Influenza ORTHOMYXOVIRIDAE Nomenclature and Classification 1 Influenza

Influenza:

ORTHOMYXOVIRIDAE Nomenclature and Classification 1. Influenza family which is subdivided into 3 genera: Influenza A, Influenza B, and Influenza C, based on antigenic differences in the nucleoprotein (NP) and matrix (M) protein. 2. Influenza viruses are further characterized within type by antigenic differences associated with the H and N glycoproteins; there at least 14 subtypes of H and 9 subtypes of N proteins in influenza A virus. 3. All subtypes have been described in birds and some of them have been found in mammals. 4. To facilitate epidemiological studies, individual viruses are coded in the following manner:

Nomenclature A/equine/Saskatoon/1/90(H 3 N 8) group species location year Isolate number • A/equine/Prague/1/56(H 7 N 7) • A/fowl/Hong Kong/1/98(H 5 N 1) • A/swine/Lincoln/1/86(H 1 N 1) Serotype of HA and N

Virus characteristics 1. Medium sized (80 -120 nm diameter), enveloped, spherical to slightly pleomorphic in shape. 2. Genome consists of 8 segments of ss RNA which code for 10 proteins (5 structural, 3 associated with polymerase and 2 non-structural); genetic reassortment can occur 3. The envelope contains 2 glycoproteins: H (hemagglutinin), and N (neuraminidase) 4 genetic reassortment occur frequently 5. Can infect humans, birds, swine, equine, seals, mink and whales

Influenza virions nucleocapsid (RNA fragments wrapped in protein) envelope haemagglutinin and neuraminidase “spikes” In envelope 100 nm

Hemagglutinin and sialic acid Neuraminidase on receptor HA receptor binding site variable loops N active site variable loops

Influenza infection: humans Influenzavirus A horses pigs birds marine mammals Influenzavirus B Influenzavirus C humans swine humans

Distribution of HA serotypes in nature HA serotype Birds HA 1 HA 3 yes HA 4 yes HA 6 HA 7 HA 8 -14 yes yes Humans yes yes HA 5 Pigs yes HA 2 Horses yes yes

Distribution of N serotypes in nature Birds N 1 yes N 2 N 3 yes N 4 yes N 5 yes N 6 N 7 N 8 N 9 yes yes Horses Pigs yes yes yes

Evolution and Spread of flu viruses pigs poultry aquatic birds fecal/oral All HA and N serotypes humans H 1 N 1 H 3 N 2 H 1 N 1 H 2 N 2 H 3 N 2 (H 5 N 1, H 9 N 2) horses H 3 N 8 H 7 N 7 respiratory

Factors that sustain epizootics/epidemics n n Antigenic drift Reassortment and antigenic shift Short term immunity Cross species transfer

ORTHOMYXOVIRIDAE Antigenic Variation 1. Periodic epidemics of influenza Type A are due to antigenic shifts in the virus which are believed to occur through genetic re-assortment between human and animal (or mammalian and avian) viruses. For example, the amino acid sequence of H 3 protein of human isolates is more similar to the amino acid sequence of any Hx of mammalian or avian isolates than to the amino acid sequence of H 2 proteins of human isolates. Prior to 1957 H 1 N 1 1957 (Asian flu) 1968 (Hong Kong) 1977 H 1 N 1 H 2 N 2 H 3 N 2 2. Between epidemics, the influenza virus undergoes minor changes in the HA and N proteins These differences are due to multiple point mutations. These minor changes are referred to as antigenic drifts.

Reassortment

Pathogenesis of Influenza Viruses - Respiratory Form Day 1 1. 2. 3. Infection initiated by aerosol route; however, birds can also be infected by fecal/oral route b. entrapment of virions in mucus and their removal by the mucociliary transport system. Non-specific neutralization of virus by receptors mimicking glycoprotein present in the mucus. Interferon production. Day 1 - 3 1. 2. 3. 4. 5. Infection of individual epithelial cells of trachea Spread to contiguous cells resulting in loss of ciliary activity Destruction of goblet cells and mucus glands further compromises the mucociliary transport system. Destruction of cells initiates a local inflammatory response which results in increased amount of exudates and transudates. The surface of the trachea becomes increasingly anaerobic which provides optimum conditions for bacterial attachment and colonization.

