1 / 57

MICR 454L

MICR 454L. Emerging and Re-Emerging Infectious Diseases Lecture 10: Influenza Viruses (Reading: Capturing a Killer Flu Virus) Dr. Nancy McQueen & Dr. Edith Porter. Overview. RNA viruses Influenza viruses Brief history Nomenclature Morphology and nature of the genome

feryal
Download Presentation

MICR 454L

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. MICR 454L Emerging and Re-Emerging Infectious Diseases Lecture 10: Influenza Viruses (Reading: Capturing a Killer Flu Virus) Dr. Nancy McQueen & Dr. Edith Porter

  2. Overview • RNA viruses • Influenza viruses • Brief history • Nomenclature • Morphology and nature of the genome • Viral replication cycle • Genetic variability • Pathogenesis and clinical symptoms • Diagnosis • Treatment • Prevention • Threat

  3. Transcription and Replication of RNA Viruses

  4. RNA viruses • Why are so many of the newly emerging infectious diseases caused by RNA viruses? • All must bring in their own RNA dependent RNA polymerases (replicases) • Are error-prone - error rate of 10-3- 10-5 • Have no proof reading function • Many quasi-species found in viral infections • Nitric oxide (NO) production by host accelerates viral mutations Rapid evolution

  5. Brief history and epidemiology • Influenza appears to have afflicted humans since ancient times. • Hippocrates in 412 BC • Numerous epidemics in the middle ages • Epidemics of influenza • Every winter • > 20,000 deaths/year • Elderly or immunocompromised individuals. • Pandemics • Irregular 10-50 year intervals. • The Spanish flu (1918-1920) - killed 20-40 million people worldwide • The Asian flu (1956-1957) - 60,000 deaths in North America.

  6. Spread of the Asian Influenza pandemic in 1957

  7. Nomenclature • Family Orthomyxoviridae • Myxo = mucus - virons bind to sialic acid residues in mucoproteins • Genus: Influenza Virus • Three groups which share a common structure and mode of replication, but differ serologically based on M and NP antigens • Type A infect humans and animals; epidemics and pandemics • Type B infects humans only; epidemics • Type C infects humans and pigs; mild disease

  8. Classification of Human Influenza Viruses • Type A or B • Geographic source • Isolate number • Year of isolation • Four HA: H0, H1, H2, H3 • Two NA: N1, N2 More on the significance of HA and NA will be discussed later on

  9. Hemagglutinin subtype Influenza type Year of isolation A/Panama/2007/99(H3N2) Isolate number Geographic source Neuraminidase subtype World Health OrganizationInfluenza Nomenclature

  10. Morphology and nature of the genome • SS (-) RNA genome • Segmented genome (8 segments encode 11 proteins) • Virions may be spherical or filamentous

  11. Influenza Virus (M2) Matrix protein (M1) (HA) (RNA + 3 polymerase proteins - PA, PB1, and PB2) (NP) (NA) medicineworld.org/images/blogs/9-2006/influenza-virus-82101.jpg

  12. Replication of Influenza Virus www.northwestern.edu/.../ pinto2/pinto_1big.jpg

  13. Why do we continue to have Influenza Virus Epidemics? • Genetic variability • Influenza virus keeps changing its structure via two different mechanisms: • Antigenic drift - changes in the antigenic determinants of the HA and NA that accumulate with time. (result in variants of the SAME NA or HA type) • Viral RNA polymerase • Error prone • No proofreading • Provides a selective advantage • Antigenic shift - major changes due to a re-assortment of genes that occurs when two different influenza viruses infect the same host. Rapid evolution

  14. Antigenic Shift Two different viruses infect the same pig and through re-assortment of the gene segments, a new virus is generated Human influenza virus Avian influenza virus New human influenza virus

  15. Genetic variability • The 1957 Asian influenza pandemic- antigenic shift (new HA, NA, and PB1) • The 1968 Hong Kong pandemic - antigenic shift (new HA and PB1) • 1918 Spanish influenza pandemic was not due to shift- new studies indicate that it arose from an avian virus by drift. • Enhanced cleavability of HA due to NA changes! • Changes in NS1 (and maybe NP and/or M) • A single mutation in HA resulted in a virus that had gained the ability to bind to sialic acid residues present in the human respiratory tract.

