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MICR 420 Emerging and Re-Emerging Infectious Diseases Lecture 8: Influenza Viruses Dr. Nancy McQueen & Dr. Edith Porter 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 What a virus? Not a living cell Acellular particle No cell membrane Consist of nucleic acid and protein only RNA or DNA, not both Protein coat : capsid Some are enveloped: membrane from host cell Infects living host cells to replicate All forms of cells can be infected by a virus Bacteria, archaea, eukaryotes Virus depends on host metabolism Viral genome subverts host cell’s machinery to reproduce Estimate of 1032 viruses on earth Transcription and replication of RNA viruses 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 What is influenza? “Influenza (the flu) is a contagious respiratory illness caused by influenza viruses. It can cause mild to severe illness, and at times can lead to death.” (http://www.cdc.gov/flu/about/disease/index.htm) Influenza Lung Normal Lung Figure 1. Gross and microscopic lesions from dog infected with highly pathogenic avian influenza (HPAI) H5N1. A) Severe congestion and edema in the lung. B) Lung histopathologic results showing severe pulmonary edema and hemorrhage with black-brown particles (hemosiderin) (magnification ×100). (cdc web site). http://www.biologyofhumanaging.com/s lides/lng120d1.jpg Brief history and epidemiology Influenza appears to have afflicted humans since ancient times. Epidemics of influenza Hippocrates in 412 BC Numerous epidemics in the middle ages 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. Spread of the Asian Influenza pandemic in 1957 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 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 World Health Organization Influenza Nomenclature Hemagglutinin subtype Influenza type Year of isolation A/Panama/2007/99 (H3N2) Geographic source Isolate number Neuraminidase subtype Morphology and nature of the genome SS (-) RNA genome Segmented genome (8 segments encode 11 proteins) Virions may be spherical or filamentous Influenza Virus Replication of Influenza Virus 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 Rapid evolution 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. Recombination by template switching Between homologous segments of two different strains More common in avian isolates 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 How do we acquire viruses from pigs? 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 made it a potent inducer of proinflammatory responses 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. Changes in PB1 enhanced viral replication PB1-F2 - promotes apoptosis 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 (not produced by all strains) 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 Death 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: Pathogenesis and clinical symptoms Fever Headache Chills Malaise Muscle aches As the fever declines runny nose coughing 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 Inactivation of eIF-4 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 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: Strain dependent differences in pathogenesis In humans variations in pathogenicity may be due to Lack, in other organs, of proteases that capable of cleaving the HA Interferon activity 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! Cytokine storm 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. 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. Hemagglutination assay Hemagglutination assay www.medicine.mcgill.ca/.../hemag_plate.jpg Serological/Immunological Methods Hemagglutination-Inhibition Assay – an indirect test for antibody against specific influenza virus types - Hemagglutination Inhibition www.cdc.gov/.../16/2/images/09-1733-Ft.gif Serological/Immunological Methods Immunofluorescence Enzyme immunoassay (EIA) Optical immunoassay 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 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 Prevention by 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. PB2 PB2 PB1 PB1 PA PA HA HA NA NA NP PB2 NP M PB1 M NS PA NS HA Attenuated Influenza Vaccine Virus NA NP M NS Attenuated Vaccine Virus To New Virus Type Virulent Wild Type Influenza Virus 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 WINTER - 2007 Should 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 