ORTHOMYXOVIRUSES POWERPOINT PRESENTATION

INFORMATIVE SLIDES

1. ORTHOMYXOVIRUSES

2. Orthomyxoviridae (features) Size- 80-120nm Shape- spherical rarely filamentous Nucleic acid- Negative sense ssRNA Segmented 8 pieces Genetic recombination and antigenic variation seen Site for RNA replication - nucleus Important human pathogens- influenza virus

3. Cont …. mucins ( myxo : meaning mucus) These viruses are major cause of morbidity and mortality and responsible for several epidemics and pandemics of respiratory diseases for last two centuries.

4. Influenza viruses Influenza viruses are divided into 3 immunogenic types: A, B, C TYPE A Isolated in 1933 by intranasal instillation of throat washing By Smith, Andrewes , Laidlaw TYPE B Isolated along with type A in cell culture in 1940 TYPE C Taylor (1949)isolated type C viruses

5. Morphology Spherical in shape , 80- 120nm in size Comprises of helical nucleocapsid (9nm), surrounded by an envelope. Viral RNA comprises of multiple segments of (- ve ) sense ss RNA. Each segments codes for specific protein Influenza A & B = 8 segments Influenza C= 7 segments Site of replication Occurs typically in nucleus ( in contrast to most other RNA viruses which replicate in cytoplasm)

6. Morphology of virus

7. Viral proteins having 8 structural proteins (PB1, PB2, PA, NP, HA, NA, M1 & M2) 2 non structural proteins (NS1 & NS2) PB1, PB2, & PA Polymerase protein Responsible for RNA transcription and replication. Nucleoprotein (NP) Major capsid protein Associated with viral RNA to form a ribonucleoprotein or nucleocapsid to form helical symmetry

8. Cont …. Matrix proteins: M1 protein: Major viral protein (40%) Forms a shell (protein layer) underneath the envelope. M2 protein: form ion channels in the envelope Help in transport of molecules. Non structural proteins: NS1 : interferon antagonist Inhibits m RNA splicing NS2 : help to export molecules across the nucleus

9. Hemagglutinin (HA) and neuraminidase(NA) are glycoprotein inserted into lipid envelop. Envelope : Lipoprotein in nature Lipid part derived from host cell membrane Protein part are virus coded. 10 nm long glycoproteins that are inserted into lipid envelope. 2 peplomers Hemagglutinin - triangular shaped peplomers , binds to mucin and sialic acid receptors on RBCs, resulting in clumping of RBC to cause hemagglutination . Bind to same receptors on respiratory epithelial cells and facilitates viral entry.

10. Neuraminidase – Mushroom shaped peplomers Present in fewer no then HA It is sialidase enzyme that degrade sialic receptors on RBC Help in Displaces HA from RBCs resulting in reversal of hemagglutination called elution. Facilitates release of virus from cell surfaces during budding by preventing self aggregation of virions to the host cells. NA helps the virus to pas through the mucin layer in the respiratory tract to reach the target epithelial cells.

11. # Primary structures of HA and NA polypeptides. # The cleavage of HA into HA1 and HA2 is necessary for virus to be infectious. # HA1 and HA2 remain linked by a disulfide bond (S–S). No posttranslational cleavage occurs with NA. Carbohydrate attachment sites (     ) are shown. # The hydrophobic amino acids that anchor the proteins in the viral membrane are located near the carboxyl terminal of HA and the amino terminal of NA.

12. Folding of the HA1 and HA2 polypeptides in an HA monomer. Five major antigenic sites (sites A–E) that undergo change are shown as shaded areas. The amino terminal of HA2 provides fusion activity (fusion peptide). The fusion particle is buried in the molecule until it is exposed by a conformational change induced by low pH. A

13. Structure of the HA trimer as it occurs on a virus particle or the surface of infected cells. Some of the sites involved in antigenic variation are shown (A). Carboxyl terminal residues (C) protrude through the membrane.

14. Structure of the NA tetramer. Each NA molecule has an active site on its upper surface. The amino terminal region (N) of the polypeptides anchors the complex in the membrane.

