequence changes can alter antigenic sites on the molecule such that a virion can escape recognition by the host’s immune system. • Antigenic drift variants occur very frequently, virtually every year. Key Points • Influenza A virus shows maximum antigenic variations. • Influenza B virus does not undergo antigenic shift because influenza B virus is the only human virus for which there is no animal source of new RNA segments. However, influenza B virus undergoes antigenic drift. • Antigenic variation never occurs in type C influenza virus beacuse its lack of NA. 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 birds, 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 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 influenza A virus, 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).

5 REPLICATION of Influenza virus • Viral infection initiates with the binding of a virion to cell surface receptors containing sialic acid, followed by the endocytosis of the virion. • After fusion of the viral and endosomal membranes, the viral ribonucleoproteins (vRNPs) are released into the cytoplasm and then transported into the nucleus. • In the nucleus the viral RNA polymerase transcribes the vRNA segments into mRNAs. • Viral mRNA is exported to the cytoplasm for translation by cellular mechanisms. The viral RNA polymerase also performs replication of vRNA by copying it into complementary RNA (cRNA), which serves as a template for the production of more vRNA. Newly synthesised viral polymerase and nucleoprotein are imported into the nucleus and bind to cRNA and vRNA to assemble vRNPs and cRNPs, respectively. • Following nuclear export, progeny vRNPs are transported across the cytoplasm on recycling to the cell membrane, where assembly of progeny virions takes place. • Mature virions are released by budding. Infleunza A virus replication cycle. Source: Aartjan J.W. te Velthuis and Ervin Fodor, 2017. Pathogenesis and Immunity Influenza virus is transmitted from person to person primarily in droplets released by sneezing and coughing. 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 viral envelope. Relatively few viruses are needed to infect lower respiratory tract than the upper respiratory tract. Neuraminidase of the viral envelope may act on the N -acetyl neuraminic acid residues in mucus to produce liqHaction.

6 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. In an uncomplicated case, virus can be recovered from respiratory secretions for 3–8 days. Clinical Syndrome Incubation period is short (1–3 days). The classic influenza syndrome is a febrile illness of sudden onset, characterized by tracheitis and marked myalgias. Headache, chills, fever, malaise, myalgias, anorexia, and sore throat appear suddenly. The body temperature rapidly rises to (38.3–40.0°C) and respiratory symptoms ensue. Nonproductive cough is characteristic. Sneezing, rhinorrhea, and nasal obstruction are common. Patients may also report photophobia, nausea, vomiting, diarrhea, and abdominal pain. They appear acutely ill and are usually coughing. Minimal to moderate nasal obstruction, nasal discharge, and pharyngeal inflammation may be present. Complications 1-Secondary bacterial infections: Life-threatening influenza is often caused by secondary bacterial infections with staphylococci, pneumococci, and Haemophilus influenzae. Pneumonia may develop as a complication and may be fatal, particularly in (a) Elderly persons above 60 years with underlying chronic disease. (b) In people chronic cardiorespiratory disease, renal disease, etc.) (c) Pregnant women. 2-Reye’s syndrome is a noted complication of influenza B infection. The condition is seen most commonly in young children and is associated with degenerative changes in the brain, liver, and kidney. 3- Central nervous system complications: Guillain–Barre syndrome characterized by encephalomyelitis and polyneuritis is a rare complication of influenza virus infection. Reservoir, source, and transmission of infection Infected humans are the main reservoir of infections for influenza A virus. Respiratory secretions of infected persons are the important source of infection. The virus is excreted in respiratory secretions immediately before the onset of illness and for 3–4 days thereafter. Wild aquatic birds are known reservoirs of influenza A. They secrete the viruses in their feces, which contaminates ponds and lakes. The virus is spread from person-to-person primarily by air-borne respiratory droplets released during the acts of sneezing and coughing. Influenza B virus only causes epidemics. Infection is from humans-to-humans. No animal reservoir hosts are known.

7 Laboratory Diagnosis During an epidemic of influenza, the clinical diagnosis can be made, but definitive diagnosis depends on the laboratory methods. Specimens: • Nasal or throat washings or sputum for viral antigen and viral RNA. • Throat gargles for isolation of viruses. • Serum for viral antibodies. Direct antigen detection Is made by demonstrating viral antigens directly on cells obtained from the nasopharynx. Immunofluorescence (IF) or enzyme-linked immunosorbent assay using specific monoclonal antibodies are used to detect viral antigen. The results of the rapid tests are useful to start treatment with the NA inhibitors within 48 hours of the onset of symptoms. Detection of antigens by ELISA Isolation of the virus Throat gargles are the specimen 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. Treatment Amantadine and Rimantadine are the specific antiviral agents available for treatment of influenza. Tamiflu ((Oseltamivir phosphate) Prevention This is based on the following: Immunoprophylaxis by vaccines: Influenza A subtypes H1N1 and H3N2 are most common predominate human influenza viruses.