  16. Pathogenesis and clinical symptoms • Aerosol transmission • 3 day incubation (influenza A) • Virus initially infects epithelial cells in the upper respiratory tract • Loss of the ciliated epithelium • Direct effect of virus multiplication and release • Due to toxic oxygen radical formation (host cell response) • Due to apoptosis • dsRNA and NA may trigger host cell responses that contribute to apoptosis • PB1-F2 sensitizes cells to apoptosis

  17. Pathogenesis and clinical symptoms • With loss of ciliated epithelium: • Loss in the ability of the respiratory tract to clear viruses or bacteria by mucociliary flow • Secondary bacterial infections • Virus replication induces interferons and other cytokines (IL-6, IL-8, TNF-) leading to local and systemic inflammatory responses. • This results in the symptoms that define the “flu” syndrome: Death

  18. Pathogenesis and clinical symptoms • Fever • Headache • Chills • Malaise • Muscle aches • As the fever declines • runny nose • coughing

  19. Selected virulence characteristics • HA for attachment • Inhibition of host mRNA translation (establishing control of the host) • Cap snatching • Viral mRNAs compete more effectively for initiation factors. • Inactivation of the cap binding reaction by removing the required phosphate from eIF-4E, reducing available initiation factors • NS1 interferes with host cell mRNA splicing, polyadenylation, and transport to the cytoplasm

  20. Inactivation of eIF-4

  21. Summary of virulence characteristics • Evasion of host defenses • NS1 binds to dsRNA to inhibit activation of IFN • Damage • Induction of apoptosis - dsRNA, NA, and Pb1-F2 all play a role • NO and O2- • NO enhances development of more quasi-species • Induction of cytokines

  22. Strain Dependent Differences in Pathogenesis • Strain differences may result in differences in the severity of the disease for both human and avian viruses. • Aquatic birds are the natural reservoir for avian influenza A viruses • Is usually asymptomatic in feral birds • Highly pathogenic strains may cause serious systemic infections in domestic poultry • Due to the presence of a polybasic cleavage site in HA (Cleavage of HA at a basic residue by host cell proteases is required for viral infectivity) • For human viruses, systemic spread has not been documented. This may be due to:

  23. Strain Dependent Differences in Pathogenesis • Lack, in other organs, of proteases that capable of cleaving the HA • Interferon activity • In humans variations in pathogenicity may be due to • Differences in the effectiveness of NS1 to antagonize IFN / production • Differences in NA that allow binding of host proteases that assist in HA cleavage activation or activation of apoptosis • Those most likely to succumb to the disease are usually the elderly and the very young. Why? • The 1918 strain was an exception to this rule - it caused more severe symptoms in those who were the most immunocompetent! • Due to an overdeveloped immune response (“cytokine storm”) of the host against the virus!

  24. Diagnosis • Nasopharyngeal swabs, washes, or aspirates taken early in the course of the disease are the best specimens • The virus can be grown in the amniotic or allantoic cavity of embryonated chicken eggs, or in tissue culture cells with trypsin added to cleave HA.

  25. Diagnosis • May assay directly for the virus (direct assay) • May assay for antibodies, produced in the host, against the virus (indirect assay) • Hemagglutination assay-a direct method to identify the presence of the virus and to get a rough titer of the virus. • Is based on the ability of influenza viruses to agglutinate RBCs. • Virus is titered by making serial two-fold dilutions of the virus and determining the highest dilution of virus that causes agglutination of the RBCs.

  26. Hemagglutination assay

  27. Hemagglutination assay

  28. Serological/Immunological Methods • Hemagglutination-Inhibition Assay – an indirect test for antibody against specific influenza virus types -

  29. Serological/Immunological Methods • Immunofluorescence • Enzyme immunoassay (EIA) • Optical immunoassay

  30. Treatment • Amantidine and rimantidine – targets the M2 protein, blocking the ion channel it forms and preventing uncoating of the virus. • Only effective against Group A influenza viruses

  31. Treatment • Zanamivar (Relenza) and Oseltamivar (Tamiflu) – target the neuraminidase, inhibiting its activity and, therefore, inhibiting release of the virus. • Effective against both Groups A and B

  32. Prevention • Vaccination – need a new vaccine every year because of shift and drift of the virus • Whole inactivated virus - flu shot • Live, attenuated cold adapted virus (LAIV or FluMist) • Made by combining the HA and NA genes of the targeted virus strain with the six other gene segments from mutant viruses known to have restricted growth at 370C • Nasal-spray inoculation • The reassortment viruses cannot replicate in the lung at core body temperature, but grow well in the cooler nasal mucosa where they stimulate an excellent immune response.