15. Resistance Inactivated by heating at 50 C for 30 minutes viable at 0 to 4 C for weeks and preserved for storage by freeze drying or storage at -70 C. Viable on fomites such as blanket and towel for 10-15 days and survives slow drying. Destroy infectivity by Formaldehyde Phenol Iodine Ether Salt of heavy metals

16. Antigenic structure and nomenclature Three genera (based on RNP and M proteins) A, B, C Subtype: based on (HA & NA antigens) Influenza A has distinct 16 H subtypes (H1 to H16) and 9 N subtypes ( N1- N9) Most of the subtypes infect animals and birds, but occasionally undergo genetic changes Infect human to cause major epidemics and pandemics Ex- H1,H2, H3, H5 & N1, N2 subtypes have been recovered from humans. Influenza B & C viruses though have subtypes : but are not designated.

17. The strandard nomenclature system for influenza viruses: Based on following information { influenza virus type/host/ geographical origin / strain no. / year of isolation( HA NA subtype} Human strain: influenza A/ Hong kong / 03/1968(H3N2) Nonhuman strain: influenza A/ swine/ lowa / 15/1930(H1N1)

18. Antigenic variation Changes in HA and NA occur independently. Internal proteins of the virus, such as the nucleoprotein (NP), do not undergo antigenic changes. Antigenic drift and antigenic shift account for antigenic changes in the two surface glycoproteins (HA and NA) of influenza virus.

19. Antigenic drift : It is a gradual change in antigenicity due to point mutations that affect major antigenic sites on the glycoprotein. Resulting in alteration of amino acid sequence on HA/NA Virus can escape recognition by the host immune system. New variants must contain > or = 2 mutation to become epidemiologically significant Ex- seen in influenza A and Infuenza B Result in outbreaks and minor periodic epidemics Occur more frequently every 2-3 yrs

20. Antigenic shift : it is an abrupt, major, drastic , discontinuous variation in the sequence of HA/NA change due to genetic reassortment with an unrelated strain of two or more unrelated strain infecting the same host cells Result in new strain unrelated antigenically to the predecessor strain Occur only in Influenza A viruses Results in pandemic and major epidemic Occur less frequently every 2-3 yrs

21. Epidemiology Influenza viruses occur worldwide and cause annual outbreaks of variable intensity . It is estimated that annual epidemics of seasonal influenza cause 3–5 million cases of severe illness and 250,000–500,000 deaths worldwide. The economic impact of influenza A outbreaks is significant because of the morbidity associated with infections. Influenza C is least significant; it causes mild, sporadic respiratory disease but not epidemic influenza. Influenza B sometimes causes epidemics, but influenza type A can sweep across continents and around the world in massive epidemics called pandemics. The incidence of influenza peaks during the winter. In the United States, influenza epidemics usually occur from January through April (and from May to August in the Southern Hemisphere). x

22. Influenza Virus Replication The viral multiplication cycle proceeds rapidly. There is the shut-off of host cell protein synthesis by about 3 hours postinfection , permitting selective translation of viral mRNAs. New progeny viruses are produced within 8–10 hours. There has following steps in viral multiplication Viral Attachment, Penetration, & Uncoating Transcription & Translation Viral RNA Replication Maturation

23. After receptor-mediated endocytosis, the viral ribonucleoprotein complexes are released into the cytoplasm and transported to the nucleus, where replication and transcription take place. Messenger RNAs are exported to the cytoplasm for translation. Early viral proteins required for replication and transcription are transported back to the nucleus. The assembly and budding of progeny virions occurs at the plasma membrane

24. Viral RNA Replication: The mechanisms that regulate the alternative transcription and replication are related to one or more of the viral nucleocapsid proteins . Templates for viral RNA synthesis remain coated with nucleoproteins . First step in genome replication is production of positive-strand copies of each segment. ( the 5' ends are not capped, and the 3' ends are neither truncated nor polyadenylated .) They can be recognized efficiently by the RNA-synthesizing machinery Frequencies of reassortment as high as 40% have been observed

25. Maturation: Nucleocapsids are assembled in the nucleus and move out to the cell surface. The glycoproteins, HA and NA, are synthesized in the endoplasmic reticulum modified and assembled into trimers and tetramers, inserted into the plasma membrane .