Pathogenesis of Influenza Viruses - Respiratory Form Day 5 -9 1. Infection of the lung resulting in destruction of type I and II pneumocytes 2. Increasing accumulation of exudates and transudates, loss of surfactant produced by type II pneumocytes, blockage of airways and local hypoxia. 3. Concomitant reduction of macrophage and PMN activity. Secondary bacterial infections become established resulting in bronchial pneumonia. 4. Ig. A and Ig. G begin to appear in the upper and lower respiratory tracts, respectively. 5. Secretory Ig. A appears 8 dpi, reaching a peak in 11 days but declines rapidly. 6. Serum antibodies, detectable by HI and VN tests appear 3 to 7 days after infection, peaking in 14 days; may persist up to 18 months. 7. This is followed by recovery or by systemic spread of the virus 8. Antibody response in young animals is slower and less pronounced

Pathogenesis of Influenza Viruses - Respiratory Form 1. The best model to illustrate why some influenza infections become systemic and others do not is illustrated by the avian influenza virus. Essentially virulence is determined by the ease by which the HA protein can be cleaved. 2. The pathogenesis of avian influenza is quite different from that of mammals. The virus replicates in the intestinal tract as well as the respiratory tract and is readily isolated from the cloaca. 3. Virulent strains (H 5 and H 7) of AI cause viremia and generalized infection which is often complicated by secondary bacterial or viral infections. 4. Antibody assays in birds are complicated because the adult birds may have experienced infections with many different subtypes. 5. Antibody titers are often low, especially in ducks.

Epidemiology 1. In general, orthomyxoviruses are not stable in the environment; sensitive to heat (56 C, 30 min), acid p. H, and lipid solvents. Require close contact for transmission. 2. Exceptions are avian influenza viruses. Avian influenza is unique in that it can retain its infectivity for several weeks outside of its host. 3. Can spread by the aerosol route 4. Epidemiology of AI is poorly understood because of the role of wild birds, the great variety of different strains and the variable effects in different host species. 5. Wild ducks and geese are refractory to disease but wild ducks probably represent the most important reservoir for AI viruses. Virus is shed in secretions of respiratory tract and in feces. Survives for long time in feces.

Replication of Influenza Virus (1) Adsorption: the virus interacts with sialic acid-containing cell receptors via its hemagglutinin and enters via endocytosis. (2) Fusion and uncoating: the hemagglutinin undergoes a conformational change mediated by the acid environment of the endosome, which leads to the fusion of viral and cellular membranes. The ribonucleoprotein complexes are then transported into the nucleus. (3) Transcription and replication: the viral RNA is transcribed and replicated in the nucleus by the viral RNA polymerase (Two different species of RNA are synthesized from the viral RNA template: (a) full-length, positive-sense replicative intermediate RNAs, which are used by the polymerase to produce virion RNA and (b) m. RNAs.

Influenza virus replication HA cleaved by proteases HA binds to receptor virus buds lowered p. H, HA fuses membranes virus in phagolysosome RNA released N releases virus

Cleavage of HA binds receptor HA 0 penetrates cell Clara (mucus), extracellular, serum, bacterial proteases binds receptor HA 1 HA 2

HA cleavage and virulence May ‘ 94 -> June ‘ 94 PQ--RETR Dec ‘ 94 -> P Q R K T R Jan ‘ 95 low cleavability low virulence respiratory infection high cleavability high virulence systemic infecton

Secondary effects of HA HA oxygen free radicals anti oxidants Turns on genes for TNFa activates IL-1 NFKB IL-2 IL-6 bacterial products high fever cell damage cachexia shock

Influenza species specificity Receptor binding target cell Avian influenza a 2, 3, sialic acid and gal intestinal epithelial Human influenza a 2, 6 sialic acid-gal tracheal epithelial Pigs a 2, 3 and a 2, 6 sialic acid-gal tracheal epithelial Pigs are the mixing vessel for Influenza

The big pandemic of 1918

Major influenza subtypes that have circulated in humans and swine since the 1918 “Spanish Flu” pandemic. Richard Webby and Robert Webster, “Influenza in Humans: Impact, Evolution and Surveillance. ” 2001)

Relative frequency of influenza subtypes isolated from humans in recent years. * Richard Webby and Robert Webster, “Influenza in Humans: Impact, Evolution and Surveillance. ” , 2001) * The data represent worldwide frequencies that were obtained from: HTTP: //OMS 2. B 3 E. JUSSIEU. FR/FLUNET. The WHO influenza surveillance network homepage.