  33. PB2 PB2 PB1 PB1 PA PA HA HA NA NA PB2 NP NP PB1 M M PA NS NS HA NA Attenuated Influenza Vaccine Virus Virulent Wild Type Influenza Virus NP M NS Attenuated Vaccine Virus To New Virus Type

  34. Vaccination • In development: • Subunit vaccines • Poxvirus recombinants expressing single viral proteins • Oligopeptides corresponding to the antigenic components of the HA protein • DNA-based vaccines • Target epitopes that are highly conserved in all influenza A viruses

  35. WINTER - 2007Should we be afraid of the avian (bird) flu? • Starting in 1997, a highly virulent avian form of influenza (H5N1) spread through the commercial poultry farms in Hong Kong. • It has now been found in many sites in Southeast Asia and a number of humans have been infected, with several resulting deaths • Due to apoptosis of alveolar epithelial cells and leukocytes? • Due to enhanced proinflammatory cytokine response (especially TNF)? • Fortunately, these strains have not yet shown signs of spreading efficiently among humans……….

  36. Avian Influenza • The avian viruses do not replicate well in the upper respiratory tract of humans (33º C) where the body temperature is cooler than the intestinal tract of birds (41º C) where the avian influenza virus normally replicates (may be due to polymerase proteins). • The avian influenza virus HA proteins preferentially recognize and bind to sialoligosaccharides terminated by N-acetylsialic acid linked to galactose by an α2,3 linkage. This linkage is found on the respiratory epithelium of birds while an α2,6 linkage is found on the respiratory epithelium of humans. • However, drift or shift could change this……..

  37. Nations With Confirmed Cases H5N1 Avian Influenza (February 2007)

  38. Threats • Every year in the United States, on average: • 5% to 20% of the population gets the flu; • more than 200,000 people are hospitalized from flu complications • about 36,000 people die from flu. • Research suggests that currently circulating strains of H5N1 viruses are becoming more capable of causing disease (pathogenic) in animals than were earlier H5N1 viruses. • Gambotto A, Barratt-Boyes SM, de Jong MD, Neumann G, Kawaoka Y.Human infection with highly pathogenic H5N1 influenza virus. Lancet. 2008 Apr 26;371(9622):1464-75. • Blendon RJ, Koonin LM, Benson JM, Cetron MS, Pollard WE, Mitchell EW, Weldon KJ, Herrmann MJ.Public response to community mitigation measures for pandemic influenza.Emerg Infect Dis. 2008 May;14(5):778-86. • Song D, Kang B, Lee C, Jung K, Ha G, Kang D, Park S, Park B, Oh J.Transmission of Avian Influenza Virus (H3N2) to Dogs.Emerg Infect Dis. 2008 May;14(5):741-6. • Blumenshine P, Reingold A, Egerter S, Mockenhaupt R, Braveman P, Marks J.Pandemic influenza planning in the United States from a health disparities perspective. Emerg Infect Dis. 2008 May;14(5):709-15.

  39. Take Home Message • Influenza virus epidemics and pandemics have occurred regularly since ancient times. • Influenza virus is an enveloped virus that has a SS, - RNA genome of eight segments • Influenza virus epidemics and pandemics continue to occur because of the genetic variability of the virus NA and HA due to • Drift- genetic mutations • Shift - genetic reassortment • Influenza infections are characterized by fever, headache, chills, malaise and muscle aches. • Secondary bacterial infections are common • Diagnosis is usually by immunological means • Treatment may target the HA or NA • New vaccines are needed every year • Live, attenuated cold adapted virus made by reassortment (FluMist)