26. The M 1 protein serves as a bridge, linking the nucleocapsid to the cytoplasmic ends of the glycoproteins. Progeny virions bud off the cell. During this sequence of events, the HA is cleaved into HA1 and HA2 if the host cell possesses the appropriate proteolytic enzyme. The NA removes terminal sialic acids from cellular and viral surface glycoproteins, facilitating release of virus particles from the cell and preventing their aggregation.

27. Pathogenesis & Pathology Viral NA lowers the viscosity of the mucous film in the respiratory tract, laying bare the cellular surface receptors and promoting the spread of virus-containing fluid to lower portions of the tract . Transmission- Infected aerosols generated by coughs and sneezes Rarely via contact or fomites Small particles aerosols (<10µm) are more efficient in the transmission.

28. Target cell entry Ciliated columnar cells are more commonly infected, but it may also infect other cell eg . Alveolar cells, mucous gland cells, & alveolar macrophage Multiply locally Virus replicate in local infected cells & infectious daughter virions spread to adjacent cells over several hrs.

29. Spread Rarely spread to lower respiratory tract or spills over blood stream to involve extra pulmonary site. Local damage Causes cellular destruction, Desquamation of superficial mucosa of respiratory tract In basal layer of epithelium it causes Edema and mononuclear cell infiltration cytokine influx local symptoms. Local damage predisposes to secondary bacterial invasion.

30. Host immune response Humoral immunity It is predominant immunity responsible for resistance against influenza virus Immunity is type specific , subtype specific and long lasting Antibodies against HA and NA are protective in nature, as well as subtype specific Antibodies against other viral protein are not protective Antibodies against ribonucleoprotein are type specific and are useful against typing viral isolates as influenza A,B & C. All the 3 type of influenza virus are antigenically unrelated and there is no cross protection. Immunity may be incomplete, as reinfection with the same virus can occur

31. Original antigenic sin- When a previously infected individual gets a repeated infection with a different antigenic variant of influenza virus, antibodies are produced against both the subtype, but predominant response would be against the original strain, a phenomenon called “ original antigenic sin” Component of both cell mediated immunity and innate immunity are important in providing immunity against influenza infections.

32. Clinical manifestations Incubation period It is about 18- 72 hrs. Directly depends upon the inoculum size and immune status of the host. Uncomplicated influenza ( flu syndrome) Either asymptomatic or minor upper respiratory tract ( chills, headache, and dry cough, followed by high grade fever , myalgia and anorexia) Self limiting. Complication Develop secondary bacterial pneumonia . Common agents are staphylococcus , pneumococci and haemophilus infuenzae .

33. Conti… Other pulmonary complications Worsening of chronic obstructive pulmonary disease, exacerbation of chronic bronchitis and asthma. Reyes’s syndrome Fatty degeneration of liver with acute encephalopathy occurring in children and adults following aspirin and salicylate intake Etiology is unknown Often seen with influenza B, varicella zoster and rarely influenza A viral infections. Mortality rate is 10% to 40%

34. High risk groups: Age < 2yrs and > 65yrs Pregnancy Underlying chronic lung , cardiac, renal, hepatic, and CNS conditions Low immunity ( HIV infected) Older children are at high risk of developing croup , sinusitis, otitis media , high grade fever and diarrhea.

35. Laboratory Diagnosis Specimen collections: Nasal washings, gargles, and throat swabs are the best specimens for diagnostic testing Should be obtained within 3 days after the onset of symptoms Swabs with a synthetic tip ( polyester or dacrone swabs) are best swab for collection Transport: sample immediately put under viral transport media, kept at 4ºC during transport up to 4 days , thereafter at -70ºC.