Schematic representation of the genetic reassortment events that lead to the development of the 1957 and 1968 pandemic strains of human influenza A viruses. 1918 H 1 N 1 1968 H 3 N 2 HA, N 2 NA, PB 1 Christopher Olsen, “The emergence of novel swine influenza viruses in North America, ” Elsevier Science B. V. , Article in Press, 2002. 1957 H 2 N 2 H 3 HA, PB 1

ORTHOMYXOVIRIDAE Human Influenza Viruses 1. Humans are susceptible to Types A, B, and C. 2. Type A causes the classical influenza with which we are familiar. 3. Type B causes a mild to severe influenza. Reye's syndrome which is characterized by rapidly progressive non-inflammatory encephalopathy and fatty infiltration of the liver leading to its dysfunction, has been associated with type B influenza virus (also with other viral-induced respiratory and enteric infections). 4. Type C influenza virus is associated with mild upper respiratory disease.

Clinical Signs and Symptoms of Human Influenza (1) Influenza viruses are spread from person-to-person primarily through the coughing and sneezing of infected persons. (2) The incubation period for influenza is 1 --4 days, with an average of 2 days. (3) Persons can be infectious starting the day before symptoms begin through approximately 5 days after illness onset; children can be infectious for a longer period. (4) Uncomplicated influenza illness is characterized by the abrupt onset of constitutional and respiratory signs and symptoms (e. g. , fever, myalgia, headache, severe malaise, nonproductive cough, sore throat, and rhinitis). Reported sensitivity and specificity of clinical definitions for influenza-like illness that include fever and cough have ranged from 63% to 78% and 55% to 71%, respectively, compared with viral culture. Sensitivity and predictive value of clinical definitions can vary, depending on the degree of co-circulation of other respiratory pathogens and the level of influenza activity (28). (5) Influenza illness typically resolves after several days for most persons, although cough and malaise can persist for >2 weeks. (6) In some persons, influenza can exacerbate underlying medical conditions (e. g. , pulmonary or cardiac disease), lead to secondary bacterial pneumonia or primary influenza viral pneumonia, or occur as part of a co-infection with other viral or bacterial pathogens. (7) Influenza infection has also been associated with encephalopathy, transverse myelitis, Reye syndrome, myositis, myocarditis, and pericarditis.

Equine influenza n n A 1 H 7 N 7 rare pockets in central Europe? ? A 2 H 3 N 8 annual epizootics World wide except - Australia, New Zealand, Iceland Highly contagious, rapid spread

ORTHOMYXOVIRIDAE Equine Influenza 1. Mild to acute upper respiratory disease. 2 subtypes: A/equine/1 and A/equine/2 2. Antigenic drift has been detected periodically: 3. Virus strain of avian origin was suspected in the outbreak in China. 4. Clinically, the disease is similar to that caused by equine herpesvirus type 4 (EHV-4) and equine rhinoviruses (Picornaviridae). 5. Interstitial myocarditis has been reported in horses suffering from acute influenza

Pathogenesis inhalation (infected animal or fomites) replication in epithelial cells upper RT

Clinical signs-EIV n n n Sudden onset Fever (39 -42), biphasic Loss of appetite Muscle soreness Dry cough Nasal discharge (serous ->mucopurulent) Clinical signs are similar to other respiratory diseases such as equine rhinopneumonities and viral arteritis Need lab testing

Exercise or Rest n Gross et al. 1998. Equine Vet. Jn. 30: 489 n Exercised group More severe disease n More weight loss n No difference in recovery time n Long term effects? ? n

Diagnosis n n Clinical signs Virus isolation Directagen Flu-A Serological tests n n HAI Single radial haemolysis

EIV Conventional vaccines n n n Inactivated, H 7 N 7 and H 3 N 8 isolates Adjuvant Most -> short lived protection n Revaccinate at 6 week intervals

EIV Intranasal, attenuated vaccine Heska Co. http: //www. heska. com/

Avian Influenza (AI) 1. Clinical signs/host: a. Inapparent to acute systemic disease in chickens b. Clinically affected birds may show respiratory, CNS and enteric signs of the disease c. The more severe form of the disease is sometimes referred to as "fowl plague. " d. Various species of wild birds, mainly waterfowl, constitute an important reservoir. e. Among domestic birds, chickens and turkeys are most likely to develop disease f. Pheasants, quail, guinea fowl and partridges are also susceptible

Avian Influenza (AI) 2. Virus types: a. All virulent strains that cause disease in chickens are of the H 5 and H 7 subtypes. b. However, not all subtypes of H 5 or H 7 cause the same severity of disease in chickens c. The amino acid sequence in the hinge region of the HA molecule determines virulence. A change in 1 to 4 amino acids in this region results in increased virulence. 3. Economic losses: a. Since 1980, infection of turkeys has become an economically important disease in many parts of USA. b. Losses arise from condemnation at processing plant due to air saculitis caused by secondary bacterial infections (E. coli) and losses in egg production. c. An outbreak in Pennsylvania and Virginia in 1983 -84, caused by an H 5 N 2 virus, resulted in the slaughter of > 17 million birds, with compensation and other costs in excess of $60 million.