  40. Resources • The Microbial Challenge, by Krasner; ASM Press; Washington DC; 2002. • Brock Biology of Microorganisms, by Madigan and Martinko, Pearson Prentice Hall, Upper Saddle River, NJ, 11th ed, 2006. • Microbiology: An Introduction, by Tortora, Funke and Case; Pearson Prentice Hall; 9th ed, 2007. • Fundamentals of Molecular Virology, by Nicholas Acheson; Wiley and Sons; 2007 • Suzanne L. Epstein, Terrence M. Tumpey, Julia A. Misplon, Chia-Yun Lo, Lynn A. Cooper, Kanta Subbarao, Mary Renshaw, Suryaprakash Sambhara, and Jacqueline M. Katz 2002. DNA Vaccine Expressing Conserved Influenza Virus Proteins Protective Against H5N1 Challenge Infection in Mice, Emerging Infectious Diseases • http://www.brown.edu/Courses/Bio_160/Projects1999/flu/vaccines.html • http://www.hhmi.org/biointeractive/museum/exhibit99/4_6b.html • http://www.pandemicflu.gov/ • http://www.who.int/csr/disease/avian_influenza/en/ • http://www.cdc.gov/flu/avian/outbreaks/current.htm

  41. Viral replication cycle • Attachment • The ligandon influenza virus is the hemagglutinin (HA) glycoprotein spike protein which is composed of three monomers to make a trimer. • Each monomer is composed of the 2 peptide subunits, HA1 and HA2, • HA is synthesized as a precursor, HA0 • During transport of HA to the cell surface, HA0 is cleaved into HA1 and HA2 that remain associated with each other through disulfide bonding • HA1 has a globular head that contains a conserved region that binds to the N-acetyl neuraminic acid (sialic acid) cellular receptor • HA2 spans the envelope and contains a region at its amino terminus called the fusion peptide that is released upon cleavage of HA0 into HA1 and HA2 • The fusion peptide functions to mediate fusion of the envelope of the virus with a host cell membrane during viral entry. • The fusion peptides of the three monomers are buried in the structure of the trimer and not available to mediate fusion until there is a pH dependent conformational change in the protein.

  42. The Glycoprotein Spike HA Protein of Influenza Virus Cleavage site HA1 HA2 Fusion peptide HA trimers

  43. Conformational change in HA at low pH

  44. Penetration and Uncoating • The virus HA binds to its sialic acid containing receptor • The virus enters the host cell through receptor mediated endocytosis (penetration) • When the pH in the endosome decreases, there is a conformational change in the HA of influenza which exposes the fusion peptides that were previously hidden within the trimer. • The peptides mediate fusion of the viral envelope with the endosomal envelope. • During acidification of the endosome, the M2 protein, which functions as an ion channel, allows H+ to penetrate the interior of the virion. • The low pH within the virion weakens the interaction of the matrix protein, M1, with the nucleocapsids (RNP), facilitating their release into the cytoplasm upon membrane fusion (uncoating). • The nucleocapsid (RNP) is transported into the nucleus via nuclear localization signals on the nucleoprotein and polymerase proteins

  45. Fusion after receptor mediated endocytosis

  46. pH dependent fusion

  47. Biosynthesis • Unlike most other RNA-containing viruses, influenza viruses replicate in the nucleus: • The nucleocapsids are first transported to the nucleus • Viral mRNAs are sent to the cytoplasm for translation • Viral polymerase and nucleocapsid proteins are subsequently sent back to the nucleus to direct genome replication and form nucleocapsids • Nucleocapsids are transported back to the cytoplasm for assembly • Why does influenza replicate in the nucleus?

  48. Biosynthesis • In addition to viral enzyme activities, for transcription influenza A virus requires host cell enzymatic activities that are found in the nucleus • M1 and NS2 proteins mediate movement in and out of the nucleus. • The virus requires host cell RNA polymerase II which is responsible for making host cell mRNAs – WHY? • The viral transcription complex for mRNA synthesis is composed of PB1, PB2 and PA. • PB2 recognizes the 5’ caps of host cell mRNAs and in conjunction with the viral RNA and PB1, cleaves off the 5’ cap plus 10-13 bases to use as a primer for + strand mRNA synthesis on the negative strand genomic template (cap snatching).

More Related