Due to pneumonia that progresses to ARDS and multi-organ failure Fortunately, these strains have not yet shown signs of spreading efficiently among humans………. Avian influenza Cycle of Avian Influenza Viruses in Animals & Humans Domestic birds Natural avian influenza cycle Shore birds Direct bird to human transmission is also possible. Pandemic disease cycle Water fowl Mammals (primarily swine) No human-human spread Avian HA proteins preferentially recognize and bind to sialoligosaccharides terminated by N-acetylsialic acid linked to galactose by an α2,3 linkage. found on the respiratory epithelium of birds found on lower respiratory tract of humans along with α2,6 linkage only α2,6 linkage is found on the upper respiratory epithelium of humans Pneumonia more common Shift of drift could change this……. Attachment of influenza viruses Genes involved in H5N1 pathogenesis Avian Influenza pathogenesis Changes in polymerase allow for replication at lower temperatures of humans The avian viruses normally do not replicate well in the upper respiratory tract of humans (33º C); normally replicate in intestinal tract of birds (41º C) PB1-F2 causes apoptosis in macrophages which delays immune response; also enhances proinflammatory response NS1 is a potent inducer of pro-inflammatory cytokines (TNF) through PDZ domain HA binds to sialic acid residues with α2,3 linkage Nations With Confirmed Cases H5N1 Avian Influenza (February 2007) Cumulative Number of Confirmed Human Cases of Avian Influenza A/(H5N1 ) Reported to WHO 6 May 2010 Country 2003 2004 2005 2006 2007 2008 2009 2010 Total cases deaths cases deaths cases deaths cases deaths cases deaths cases deaths cases deaths cases deaths cases deaths Azerbaij an 0 0 0 0 0 0 8 5 0 0 0 0 0 0 0 0 8 5 Bangladesh 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 Cambodia 0 0 0 0 4 4 2 2 1 1 1 0 1 0 1 1 10 8 China 1 1 0 0 8 5 13 8 5 3 4 4 7 4 0 0 38 25 Djibouti 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 Egypt 0 0 0 0 0 0 18 10 25 9 8 4 39 4 19 7 109 34 Indonesia 0 0 0 0 20 13 55 45 42 37 24 20 21 19 3 2 165 136 Iraq 0 0 0 0 0 0 3 2 0 0 0 0 0 0 0 0 3 2 Lao People's 0 Democratic Republi c 0 0 0 0 0 0 0 2 2 0 0 0 0 0 0 2 2 Myanmar 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 0 Nigeria 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 1 1 Pakistan 0 0 0 0 0 0 0 0 3 1 0 0 0 0 0 0 3 1 Thail and 0 0 17 12 5 2 3 3 0 0 0 0 0 0 0 0 25 17 Turke y 0 0 0 0 0 0 12 4 0 0 0 0 0 0 0 0 12 4 Viet Nam 3 3 29 20 61 19 0 0 8 5 6 5 5 5 7 2 119 59 Total 4 4 46 32 98 43 115 79 88 59 44 33 73 32 30 12 498 294 Total number of cases includes number of deaths. WHO reports only laboratory-confirmed cases. All dates refer to onset of illness. Indonesia numbers indicate cumulative total of sporadic cases and deaths which occurred during 2009. Amantadine (Brand name Symmetrel: Treatment of influenza type A-2, but not type B). This drug cannot treat the cytokine storm associated with avia n influenza The pandemic H1N1 influenza virus In 2009 a new H1N1 strain was passed from pigs to humans The virus rapidly dispersed worldwide in a few months Ultimately found to not be more virulent in humans than the seasonal strains No genetic markers for severe disease detected HA - has only 1 arginine at the HA cleavage site - no enhanced cleavability PB1 - gene encodes a truncated, and most probably non-functional PB1-F2 protein PB2 lacks mutation in 627 and 701 that contribute to better replication in mammalian cells NS1 - has deletion of 11 carboxy-terminal encoding the PDZ domain H1N1 Pandemic 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. 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 Recombination by template switching 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) 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, et al. 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 Arias, Carlos F, et al. 2009. Molecular anatomy of 2009 influenza virus A (H1N1), Archives of Medical research 40:643-654. Thanh, Tran T., et al. (2008) Human H5N1 influenza: Current insight into pathogenesis. The Int’l J. of Biochem. and Cell Biol 40:3671-2674. Kuiken, Thijs and Taubenberger, Jeffery K. (2008) Pathology of Human Influenza revisited. Vaccine 26S:D59-D66. Basler, Christopher and Aguilar, Patricia V. (2008) Progress in identifying virulence determinants of the 1918 H1N1 and the Southeast Asian H5N1 influenza A viruses. Antiviral Research 79:166-178.