Avian influenza n n n Pennsylvania - 1983 - $61, 000 Mexico - 1993 -4 - $$? Asymptomatic to fatal (sudden death) Kristi Askin, Tina Tuason, Elisabeth Ludlage http: //duke. usask. ca/~misra/virology/AVFLU/INFLUVIR. HTM

The emergence of H 5 N 1 influenza in Hong Kong. Robert G. Webster, “Influenza: An Emerging Disease, ” Emerging Infectious Diseases, ” Vol. 4 No. 3, July-Sept. , 1998 (URL: http: //www. cdc/gov/ncidod/eid/vol 4 no 3/webster. htm)

Swine Influenza 1. One principal subtype (H 1 N 1) but 2 variants within this subtype in United States up until 1998; one is common in Europe and the other in the USA. 2. Swine were infected with H 3 N 2 strains – 1998, reassorted virus from humans and birds 3. Clinically, upper respiratory disease which generally runs its course within a week. 4. Avoiding stress during infection usually results in lower mortality rate (< 1%). 5. Recovered animals either lose weight or their weight gains are reduced, attributing to economic loss to producers. 6. Outbreaks in swine occur in late fall and early winter. 7. Swine influenza virus (H 1 N 1) can infect turkeys and humans. In turkeys, it causes drop in egg production and increased number of abnormal eggs.

SIV in North America 1918 Influenza recognized clinically in pigs (Koen al 1918) 19191930 First swine influenza isolated. classical H 1 N 1(Shope 1931) 19201930 -1990’s Classical H 1 N 1 in North America (Hinshaw 1976. . , ) 19211976 Swine influenza vaccine in humans 19221997 -98 Appearance of H 3 N 2 (Zhou et al 1999) 1923 seropositive to H 3 (Olsen 2000) 19242000 H 1 N 2 reassortmant of H 1 N 1 and H 3 N 2 (Karasin 2000) 19252000 H 4 N 6 avian isolated from pigs (Karasin 2000)

Swine influenza n n n H 1 N 1 - two variants H 3 N 2 Ontario (1989 -92) n n n 53% H 1 N 1 17% H 3 N 2 (similar to human virus) 4% H 1 N 2 (similar to human virus)

Genotypes of H 3 N 2 influenza A viruses isolated from pigs in North America since 1997. Christopher Olsen, “The emergence of novel swine influenza viruses in North America, ” Elsevier Science B. V. , Article in Press, 2002.

Genotype of the H 1 N 2 influenza A viruses isolated from pigs in the United States since 1999. Christopher Olsen, “The emergence of novel swine influenza viruses in North America, ” Elsevier Science B. V. , Article in Press, 2002.

Swine Influenza: Zoonosis • 20 million deaths 1918 -19, due to swine influenza • Continued reports of humans with swine influenza death by H 1 N 1 strain of swine influenza (Kimura et al 1998 Mayo Clin Proc 73: 243) • All 10 islet recipients had antibodies to swine influenza (Butler Nature 391: 320. 1998)

Swine Influenza-zoonosis Classical swine influenza viruses can also be directly transmitted to humans as zoonotic infections, sometimes with fatal consequences. Human infections with swine influenza viruses have been documented in the U. S. at least 10 times since 1974, including fatal infections, as well as in Europe and in New Zealand. In addition, data suggest that zoonotic swine influenza virus infections may actually occur more routinely among people in regular contact with pigs than the relatively small number of documented cases would suggest.

Swine Influenza Disease n Respiratory Disease in epizootic n n Fever, lethargy, coughing, nasal/ocular discharge, off feed PRDC porcine respiratory disease complex n PRRSV, Mycoplasma

Swine Influenza-disease 8. The pathogenesis of swine and equine influenza virus infections resembles that in man. Influenza Virus-Induced Pneumonia in a Pig

Body temperature course after intratracheal inoculation with. H 1 N 1, H 3 N 2 or H 1 N 2 subtypes of SIV Kristen Van Reeth, “A New Look at Swine Influenza in Europe. ” 2001)

Relationship between H 1 antibody titer and protection against lung lesions. * Terri Wasmoen, “Immune Response to Swine Influenza Vaccination. ” 2001) * No data is available for H 3 N 2 at 10 and 20 or H 1 N 1 at 320 and 640 antibody titers (at publication. )