36. Isolation of virus: Embryonated eggs and primary monkey kidney cell lines best method for isolation. Amniotic cavity of embryonated egg is preferred sit for inoculation. Allantoic cavity only support influenza A virus growth

37. Detection of viral growth in embryonated egg or cell lines : Hemadsorption test: detect the adsorption of RBCs onto the surfaces of infected cell lines that are coated with viral HA antigen; within 3-5 days of inoculation. Hemagglutination : detect in the culture fluid 5-7 days of inoculation by adding fowl or guinea pig RBC Type A virus: agglutinates guinea pig RBCs Type B : agglutinate both fowl and guinea pig RBCs. Type C : agglutinates fowl RBCs at 4ºC

38. Direct immunofluorescence test: Viral antigens directly detected in nasal aspirates by using fluorescent tagged antibodies. Rapid and less sensitive Molecular method: RT-PCR- sensitive , specific rapid method . detect specific HA and NA genes. Real time – RTPCR: It is a quantitative method Designed for the diagnosis of avian flu ( H5N1) & H1N1 flu infections.

39. Antibodies detection: HAI test ( hemagglutination inhibition test) Neutrilization test ELISA

40. TREATMENT : SPECIFIC ANTIVIRAL THERAPY IS AVAILABLE FOR INFLUENZA VIRUS INFECTIONS Neuraminidase inhibitors: Administered for Infuenza A and influenza B infections. DOC for A/ H1N1 2009 flu , A/ H5N1 avian flu and influenza B Dosage- oseltamivir ( Tamiflu 75 mg tablets) zanamivir (10 mg , inhalation form) Schedule: for treatment – given twice a day for 7 days for chemoprophylaxis – given OD . Duration depend upon clinical settings

41. Matrix protein M2 inhibitors: Amantadine & rimantadine Strain of A/H1N1 2009 flu and H5N1 avian flu and influenza B can develop resistance.

42. Preparation of Inactivated Viral Vaccines Inactivated influenza A and B virus vaccines are licensed for parenteral use in humans. Federal bodies and the World Health Organization make recommendations each year about which strains should be included in the vaccine . Selected seed strains are grown in embryonated eggs, the substrate used for vaccine production. Sometimes the natural isolates grow too poorly in eggs to permit vaccine production, in which case a reassortant virus is made in the laboratory. The reassortant virus, which carries the genes for the surface antigens of the desired vaccine with the replication genes from an egg-adapted laboratory virus, is then used for vaccine production.

43. Virus is harvested from the egg allantoic fluid, purified, concentrated by zonal centrifugation, and inactivated with formalin or   β  - propiolactone The quantity of HA is standardized in each vaccine dose (approximately 15  g of antigen), but the quantity of NA is not standardized, as it is more labile under purification and storage conditions. Each dose of vaccine contains the equivalent of about 10 billion virus particles. Vaccines are either whole virus (WV), subvirion (SV), or surface antigen preparations

44. Live-Virus Vaccines cold-adapted donor virus, able to grow at 25°C but not at 37°C—the temperature of the lower respiratory tract—should replicate in the nasopharynx , which has a cooler temperature (33°C). A live attenuated, cold-adapted, temperature-sensitive, trivalent influenza virus vaccine administered by nasal spray was licensed in the United States in 2003. It was the first live-virus influenza vaccine approved in the United States, as well as the first nasally administered vaccine in the United States

45. Use of Influenza Vaccines Annual influenza vaccination is recommended for all children aged 6 months to 18 years and for high-risk groups. These include individuals at increased risk of complications associated with influenza infection (those with either chronic heart or lung disease, including children with asthma, or metabolic or renal disorders; residents of nursing homes; persons infected with the human immunodeficiency virus [HIV]; and those 65 years of age and older) and persons who might transmit influenza to high-risk groups (medical personnel, employees in chronic care facilities, household members).

46. REFERENCES MEDICAL MICROBIOLOGY, Jawetz , Melnick & Adelberg’s , 27 Edition ESSENTIALS OF MEDICAL MICROBIOLOGY, Apurba Sankar Sastry , Sandhya Bhat K