Diagnosis of SIV Virus detection Virus isolation egg inoculation (EI) Virus isolation-cell culture Membrane enzyme immunoassay-hu flu (EIA) Microwell enzyme immunoassay-hu flu IFA Immunohistochemistry(IHC) PCR Antibodies Hemagglutination Inhibition ELISA to H 1 or H 3

Maxi. Vac®-FLU Pharmaceutical Name Swine Influenza Vaccine, Killed Virus. Product Features and Benefits • Inactivated virus vaccine provides strong, durable immunity against swine flu. • Special oil-in-water adjuvant system helps stimulate strong immune response. • Protection against clinical disease shown in vaccinates challenged with highly virulent SIV. • Significantly reduces circulating virus in lungs after severe challenge which helps prevent costly setbacks due to lung tissue damage, consolidation, and opportunistic bacterial infections. • Reduced viral shedding helps prevent virulent virus spread. • Convenient vaccination schedule fits easily into any herd health program.

MMWR Recommendations and Reports April 20, 2001 / 50(RR 04); 1 -46 Prevention and Control of Influenza Recommendations of the Advisory Committee on Immunization Practices (ACIP) Advisory Committee on Immunization Practices Membership List, February 2001 CHAIRMAN John F. Modlin, M. D. Professor of Pediatrics and Medicine Dartmouth Medical School Lebanon, New Hampshire EXECUTIVE SECRETARY Dixie E. Snider, Jr. , M. D. , M. P. H. Associate Director for Science Centers for Disease Control and Prevention Atlanta, Georgia

Process for human influenza vaccines n n surveillance February meeting n n n Commonwealth Serum Labs (Australia) CDC (USA) Natl. Inst. For Medical Research (UK) European Inst. For Biological Standardization (EU) Food and Drug Admin. (USA)

Process for human influenza vaccines n March-April n n Genetic and antigenic characterization of approved strains Distribution by WHO to manufacturers Production of seed stock Tests for contaminants (bacteria, mycoplasma, viruses)

Process for human influenza vaccines n April-August n n n Vaccine production License application made Clinical trials (to be submitted before vaccination season)

Process for human influenza vaccines n August-September n Distribution begins

Human Influenza Vaccine Composition (20012002) 1. 2. 3. 4. Influenza vaccine contains three strains (i. e. , two type A and one type B. The vaccine is made from highly purified, egg-grown viruses that have been made noninfectious (i. e. , inactivated). Subvirion and purified surfaceantigen preparations are available. The trivalent influenza vaccine prepared for the 2001 --2002 season will include A/Moscow/10/99 (H 3 N 2)-like, A/New Caledonia/20/99 (H 1 N 1)-like, and B/Sichuan/379/99 -like antigens. To be administered IM

Flu vaccine recommendations 1. Persons at Increased Risk for Complications Vaccination is recommended for the following groups of persons who are at increased risk for complications from influenza: • persons aged >65 years; • residents of nursing homes and other chronic-care facilities that house persons of any age who have chronic medical conditions; • adults and children who have chronic disorders of the pulmonary or cardiovascular systems, including asthma; • adults and children who have required regular medical follow-up or hospitalization during the preceding year because of chronic metabolic diseases (including diabetes mellitus), renal dysfunction, hemoglobinopathies, or immunosuppression (including immunosuppression caused by medications or by human immunodeficiency [HIV] virus); • children and teenagers (aged 6 months--18 years) who are receiving long-term aspirin therapy and, therefore, might be at risk for developing Reye syndrome after influenza infection; and • women who will be in the second or third trimester of pregnancy during the influenza season. 2. Persons Aged 50 --64 Years Vaccination is recommended for persons aged 50 --64 years because this group has an increased prevalence of persons with high-risk conditions. 3. Persons Who Can Transmit Influenza to Those at High Risk Persons who are clinically or subclinically infected can transmit influenza virus to persons at high risk for complications from influenza. . The following groups should be vaccinated: • physicians, nurses, and other personnel in both hospital and outpatient-care settings, including emergency response workers; • employees of nursing homes and chronic-care facilities who have contact with patients or residents; • employees of assisted living and other residences for persons in groups at high risk; • persons who provide home care to persons in groups at high risk; and

Chemotherapy-humans n Prevent membrane fusion n Amantidine (Symmetrel) Remantidine (Flumadine) Neuraminidase inhibitors n n Zanamivir (Relenza) Oseltamivir (Tamiflu)

Human flu vaccine used Pasteur Merieux Connaught